Morales J. The Innate Immune System in Health... Vol 2. 2022

Immunology and
Immune System Disorders
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Immunology and
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The Innate Immune System in Health and Disease:

From the Lab Bench Work to Its Clinical Implications. Volume 2
Jorge Morales-Montor (Editor)
2022. ISBN: 978-1-68507-510-1 (Hardcover)
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Jorge Morales-Montor (Editor)
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Jorge Morales-Montor
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The Innate Immune System

in Health and Disease
From the Lab Bench Work to
Its Clinical Implications. Volume 2
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Contents
Preface .......................................................................................... vii

Section 1 Innate Immunity and Inflammation ................................1
Chapter 1 The Role of Innate Lymphoid Cells
in Homeostasis and in the Pathogenesis
of Chronic Inflammatory Diseases ...................................3
Bibiana Patricia Ruiz-Sánchez
and Isabel Wong-Baeza
Chapter 2 The Innate Immune Response
and Its Modulation by Sex Hormones During
Chronic Lung Inflammation...........................................31
Mireya Becerra-Díaz
Chapter 3 Asthma beyond Adaptive Immunity:
Fighting Corticosteroid Resistance ................................61
Claudia Andrea Morales-Garay,
Claudia Hallal-Calleros
and Claudia A. Garay-Canales
Section 2 Innate Immunity and Non-Classic
Immunomodulators .........................................................87
Chapter 4 Arthritis Rheumatoid Onset
and Development: The Gaze of TLRS ...........................89
Laura del Carmen Sánchez García
Chapter 5 Modulation of the Innate Immune System
by Extracellular Vesicles ...............................................129
César Díaz-Godínez, Alejandra Garduño-Nieto,
Raúl Bobes-Ruíz and Julio César Carrero
vi Contents
Chapter 6 Enhanced Innate Immune Response:

A Novel Strategy to Interrupt the
Transmission of Diseases by Mosquitoes .....................235
Jorge Cime-Castillo, Fabiola Claudio-Piedras,
Valeria Vargas-Ponce de León
and Humberto Lanz-Mendoza
Chapter 7 Modulation of the Innate Immune System
by the Endocrine Disrupting Compounds
Bisphenol A (BPA) and Phtalathes ..............................271
Karen Elizabeth Nava-Castro,
Margarita Isabel Palacios-Arreola,
Victor Hugo Del Río-Araiza,
Mariana Segovia-Mendoza
and Jorge Morales-Montor
About the Editor .......................................................................................295
Index .........................................................................................297
Preface
The aim of this book, The Innate Immune System in Health and Disease: From
the Lab Bench Work to Its Clinical Implications – Volume 2, is to provide
updated information to scientists and clinicians that is valuable in their quest
to gather information, carry out new investigations, or to check on clinical
implications of the innate immune system function during disease. This book
is of high priority to people interested in an update on innate immunity.
Volume 2 examines topics such as the participation of the innate immune
system in homeostasis and in the pathogenesis of chronic inflammatory
diseases, the innate immune response and its modulation by sex hormones
during chronic lung inflammation, and asthma beyond adaptive immunity.
Moreover, the role of TLRS during arthritis rheumatoid onset and
development is discussed as well as the modulation of the innate immune
system by extracellular vesicles. Furthermore, a novel strategy to interrupt the
transmission of diseases by mosquitoes and the modulation of the innate
immune system by the endocrine disrupting compounds bisphenol A (BPA)
and phthalates are discussed.
The Innate Immune System in Health and Disease: From the Lab Bench
Work to its Clinical Implications – Volume 2 promises to be a must-have book
on the shelf of all people who want to know about the role of the basic
functioning of the innate immune system in several diseases of actual
relevance to human health.
Chapter 1 - Innate lymphoid cells (ILCs) are lymphocytes that lack
antigen-specific receptors. ILCs include NK cells, which were described in
1975, and lymphoid tissue inducer (LTi) cells, which were described in 1992,
but it also includes the ILC1, ILC2 and ILC3 subsets, which were described
in the late 2010s and produce cytokine profiles that are similar to those
produced by conventional Th1, Th2 and Th17 cells. NK cells are cytotoxic
lymphocytes that participate in the immune response to tumors and virus, and
LTi cells are involved in the early development of secondary and tertiary
lymphoid organs. In contrast, ILC1, ILC2 and ILC3 have been described as
viii Jorge Morales-Montor
helper ILCs, because their main effector function is the production of

cytokines that regulate other immune cells. ILC1 produce IFN-γ, which
activates macrophages, Th1 cells and NK cells. ILC2 produce IL-4, IL-5, IL-
9 and IL-13 and are thus involved in the pathogenesis of allergy and obesity.
ILC3 produce IL-17 and IL-22, and are important for intestinal homeostasis.
These three ILC subsets participate in the innate immune response to
infections, and in the immunopathology of many chronic inflammatory
diseases. Here the authors describe the role of ILCs during homeostasis, in the
immune response to pathogens and in the pathogenesis of obesity, cancer,
asthma, psoriasis and inflammatory bowel disease.
Chapter 2 - Historically, both basic and clinical biomedical research have
mainly focused on the study of male animals and human subjects, neglecting
important physiological differences between the two biological sexes. Now it
is known that sex hormones are involved in a number of events that mediate
functional differences between females and males, and that these differences
are important in lung diseases such as infections, asthma, and COPD. Sex bias
excluding females as research subjects has resulted in the poor development
of effective therapies for sick women. Here, the authors analyze the
differences in the innate immune response mediated by sex and sex hormones
during chronic lung inflammation. Moreover, the authors address the
importance of studying females and males in animal models, the translation to
women and men in these diseases, and the challenges that include sex and sex
hormones as a biological variable implies.
Chapter 3 - Asthma is a multifaceted heterogeneous syndrome
characterized by hyper-responsiveness of the respiratory tract, bronchial
obstruction, mucus overproduction, and airway remodeling. It is one of the
most rapidly growing disorders, which affects around one-third of the world’s
population. According to WHO statistics, around 461,000 deaths annualy are
attributed to severe disease annually, and most asthma-related deaths occur in
low and middle-income countries. There are increasing reports of resistance
to conventional therapies, such as steroids or bronchodilators. Misdiagnosed
or inadequate treatment leads to poor quality of life and creates a burden on
families, societies, and countries. Its heterogeneity is linked to its
multifactorial nature, which leads to various mechanistic inflammatory
pathways. The common misconception is that asthma is mainly set off by
common allergens like pollen or dust. Furthermore, asthma has been
commonly classified into two main types: allergic and nonallergic.
Nonetheless, this classification appears to be an oversimplification.
Nowadays, asthma can be classified in different endotypes, based on the
Preface ix
source of the stimulus that triggers the immunopathology; pollution particles,

fungus or viral infections, work environment, aspirin, etc. The pathogenesis
can also be related to the age of onset of the disease (childhood vs. late-onset
asthma), obesity, or even menstruation. Traditionally, airway inflammation in
asthma was considered exclusively part of adaptive immunity. Nonetheless,
the identification of innate lymphoid cells, the difference in the pathogenesis
according to the presence of eosinophils vs. neutrophils, an increased presence
of monocytes or dendritic cells, the secretion of different cytokines, and their
potential role in atopic disorders significantly contributes to determining the
inflammatory response that characterizes the different asthma phenotypes. In
fact, the pathophysiology of asthma shares common cascades and signaling
molecules in both types of immunity. Therefore, any immunological
mechanism leading to asthma also involves the activation of the innate
immune system. In this chapter, the authors address the innate immune
mechanisms of different endotypes involved in the diversity of phenotypes of
asthma, the approaches for an accurate diagnosis, and the current options for
its proper management. The deeper knowledge of the role of different cells of
the innate immune system, the mechanisms involved, and the identification of
asthma endotypes will lead to a better understanding of the pathophysiology
of this heterogeneous condition. Additionally, biomarkers involved in the
disease will provide a better diagnosis, which will help physicians offer a
safer, more precise, and more effective treatment focused on targeted
medicine.
Chapter 4 - Rheumatoid arthritis (RA) development as a chronic
inflammatory disease involves the early activation of innate immunity. RA
patient monocytes strongly express TLR2, TLR4, TLR5, TLR7, and TLR8
induced by their exogenous and endogenous ligands in synovial tissue that
activated the initial production of inflammatory cytokines, chemokines, and
destruction of the cartilage and bones by destructive enzymes. Fibroblast
hyperplasia is an anticipatory process during the development of RA, followed
by the persistence of inflammatory cytokine that recruits cells by enhancing
the expression of a broad range of inflammatory chemokines and TLRs.
Moreover, macrophages and monocytes contribute significantly to the release
of cytokines such TNF-alpha, IL1, and GM-SCF and the production of
proteases collagenase, gelatinase B, stromelysin, and leucocyte elastase that
contribute to the accelerated reabsorption bone process. Besides, some pieces
of evidence indicated that miRNAs act as key immunoregulators of TLRs and
their multiple components of the signaling pathways, including signaling
proteins, transcription factors, regulatory molecules, and cytokines. Thus,
x Jorge Morales-Montor
innate immunity control of RA is beyond what was expected before and opens
an extended therapeutic option that can control the pathology at very early
steps. This chapter reviews the innate central actors such as TLRs that act as
a trigger to develop non-clinical manifestations of RA.
Chapter 5 - Extracellular vesicles (EVs) include a wide range of structures
delimited by a lipid bilayer that are released into the extracellular space by
virtually any cell studied. EVs are classified according to their subcellular
origin and diameter into exosomes, microvesicles, apoptotic bodies and other
uncharacterized EVs. EVs are key structures in intercellular communication
allowing the exchange of numerous molecules that constitute their cargo,
including soluble and membrane proteins, peptides, carbohydrates, lipids,
DNA, and diverse RNA types. When EVs reach a target cell, they are usually
internalized by endocytosis, phagocytosis, or by fusion with the acceptor
membrane to deliver their cargo into the cytosol, thereby inducing changes in
the recipient cell. In the case of innate immune cells, EVs can influence many
processes, including maturation, activation, migration, cytokine and
chemokine release, antigen presentation and effector functions. The effects
can result in either activation or suppression of their function depending on
the source of EVs and the context in which they are produced. During a
pathological process, host cell EVs tend to promote inflammatory responses
that activate the innate and adaptive immunity to control the disease. In
contrast, EVs released by pathogens during infection diseases or EVs from
tumor cells during cancer tend to exert an important immunosuppressive role
on innate immune cells that prevent the resolution of the infection or favor the
development of tumors and metastatic niches, respectively. On the other hand,
during autoimmune diseases, EVs tend to promote the development and
maintenance of inflammation. However, due to their immunomodulatory
effects, EVs are promising tools for the treatment of many diseases in humans
and animals. Thus, immunotherapy based on EVs is a field that is gaining
popularity recently and it is envisioned that it may have a great impact on
processes such as transplantation and the treatment of acute inflammation.
Therefore, EVs play an important role in the maintenance of innate immune
homeostasis and their study will make an important contribution to
understanding the intricate communication of innate immune cells in health
and disease.
Chapter 6 - The immune system is an interweaving of molecules,
signaling pathways, transcription, and modulation of effectors whose purpose
is to control, mitigate or eradicate what is not proper. In vector-borne diseases
mosquitoes, the innate immune system has been characterized as highly
Preface xi
efficient in combating foreign organisms but sufficiently benevolent to

tolerate vector-borne diseases. These implications lead to replication,
dissemination, and ultimately the transmission of the pathogenic organisms
when feeding on a host. In recent years, it has been discovered that the innate
immune response of insects, including mosquitoes, can trigger an enhanced
immune response to the stimulus of a pathogen that it previously had an
encounter. This phenomenon has been called immune priming. This defense
mechanism allows effector molecules of the immune response to be alert and
act efficiently as an immune boost, which has earned it the name of "innate
immune memory." In this chapter, the authors will describe immune priming
and its implications as an enhancer of the immune response to control diseases
transmitted by mosquitoes, vectors of diseases of medical importance, and
reduce the tolerance of mosquitoes to the main organisms that they transmit.
Likewise, the authors emphasized its use as a possible tool for protecting
mosquitoes against a second encounter with a pathogen. The differences
between immune priming, classical immune memory, and the finding of
trained immunity in vertebrates will be put into context.
Chapter 7 - As a result of human socio-economic activity, industrial
wastes have increased distressingly. Plastic pollution is globally distributed
across the world due to its properties of buoyancy and durability. A big health
hazard is the sorption of toxicants to plastic while traveling through the
environment. Two broad classes of plastic-related chemicals are of critical
concern for human health—bisphenols and phthalates. Bisphenol A (BPA) is
an endocrine-disruptor compound (EDC) with estrogenic activity. It is used in
the production of materials that are used daily. The endocrine modulating
activity of BPA and its effects on reproductive health has been widely studied.
BPA also has effects on the immune system; however, they are poorly
investigated and the available data are inconclusive. Phthalates are also EDCs
used as plasticizers in a wide array of daily-use products. Since these
compounds are not covalently bound to the plastic matrix, they easily leach
out from it, leading to high human exposure. These compounds exert several
cell effects through modulating different endocrine pathways, such as
estrogen, androgen, PPARγ and AhR pathways. The exposure to both classes
of plastic derivatives during critical periods of development has detrimental
effects on human health. In this chapter, the authors have compiled the most
important of their effects on the function of the innate immune system.
Section 1. Innate Immunity and Inflammation
Chapter 1
The Role of Innate Lymphoid Cells

in Homeostasis and in the Pathogenesis
of Chronic Inflammatory Diseases
Bibiana Patricia Ruiz-Sánchez1,2,

and Isabel Wong-Baeza3
1Departamento de Bioquímica, Facultad de Medicina,
Universidad Nacional Autónoma de México, Mexico City, Mexico
2Facultad de Medicina, Universidad Westhill, Mexico City, Mexico
3Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas,
Instituto Politécnico Nacional, Mexico City, Mexico
Abstract
Innate lymphoid cells (ILCs) are lymphocytes that lack antigen-specific

receptors. ILCs include NK cells, which were described in 1975, and
lymphoid tissue inducer (LTi) cells, which were described in 1992, but it
also includes the ILC1, ILC2 and ILC3 subsets, which were described in
the late 2010s and produce cytokine profiles that are similar to those
produced by conventional Th1, Th2 and Th17 cells. NK cells are
cytotoxic lymphocytes that participate in the immune response to tumors
and virus, and LTi cells are involved in the early development of
secondary and tertiary lymphoid organs. In contrast, ILC1, ILC2 and
ILC3 have been described as helper ILCs, because their main effector
function is the production of cytokines that regulate other immune cells.
ILC1 produce IFN-γ, which activates macrophages, Th1 cells and NK

Corresponding Author’s E-mail: mwongb@ipn.mx.
In: The Innate Immune System in Health and Disease

Editor: Jorge Morales-Montor
ISBN: 978-1-68507-510-1
© 2022 Nova Science Publishers, Inc.
4 Bibiana Patricia Ruiz-Sánchez and Isabel Wong-Baeza
cells. ILC2 produce IL-4, IL-5, IL-9 and IL-13 and are thus involved in
the pathogenesis of allergy and obesity. ILC3 produce IL-17 and IL-22,
and are important for intestinal homeostasis. These three ILC subsets
participate in the innate immune response to infections, and in the
immunopathology of many chronic inflammatory diseases. Here we
describe the role of ILCs during homeostasis, in the immune response to
pathogens and in the pathogenesis of obesity, cancer, asthma, psoriasis
and inflammatory bowel disease.
Keywords: innate lymphoid cells, pathogen-associated molecular patterns,

NK cells, Th1, Th2, Th17
Introduction
The immune system protects against infections, and the innate immune system
is activated when it recognizes pathogen-associated molecular patterns
(PAMPs) through germline-encoded pattern recognition receptors (PRRs)
(Rajamuthiah and Mylonakis 2014). On the other hand, the T and B cells of
the adaptive immune system have highly specific antigen receptors (TCR and
BCR) that are generated by somatic DNA recombination and, in the case of
the BCR, are released as antibodies (Netea et al. 2015). The cells of the innate
immune system are activated at the site of infection, and a subset of these cells
(known as dendritic cells) migrates to the draining lymph nodes and processes
microbial antigens that are then presented to T cells. Dendritic cells also
express costimulatory molecules and cytokines that activate cytotoxic T cells,
and activate and promote the differentiation of helper T cells to specific
lineages (Inaba and Steinman 1985). Th1, Th2 and Th17 cells are
characterized by the expression of specific transcription factors (T-bet,
GATA-3 and RORt, respectively), and by the production of specific cytokine
profiles. Th1 cells produce a type 1 profile dominated by IFN, Th2 cells
produce a type 3 profile with IL-4, IL-5 and IL-13, and Th17 cells produce a
type 3 profile with IL-17 and IL-22 (Dumonde et al. 1969). Cytotoxic T cells
lyse their target cells, which express their activating epitope (Rosenau and
Moon 1961), while B cells release the main soluble effectors of adaptive
immunity: antibodies (Nossal and Lederberg 1958; Jerne and Nordin 1963).
In 1975, Kiessling et al. discovered splenic lymphocytes that eliminated
tumor cells in less than four hours, without requiring antigen presentation
(Kiessling, Klein, and Wigzell 1975). He named these lymphocytes, which
lacked TCR or BCR, as natural killer (NK) cells (Kiessling et al. 1976). In
The Role of Innate Lymphoid Cells in Homeostasis … 5
1992, Kelly et al. demonstrated that the lymphocytes that were found in the
secondary lymphoid organs during the early stages of embryogenesis lack the
expression of CD3, a TCR-associated molecule that is essential for T cell
activation (Kelly and Scollay 1992). These cells were classified as lymphoid
tissue inducer (LTi) cells (Kelly and Scollay 1992; Finke 2005). In 2008,
Satoh-Takayama et al. and Cella et al. identified intestinal lymphocytes that
lacked antigen-specific receptors and produced IL-22 (Satoh-Takayama et al.
2008; Cella et al. 2009), and so were classified as NK22 cells (Satoh-
Takayama et al. 2008). Other contemporary studies described lymphocytes
without characteristic T-cell lineage markers that nevertheless produced T-cell
associated cytokines, and these cells were called nuocytes (Neill et al. 2010),
innate helper cells (Price et al. 2010) and natural lymphocytes (Moro et al.
2010). Flow cytometry and genomic analysis led to the classification of these
cells into three subsets, each one expressing one of the transcription factors
that characterize Th1, Th2 and Th17 cells: T-bet, GATA-3 y RORt (Satoh-
Takayama et al. 2008). In 2010, Spits et al. coined the term innate lymphoid
cells (ILCs), which included IFN-producing ILC1, IL-4-, IL-5- and IL-13-
producing ILC2, and IL-17- and IL-22-producing ILC3. This initial
classification included NK cells as ILC1, and LTi cells as ILC3 (Spits et al.
2013) (Figure 1).
Figure 1. Discovering ILCs. Timeline of the main discoveries leading to the current
classification of ILCs. The image was created with BioRender.com.
Classification of ILCs
Single-cell RNA sequencing studies indicate that ILCs contain at least 50 cell
clusters (Gury-BenAri et al. 2016), but the current classification defines 5
groups: NK cells, ILC1, ILC2, ILC3 and LTi cells (Vivier et al. 2018). NK
cells express the transcription factor Eomes, and are activated by natural
cytotoxicity receptors (NCR) that recognize molecules expressed on infected
or tumor cells, or by the absence of MHC class I molecules that normally
engage killer-cell inhibitory receptors (KIR) (Erick and Brossay 2016).
Activated NK cells release perforin and granzymes, which cause cell lysis and
apoptosis (Montel et al. 1995). ILC1 express T-bet, are activated by IL-12,
and produce type 1 cytokines, including IFN, TNF and lymphotoxin
(C.S.N. Klose et al. 2014). ILC2 express GATA-3 (Hoyler et al. 2012), are
activated by IL-25, IL-33 and TSLP (Price et al. 2010; C.S.N. Klose et al.
2014) and produce type 2 cytokines, including IL-4, IL-5 and IL-13 (Neill et
al. 2010), but they can also produce IL-9, IL-2 (Sasaki et al. 2019) and other
molecules that are involved in tissue repair, like amphiregulin (C.S.N. Klose
et al. 2014). ILC3 express RORt (Takatori et al. 2009), are activated by IL-
1, IL-6 and IL-23 (Xu et al. 2012), and produce type 3 cytokines, including
IL-17 and IL-22 (Guo et al. 2015). ILC3 are classified according to their
expression of the chemokine receptor CCR6: CCR6+ ILC3 are LTi-like cells
(Takatori et al. 2009) and produce IL-22 (Hepworth et al. 2015; Hepworth et
al. 2013; Farkas and Ivanov 2015), while CCR6- ILC3 express NCR
(Mjosberg et al. 2012). ILC1, ILC2 and ILC3 have been described as helper
ILCs, because their main effector function is the production of cytokines that
regulate other immune cells.
Development and Differentiation of ILCs
The evolutionary origin of ILCs is not known exactly, but it is suggested that
NK cells and ILC2 arose with T cells and B cells in jawless vertebrates, while
ILC1 and ILC3 arose in bony fish and LTi cells arose more recently, in
amphibians (Vivier et al. 2016). ILCs are produced during hematopoiesis,
from a common lymphoid progenitor (CLP) cell (Miller et al. 2018), and thus
express CD45 (Scoville, Freud, and Caligiuri 2019). In mice, CLPs develop
into -lymphoid progenitors, which express Nfil3, a transcription factor that
drives the expression of Id2, an inhibitor of the E proteins that are required for
somatic recombination and T cell development (De Obaldia and Bhandoola

2015). -lymphoid progenitors develop into early innate lymphoid cell
progenitors (EILP), which differentiate into NK progenitors and common
helper innate lymphoid cell progenitors (CHILP). CHILP, in turn, develop into
LTi cell progenitors, and into CD127 (IL-7R)+ progenitors that give rise to
the helper ILCs: ILC1 (C.S.N. Klose et al. 2014), ILC2 (Hoyler et al. 2012)
and ILC3 (Scoville, Freud, and Caligiuri 2019). In humans, CLPs differentiate
into common ILC progenitors (CILPs) and into ILC progenitors (Scoville,
Freud, and Caligiuri 2019), which can be found in the fetal liver, the umbilical
cord and the peripheral blood and give rise to ILC1, ILC2 and ILC3 (Lim et
al. 2017) (Lim and Di Santo 2019). Human ILC1 require T-bet for their
development, but they also require the transcription factors Nfil3 and Runx3.
Similarly, ILC2 require ROR and Bcl11b in addition to GATA-3 (Hoyler et
al. 2012), and ILC3 require the transcription factors aryl hydrocarbon receptor
(AHR) and Id2 in addition to RORt (Vivier et al. 2018). Other transcription
factors, such as STATs and SMADs, are activated by extracellular signals and
influence ILC differentiation (Fernando et al. 2021) (Figure 2).
Figure 2. Development and differentiation of ILCs. The development of ILCs in

mice and in humans is shown. In both species, ILCs develop from a common
lymphoid progenitor (CLP), but the subsequent stages involve different precursor
cells in each species. The image was created with BioRender.com.
ILC Receptors and Other Surface Markers
ILCs detect environmental signals through their surface receptors, and these
receptors and other surface markers are used to identify and characterize each
ILC subset. Helper ILCs are CD45+, CD127 (IL-7R)+ and negative for
myeloid and lymphoid lineage markers. Murine ILCs express CD90 (Thy1),
and murine ILC1 express CD49a, CD253 (TRAIL), NK1.1, CD200R, CD69,
CD122 and Ly49. Murine ILC2 are characterized by the expression of IL-33R
(ST2), and they can also express CD278 (ICOS), IL-25R, the prostaglandin
D2 receptor CRTH2 (CD294) and KLRG1. Murine ILC3 can express many
NCR (NKp46, NKG2D) and the integrin CD49d (47) (Vivier et al. 2018).
In addition, intestinal ILC2 and ILC3 express CD117 (c-KIT), MHC class II
molecules, CD1d and the costimulatory molecules OX40L and CD30L
(Willinger 2019).
Human ILC1 express CD49a, CD253 (TRAIL), NKp44, NKp46 and
variable levels of CD69 (Vivier et al. 2018). Human ILC2 express CRTH2,
CD161, ICOS, IL-25R, KLRG1 and CD25, and human ILC3 express CD254
(RANKL), CD56 and NKp44 (Vivier et al. 2018); both ILC2 and ILC3
express CD117 (c-KIT). ILCs also express chemokine receptors, which
determine their anatomical distribution. In humans, ILC1 express CXCR3 and
ILC1 and ILC2 express CCR4. ILC2 can express CCR8, CCR10 and the
cutaneous leukocyte-associated antigen (CLA), which are associated with skin
homing; and CCR7 and CXCR5, which are associated with lung homing
(Ardain et al. 2019). ILC3 can express CCR6 and CXCR6 (Willinger 2019),
while LTi cells express CXCR5 and neuropilin (Vivier et al. 2018).
ILCs express PRRs that enable them to respond to microorganisms
(through PAMPs) and to damage-associated molecular patterns (DAMPs); the
main PRRs described on ILCs are Toll-like receptor (TLR)1, TLR2, TLR4
and TLR6 (Crellin et al. 2010) (Xu et al. 2015). ILCs also express receptors
for neurotransmitters, including 2 adrenergic receptors, which respond to
adrenaline and noradrenaline, and muscarinic receptors, which respond to
acetylcholine. In addition, ILCs can express receptors for many neuropeptides,
such as vasoactive intestinal peptide, neuromedin U and calcitonin gene-
related peptide, and receptors for neurotrophic factors. ILCs are regulated by
glucocorticoids, estrogens and androgens, by metabolites that activate AHR,
and by prostaglandins and leukotrienes that activate CRTH2 and CYSLTR1
on ILC2 (Jacquelot, Luong, and Seillet 2019). The receptors and surface
markers that are found on each ILC subset determine their activation,
anatomical distribution, regulation and functions.
Activation of ILCs
ILCs, through their many receptors, are activated in response to

microorganisms and tissue damage, and also in response to cytokines,
neuropeptides and hormones (Spits et al. 2013; Spits and Di Santo 2011; Eberl
2012). ILCs integrate all these signals, and so ILCs can respond in a tailored
way to infections and tissue alterations, even without antigen-specific
receptors.
In 2009, Takatori et al. found that IL-23 increases the production of IL-17
in Rag-/- mice, which lack T cells and B cells, but not in Rag-/- mice that are
also deficient in the common gamma chain (c-/-), required for IL-7 signaling.
As mentioned above, helper ILCs express CD127 (IL-7R) and are dependent
on IL-7 signaling for their development and survival, so Rag-/- c-/- mice lack
T cells, B cells and ILCs. This study demonstrated that ILCs produce IL-17, a
cytokine that is important for the antifungal immune responses (Takatori et al.
2009). A year later, Crellin et al. demonstrated that human ILCs isolated from
tonsils produced IL-5, IL-13 and IL-22 after activation with TLR2 ligands and
IL-2 (Crellin et al. 2010). These first studies established that ILCs produce
cytokines that were previously considered T-cell specific.
T-cell activation requires the activity of the transcription factors NF-B,
AP.1 and NFAT, while STAT transcription factors are responsible for T-cell
polarization. During ILC activation, the same transcription factors are
activated through different pathways. For example, in ILC2, DAMP receptors
activate NF-B, the leukotriene D4 receptor CYSLTR1 activates NFAT and
cytokine receptors activate STAT-6. These transcription factors act
synergistically with the lineage-determining transcription factor GATA-3 to
drive the expression of a type 2 cytokine profile (McGinty and von Moltke
2020). In general, ex vivo activation of ILCs with IL-12 and IL-15 leads to a
type 1 cytokine profile (IFN and TNF), while activation with IL-25 and IL-
33 leads to a type 2 profile (IL-5 and IL-13), and activation with IL-1 and
IL-23 leads to a type 3 profile (IL-17 and IL-22) (Bonne-Annee, Bush, and
Nutman 2019) (Table 1). Cytokines are essential for ILC activation, but the
effector function of these cells depends on their anatomical distribution and
on the specific tissue microenvironments that they experience.
Table 1. General characteristics of ILCs
ILCs Phenotype (in humans) Main activating cytokines Main effector cytokines
ILC1 CD49a IL-12 IFN 𝛄
CD253 (TRAIL) IL-15 TNF𝛂
NKp44
CD69
CXCR3
CCR4
ILC2 CRTH2 IL-25 IL-5
CD161 IL-33 IL-13
ICOS
IL-25R
KLRG1
CD25,
CD117
CCR4
CCR8
CCR10
CCR7
CXCR6
ILC3 CD117 IL-1𝛃 IL-17
CD254 (RANKL) IL-23 IL-22
CD56
CCR6
CXCR6
Anatomical Distribution of ILCs
ILCs and ILC progenitors are found in circulation even before birth (Miller et
al. 2018), and they are distributed to all organs, particularly to mucosal tissues
(Willinger 2019). ILCs are abundant in the umbilical cord, and the frequency
of these cells in circulation decreases with age (Vely et al. 2016; Ruiz-Sanchez
et al. 2017). In mice, ILCs can be transferred from the maternal milk to the
neonatal organs, mainly to the thymus, lung, stomach and intestine (Yu et al.
2020), although the role of these allogeneic cells in the mouse immune system
is still to be determined.
ILCs are more frequent in tissues than in the circulation. Initially, ILCs
were described as tissue-resident cells that were maintained through local
proliferation. However, the discovery of ILC progenitors in circulation
indicates that tissue ILCs can be replenished by these progenitors, which are
recruited by inflammatory signals and complete their differentiation in
response to the tissue microenvironment (Lim and Di Santo 2019). ILCs can
enter the lymph nodes through CD62L and CCR7, while the sphingosine-1
phosphate receptor-1 (S1PR1) promotes their entrance to the circulation
(Willinger 2019). The intestinal homing of ILCs is associated with retinoic
acid, which induces the expression of 47 and CCR9 in ILC1 and ILC3
(Willinger 2019; Spencer et al. 2014). ILCs are found in the mucosa of all the
gastrointestinal tract in mice and in non-human primates (Sonnenberg 2014);
LTi cells are found in the mucosa-associated lymphoid tissues, while NK cells,
ILC1, ILC2, and NCR+ CXCR6+ ILC3 are found in the lamina propria and
can migrate to Peyer patches during inflammation. ILC1 are also found in
salivary glands and in the epithelium, as intraepithelial ILC1 (Willinger 2019);
they are also found in the liver and in the peritoneal cavity (Krueger et al.
2017), in the uterus and in the adipose tissue (S. Wang, Wu, et al. 2020). ILC2
are found in the perivascular regions of the lung (Cortez, Robinette, and
Colonna 2015; Monticelli et al. 2011) and in the adipose tissue (Moro et al.
2010), while ILC3 are found in the lung (Carrega et al. 2015) and the vaginal
mucosa (Xu et al. 2015).
Role of ILCs in Tissue Homeostasis
ILCs are preferentially located in mucosal tissues, and their many receptors
allow them to respond to cytokines, hormones, neuropeptides and metabolites,
even in the absence of infection or tissue damage. Thus, ILCs are integrating
hubs that regulate the homeostatic functions of many tissues (Figure 3).
Several studies have explored the regulation of ILC2 by neuro-
transmitters, lipids and hormones. Noradrenaline decreases ILC2 proliferation
while neuromedin U promotes the secretion of IL-5, IL-13 and amphiregulin
by these cells (Jacquelot, Luong, and Seillet 2019). In mice that lack the
neuromedin U receptor (Nmur1), a challenge with house dust mite in a mouse
model of allergic airways inflammation does not cause immunopathology
(Wallrapp et al. 2017). Vasoactive intestinal peptide, which controls intestinal
relaxation after feeding, stimulates type 2 cytokine production by intestinal
and lung ILC2 (Jacquelot, Luong, and Seillet 2019). Intestinal ILC2 produce
IL-5 in response to the vasoactive intestinal peptide that is produced after food
ingestion, and this leads to eosinophil recruitment (Nussbaum et al. 2013).
ILC2 are also regulated by arachidonic acid-derived lipids: leukotriene D4,
through its receptor CYSLTR1, stimulates the production of IL-4, IL-5 and
IL-13 by these cells, and prostaglandin D2, through its receptor CRTH2,
stimulates ILC2 migration and accumulation in the lung. Prostaglandins E2
and I2 inhibit ILC2 and ILC3 cytokine secretion (Jacquelot, Luong, and Seillet
2019). Uterine ILC2 express estrogen receptors, and testosterone inhibits
ILC2 activation (Jacquelot, Luong, and Seillet 2019), indicating that ILC2 are
also regulated by hormones.
ILC2 are located in the intestine, the lungs and the visceral adipose tissue.
The absence of vitamin A leads to an increase of intestinal ILC2 (Spencer et
al. 2014), and a subset of small intestine ILC2 are required for the development
of glucose intolerance and diet-induced obesity (Sasaki et al. 2019). Lung
ILC2 participate in tissue repair after infection with H1N1 influenza virus,
through the production of IL-13 and amphiregulin (Monticelli et al. 2011). In
the visceral adipose tissue, ILC2 produce methionine-enkephalin peptides that
promote beiging of the adipose tissue, improve glucose metabolism and
reduce obesity (Vivier et al. 2018; Brestoff et al. 2015). In contrast, an excess
of NK cells and ILC1 in the adipose tissue promotes insulin resistance (Lee et
al. 2016; Vivier et al. 2018).
ILC3 are abundant in the intestine, and play an important role in the
interaction with the microbiota. Intestinal ILC3 produce IL-22, a cytokine that
induces colonocyte proliferation and IL-10 production (Satoh-Takayama et al.
2008). These ILC3 require AHR activation to produce IL-22 (Spencer et al.
2014; Vivier et al. 2018); AHR is activated by tryptophan derivatives and
dietary carotenoids, and by some microbial metabolites (Jacquelot, Luong, and
Seillet 2019). Intestinal ILC3 express the neuroregulatory receptor RET,
which is activated by glial cell-derived neurotrophic factors and is also
necessary for IL-22 production (Ibiza et al. 2016). In Rag-/- mice, ILC
depletion leads to an increase in bacterial translocation from the intestine to
circulation, which leads to neutrophilia, splenomegaly and increased serum
concentrations of IL-6 and TNF. The bacterial translocation is reversed by
recombinant IL-22, which promotes the production of the antimicrobial
peptides Reg3B, Reg3g, S100a8 and S100a9 in the intestine (Sonnenberg et
al. 2012). Another mechanism that promotes intestinal tolerance of the
commensal microorganisms occurs because ILC3 express MHC class II
molecules; these cells present antigens to memory T cells that are specific for
microbiota-derived epitopes and induce their apoptosis (Hepworth et al.
2015). Similarly, ILC3 cells from lymph nodes and mucosa-associated
lymphoid tissues express MHC class II, CD80, CD86, CD40, ICOSL and
PDL-1, and also induce the negative selection of T cells in the periphery
(Yamano et al. 2019).
ILCs are decreased in patients with common variable immunodeficiency,
while naïve B cells, but not other B cells, are increased; this observation
suggests that ILCs are important for B cell activation (Geier et al. 2017). In
fact, ILC3 provide B-cell activating factor (BAFF) to marginal zone B cells
(Ebihara 2020), and ILC3 can also induce the differentiation of regulatory T
cells, through the OX40L-OX40 signaling pathway (Deng et al. 2020)
(Ebihara 2020).
Figure 3. The role of ILCs in tissue homeostasis. The role of ILCs during
homeostasis depends on their anatomical distribution and on the particular
combination of cytokines, hormones, neuropeptides and metabolites that is present in
a given tissue. The image was created with BioRender.com.
Role of ILCs in the Immune Response to Pathogens
During infections, the composition of the tissue ILC pool is altered by their
proliferation, redistribution, differentiation, and death. Newly-recruited ILCs
adapt to the tissue microenvironment and change their phenotype accordingly,
in a process known as plasticity or trans-differentiation (Schulz-Kuhnt et al.
2020) (Figure 4).
ILC2 have been extensively studied in the immune response against
parasites and fungi. These cells are the mayor producers of IL-13 in response
to an infection with Nippostrongylus brasiliensis (Neill et al. 2010). In Rag-/-
mice, but not in Rag-/- c-/- mice that lack ILCs, the administration of IL-25
leads to the elimination of this nematode (Price et al. 2010). IL-13 eliminates
the nematode by inducing mucus production in the intestine (Hoyler et al.
2012), and this cytokine is also associated with smooth muscle contraction
(Spencer et al. 2014). In patients infected with the nematodes Loa loa,
Wuchereria bancrofti or Onchocerca volvulus, the numbers of IL-4-, IL-5-
and IL-13-producing ILCs are increased (Boyd, Ribeiro, and Nutman 2014).
However, in the case of Toxoplasma gondii, an intracellular parasite, IFN and
TNF production by ILC1 and ex-ILC3 is required to control the infection
(C.S.N. Klose et al. 2014). ILC2 promote eosinophil infiltration to the lungs
in response to the ascomycete Alternaria spp., and this infiltration is
associated with bronchoconstriction and increased mucus production (Kita
2015). In contrast, ILC3 are important for the immune response against
Candida spp. in a mouse model of oropharyngeal infection (Gladiator et al.
2013).
ILCs also participate in the immune response against bacteria. ILC3 are
required to control infections with Citrobacter rodentium in mice (Satoh-
Takayama et al. 2008), and the resistance to C. rodentium colonization is
mediated by IL-22-dependent modulation of the intestinal microbiota (Guo et
al. 2015). ILC3 are also required to control infections with Salmonella
typhimurium in mice, but these ILC3 co-express the transcription factors
RORt and T-bet (“ex-ILC3”) and produce IFN (C.S. Klose et al. 2013). In
these intestinal infections, IFN is required to control the infection, but IL-22
is required to promote tissue repair; this combined response promotes survival
in mice (Abt et al. 2015). ILC3 also participate in the immune response against
Mycobacterium tuberculosis. In a mouse model of this infection, ILC3 are the
first lymphocytes recruited to the lung, through CXCR5, and these cells are
associated with increased numbers of alveolar macrophages. The production
of IL-17 and IL-22 by ILC3 is necessary to control the bacterial load in the
lungs of infected mice (Ardain et al. 2019). Finally, ILCs also participate in
the pathogenesis of sepsis, which is caused by a systemic inflammatory
response to infection. During sepsis, circulating ILC1 and ILC3 express
caspase-3, which suggests that they undergo apoptosis. These ILCs decrease
their expression of MHC class II molecules, but increase their expression of
activating molecules like NKp46 and NKp44, and maintain their capacity to
produce TNFα after stimulation with TLR ligands (Cruz-Zarate et al. 2018).
In non-human primates infected with the simian immunodeficiency virus,
ILCs are significantly reduced during the acute and chronic infection phases
(Xu et al. 2012). The loss of ILCs is caused by apoptosis and is reflected in a
reduced production of IL-17 and an increased production of IFN, TNF and
MIP1 (H. Li, Richert-Spuhler, et al. 2014). ILC3 undergo apoptosis in

response to TLR ligands, and the loss of these cells leads to alterations in the
intestinal barrier function and to increased bacterial translocation from the gut
to the circulation. Early administration of highly active antiretroviral therapy
limits the loss of mucosal ILCs, which decreases opportunistic infections and
delays the appearance of the acquired immunodeficiency syndrome (Mudd
and Brenchley 2016).
Role of ILCS in the Immunopathology of Chronic

Inflammatory Diseases
ILCs are activated in response to pathogens, but they can also be activated in
non-infectious conditions (C.S. Klose and Artis 2016; Russell and Walsh
2012), as occurs during intestinal inflammation (Sonnenberg and Artis 2015),
obesity and cancer (Vivier et al. 2018; Lee et al. 2016), and also during allergy,
atopic dermatitis and chronic sinusitis (Ruiz-Sanchez et al. 2017; Ebbo et al.
2017) (Figure 4).
ILC1-Associated Diseases
Obesity is a multifactorial disease with many complications. The chronic

inflammation that is observed in these patients is associated with a loss of
ILC2 in subcutaneous adipose tissue, which alters thermogenesis and glucose
metabolism (Brestoff et al. 2015; Flach and Diefenbach 2015). The loss of
ILC2 promotes a pro-inflammatory microenvironment in the adipose tissue,
and an infiltration of M1 macrophages that, in a positive feedback loop, release
pro-inflammatory cytokines and promote the infiltration of NK cells and
ILC1. All these changes in the adipose tissue lead to insulin resistance (Lee et
al. 2016; Vivier et al. 2018). Obesity and insulin resistance can lead to the
development of metabolic syndrome and its associated cardiovascular risks.
ILC1 have been implicated in the progression of atherosclerosis, because IFN
promotes lipid deposits, which increase inflammation, and leukocyte
infiltration, which contributes to arterial wall thickening (Gong, Xia, and Su
2021). ILC2 can limit this atherosclerosis progression, at least in mice
(Engelbertsen et al. 2015).
Inflammation is often associated with cancer progression. In a mouse

model of hepatic carcinoma, the presence of Helicobacter hepaticus promotes
the infiltration of ILC1-like cells (CD3- NK1.1+) that produce IFN. This
cytokine decreases the expression of E-cadherin and contributes to the
transition of epithelial cells to mesenchymal cells that participate in tumor
invasiveness and metastasis (Han, Huang, and Han 2019).
ILC2 have been associated to the immunopathology of type I hypersensitivity

(Ebbo et al. 2017; Walker, Barlow, and McKenzie 2013). Asthma is a chronic
disease that is a consequence of this hypersensitivity in the airways; it is
caused by the thickening and inflammation of the bronchial mucosa and by
Goblet cell hyperplasia that leads to mucus hypersecretion. In the chronic
phases, fibrosis and neutrophil and eosinophil infiltration are also present. In
these patients, lung ILC2 have increased levels of the prostaglandin D2
receptor CRTH2, and this leads to increased production of IL-5, IL-9 and IL-
13, three cytokines that are directly implicated in asthma immunopathology
(Klein Wolterink et al. 2012; Wilhelm et al. 2011). Leukotriene D4, through
its receptor CYSLTR1, contributes to tissue damage by recruiting eosinophils
(Kim, Wojno, and Artis 2013). Pediatric patients with severe therapy-resistant
asthma have increased numbers of ILC2, eosinophils and Th2 cells, compared
to patients with difficult to control asthma and to healthy individuals, but ILC2
are the cells that produce the highest levels of IL-13 and are associated with
severity (Nagakumar et al. 2019). IL-5- and IL-13-producing ILC2 are also
associated with atopic dermatitis, a disease that is characterized by chronic
inflammation of the skin. In this case, ILC2 are activated by DAMPs (Salimi
et al. 2013) and by nociceptive stimuli (Park et al. 2013).
ILC2 activation can be associated with the resolution of inflammation or
with fibrotic processes (S. Wang, Wu, et al. 2020). During heart inflammation,
cardiac fibroblasts release IL-33, which activates ILCs and induces their
release of IL-5 and IL-33, and this type 2 profile attenuates the inflammatory
response (Gong, Xia, and Su 2021). IL-33 is significantly increased in the
blood of patients with biliary atresia, and this cytokine stimulates the
proliferation of cholangiocytes, which is associated with the development of
cholangiocarcinomas. A depletion of ILC2 decreases this proliferation (J. Li,
Razumilava, et al. 2014). ILC2 are increased in the blood of patients with
gastric carcinoma, and these cells promote tumor infiltration with M2
macrophages and with myeloid-derived suppressor cells (Bie et al. 2014).

ILC2 have been associated with carcinogenesis in breast cancer (Jovanovic et
al. 2014), gastric cancer (Chan et al. 2014) and prostate and bladder cancer (S.
Wang, Wu, et al. 2020).
ILC3 are particularly abundant in the skin and the intestine. Psoriasis is an
auto-inflammatory skin disease characterized by erythematous and
desquamative plaques. ILC3 are increased in the blood of patients with
psoriasis, and they also infiltrate the affected skin (Villanova et al. 2014).
Inflammatory bowel disease is characterized by a chronic inflammation that
damages the gastrointestinal tract, and is characterized by persistent diarrhea,
weight loss, abdominal pain and fatigue; this disease is associated with ILC3
dysfunction (Hepworth et al. 2015). In patients with Crohn’s disease, a form
of inflammatory bowel disease, ILC3 have decreased expression of MHC
class II molecules, and this is associated with increased numbers of
microbiota-specific Th1 cells that produce IFN and TNF. These Th1 cells
are associated with inflammation and with the loss of the intestinal
architecture (Hepworth et al. 2015). However, ILC3-derived IL-17 can
promote chronic inflammation and the development of adenomas (Chan et al.
2014). ILC3 are located in the margins and the interior of colon cancers, and
these cells express MHC class II molecules and can activate specific T cells in
the presence of IL-2 and IL-1, but not in the presence of TGF (Rao et al.
2020).
ILC3 have been associated with other pathologies. In the heart, ILC3
release IL-17 in response to the IL-1 that is produced by macrophages that
ingest oxidized low-density lipoproteins, and this pathway increases cardiac
inflammation (Gong, Xia, and Su 2021). In patients with autoimmune
thrombocytopenia, treatment with intravenous immunoglobulin improves the
platelet counts and increases the numbers of regulatory T cells, which limit
ILC1 and ILC3 proliferation (S.C. Wang, Yang, et al. 2020). In some cancers,
such as breast cancer, squamous cell carcinoma of the lung (Carrega et al.
2015) and melanoma, the role of ILC3 remains controversial (S. Wang, Wu,
et al. 2020; Mattner and Wirtz 2017).
Figure 4. The role of ILCs in the immune response to pathogens and in chronic
diseases. During infections and chronic diseases, the tissue microenvironment is
modified by the response to PAMPs and DAMPs. The tissue ILC pool is altered by
proliferation, redistribution, differentiation, and death, and ILCs adapt to the new
tissue microenvironment by changing their phenotype and their secreted cytokines,
thus affecting the recruitment and response of other immune cells. The image was
created with BioRender.com.
Conclusion
ILCs were formally described only 10 years ago, as a cell group that is found
in vertebrates and has some functional redundancy with the T cells of adaptive
immunity. These cells lack antigen-specific receptors, but nevertheless can
provide tailored responses to many infectious and non-infectious threats,
through their many receptors for PAMPs, DAMPs, cytokines, hormones,
neuropeptides and metabolites that allow a very precise sensing of the
microenvironment. Helper ILCs include ILC1, ILC2 and ILC3, and each
subset has a particular tissue distribution and contributes to tissue homeostasis
through different mechanisms. However, immunological, neurological,
metabolic and endocrinological alterations impact ILCs, and condition the
progression of many chronic inflammatory diseases.
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705.
Chapter 2
The Innate Immune Response

and Its Modulation by Sex Hormones During
Chronic Lung Inflammation
Mireya Becerra-Díaz*
Johns Hopkins University, School of Medicine,
Baltimore, Maryland, USA
Abstract
Historically, both basic and clinical biomedical research have mainly

focused on the study of male animals and human subjects, neglecting
important physiological differences between the two biological sexes.
Now it is known that sex hormones are involved in a number of events
that mediate functional differences between females and males, and that
these differences are important in lung diseases such as infections,
asthma, and COPD. Sex bias excluding females as research subjects has
resulted in the poor development of effective therapies for sick women.
Here, we analyze the differences in the innate immune response mediated
by sex and sex hormones during chronic lung inflammation. Moreover,
we address the importance of studying females and males in animal
models, the translation to women and men in these diseases, and the
challenges that include sex and sex hormones as a biological variable
implies.
*
Corresponding Author’s E-mail: ayerim_mbd@hotmail.com.

ISBN: 978-1-68507-510-1
32 Mireya Becerra-Díaz
Keywords: cancer, inflammation, metastasis, tumor-associated

neutrophil, elastase, neutrophil extracellular traps, immunotherapy
Introduction
Beginning in 2016, the US National Institutes of Health (NIH) called for the
consideration of sex as a biological variable (SABV) in NIH-funded research
on vertebrate animals and humans, meaning including male and female cells
and subjects in basic and preclinical investigations. This requirement implied
including male and female research subjects in analysis and reporting or
providing strong justification for single-sex investigations. This new policy
aimed to enhance the value of biomedical science and correct the historical
negligence of not considering this variable, masking important differences in
many reports. Before the establishment of this policy, less than 30% of
biomedical reports analyzed data by sex, included sex in their statistical
analyses, or justified the lack of this analysis.
The consequences of not including both males and females in an
experimental design, and the lack of segregation by sex, had resulted in a
heterogeneity of data and poor reproducibility of results since different
researchers could use different male/female ratios in the study subjects without
reporting it. In general, female subjects have been the big absent in the
majority of preclinical studies in the previous decades. This absence has
impacted women´s health: up to 8 out of 10 drugs withdrawn from the US
market from 1997–2000 had greater adverse effects in women, either due to
unanticipated gender-prescribing trends or sex-specific adverse drug
reactions.
Men and women, as well as male and female animals used in animal
models, are quite different between sexes, with sex hormones mediating a
variety of these differences. Sex hormones, also known as sex steroids or
gonadal steroids, are cholesterol-derived molecules that act as
major modulators of the sexual phenotype. Besides driving sexual
development, sex hormones are responsible for the development of male and
female secondary sexual characteristics during puberty: breast and hips
development in females and growth of facial hair and deep voice in males, to
name a few examples. Androgens, the main male sex hormones, and estrogen
and progesterone, the female sex hormones, are predominately produced in
the gonads and partly in the adrenal gland cortex. Although sex hormones are
The Innate Immune Response and Its Modulation … 33
found in circulation in both men and women, they are present in different
concentrations, and therefore mediate important differences in physiological
responses and processes between sexes. There are reports highlighting
significant sex-based differences in immune responses. However, less than
10% of immunology reports analyze data by sex, thus, ranking the lowest of
ten biological disciplines for reporting the sex of the animal or human subjects
in published papers. Therefore, in this chapter, we will focus on analyzing how
sex hormones modulate the innate immune response in the context of lung
inflammation.
The innate immune response initiates upon recognition of pathogens,
pathogen-associated molecular patterns (PAMPs), danger-associated
molecular patterns (DAMPs), and damaged tissues by a group of conserved
molecules named pattern recognition molecules (PRMs). The PRMs can be
expressed or released by innate immune cells and other cell types and
participate in discriminating self versus non-self molecules. The innate
immune system is compounded by a network of immune cells, including
innate myeloid cells, natural killer cells (NKs), and innate lymphoid cells
(ILCs). Innate immune cells are able to detect unique and conserved pathogen-
associated molecular patterns through the expression of pattern recognition
receptors (PRRs) and represent the first line of defense against pathological
conditions such as infection, cancer, or autoimmunity, among other diseases.
Both the innate immune response and the adaptative immune response present
important differences depending on the biological sex, understanding
biological sex as the set of chromosomes, genes, and genitalia that
differentiate between females and males. Particularly in humans, besides sex,
gender, which involves social activities and behaviors, also impacts the
immune response. It can influence decisions that could impact a person's
health status by modulating the exposure to infections, as wells as the seeking
for healthcare. Although gender also participates as a modulator of the
immune response, in this chapter, we will focus on the role of sex and sex
hormones and their importance as modulators of the innate immune response
during chronic lung inflammation. In addition, we will analyze the
repercussions of the majority use of males in both basic and clinical research
and the neglect of not reporting or not segregating by sex in many scientific
reports focused on understanding the immune response.
Sex and Sex Hormones as Modulators

of Immune Cells Responses
There are notorious and critical differences in the immune response between
women and men. Since the 1940s, it has been reported that females have a
more robust antibody (Ab) response than males do, that 80% of autoimmune
disease occurs in females, that males have a greater susceptibility (almost
twofold higher) to die due to malignant cancer than women, and that in
general, that women have more robust innate and adaptive immune responses
than males. Sex dimorphism has been observed in a significant number of
innate immune cells. These differences may be in part mediated by differences
in the expression of pattern recognition receptors and functional responses of
phagocytes and antigen-presenting cells. In mice, innate cell receptors such as
toll-like receptors (TLRs), which participate in the recognition of conserved
pathogen-associated molecular patterns of microbes, are differentially
expressed between sexes, with female mice having a higher expression of
TLR3, 7, and 9, and male mice having a greater expression of TLR2 and
TLR4. This differential expression of TLRs modulates the immune response
against different infectious pathogens as TLR3, 7, and 9 are intracellular
receptors that recognize viral RNA or DNA, while TLR2 and TLR4 recognize
bacterial cell wall proteins. In humans, similar observations have been
reported on peripheral blood mononuclear cells (PBMCs) from healthy donors
when exposed to TLR7 ligand, where female PBMCs produce higher amounts
of interferon (IFN)α in response to TLR7 compared to male PBMCs, to name
a few examples.
Sex hormones are known to participate in the sex dimorphism observed
in innate immune cells. Estrogen (E2), for example, downmodulates the
expression of interleukin (IL)-6 in Kupffer cells. In neutrophils, E2 modulates
apoptosis, chemotaxis, and recruitment; it augments the response of
plasmacytoid dendritic cells by increasing the production of IFNα after
stimulation with TLR7 ligands as well as increases the differentiation of
dendritic cells (DCs) from bone marrow precursors and their capability to
activate CD4+ T cells. In macrophages, E2 increases the expression of TLR4
and CD14 and enhances the IL-4-induced M2 gene expression. Progesterone,
another female sex hormone, has both pro-inflammatory and regulatory
effects. While it enhances the chemotaxis of neutrophils, progesterone also
inhibits TLR4-mediated innate immune response by suppressing NF-κB
activation and enhancing SOCS1 expression in macrophages. On the other
hand, testosterone presents overall immunoregulatory effects. Among the
immunosup-pressive effects of this androgen, there is the dampening of

cytokine production and the proliferation of lymphocytes and group 2 innate
lymphoid cells (ILC2s), the induction of neutropenia during microbial
infections in mice lacking androgen receptor (AR), and the reduction of the
expression of TLR4 in macrophages. However, it is essential to mention that
the role of androgens is not as simple as it looks, as 5α-dihydrotestosterone
(DHT), a non-aromatizable androgen, is involved in modulating the activation
profile of macrophages.
Sex- and Sex Hormone-Induced Differences

in Innate Immune Cells
As mentioned above, the innate immune system consists of a diverse network

of immune cells, and sex and sex hormones are known to modify the function
of these cells.
Natural Killer Cells
In humans, NK cells are commonly defined as CD3−CD56+ cells and

constitute one of the main front-line defense systems of our bodies, as they do
not require pre-stimulation to perform their effector functions. These cells are
present in blood and as tissue-resident lymphocytes in the peritoneal cavity,
spleen, liver, lung, lymph nodes, thymus, and in the uterus during gestation.
NK cells participate in the early responses against tumors and viral infections.
Human and murine NK cells express estrogen receptors (ERs) and the
progesterone receptor (PR). It is unclear if they express androgen receptors.
However, some studies indicate higher numbers and cytotoxic activity of
blood NK cells in men compared to women and that this sex difference is
reversed in old age, correlating with a decrease in androgen levels. The most
obvious example of how sex hormones affect the physiology of NK cells is
observed during pregnancy. NK cells are the most abundant subset of
lymphocytes in the uterus, reaching up to 70 to 80% of the total leukocytes in
the first trimester of pregnancy, with a subsequent decline and return to basal
levels at the end of pregnancy. Moreover, the menstrual cycle also correlates
with an increase in number in the proliferative phase and peaking in the late
secretory phase in response to hormone-induced decidualization. However,
the main function of decidual NK cells is not against infections as their

cytotoxicity is poor. Instead, they are crucial for the normal development of
the placenta and uterine tissue remodeling due to their production of cytokines,
chemokines, and angiogenic factors. Notably, the increased migration
observed in NK cells and their chemokine receptor profile is mediated by
progesterone. Nonetheless, further work is needed to fully understand the
effect of progesterone on NK cells, as some other reports suggest that
progesterone and estrogen could decrease NK cell activity while others found
no effect.
Natural killer cells are crucial in various lung diseases where chronic
inflammation is key such as lung cancer, chronic obstructive pulmonary
disease (COPD), asthma, and infections. Besides, sex hormones can modulate
NK cell function. However, important gaps still exist in understanding how
NK cells mediate sex differences in chronic lung inflammation. Further work
is necessary in order to decipher how to prevent or ameliorate NK cell-induced
inflammation efficiently in men and women.
Group 2 Innate Lymphoid Cells
First identified as non-B, non-T (NBNT), IL-13-producing, and IL-25-

dependent cells, ILC2s are tissue-resident cells and a major component of type
2 inflammation due to their ability for rapid secreting large amounts of IL-4,
IL-5, IL-9, and IL-13. In steady conditions, ILC2s are the main ILC subset in
the lung in mice, and along with ILC3s, they are the most abundant in the
human lung. Interestingly, ILC2s present a sex bias: in homeostasis, female
mice present a higher number of ILC2s in the lung compared to male mice.
ILC2s express abundant levels of AR, suggesting a modulatory role for
androgens in this population. Several studies have demonstrated that
androgens are an important regulator of ILC2s: androgens can suppress ILC2
proliferation, and male mice have fewer lung ILC2s than females. Moreover,
DHT potently suppresses the gene expression of Il13 in ILC2 cells, but poorly
is known about mechanisms by which androgens regulate pro-inflammatory
gene expression and activation of ILC2s, and further work is needed to
understand this physiology better. The role of estrogen on ILC2s is less clear.
A recent study suggests that estrogen can also have modulatory effects on
ILC2 cells, as demonstrated by the estrogen-mediated reduction of the ratio of
ILC2-to-CD45 in the liver. On the other hand, a study using gonadectomized
female mice that lack estrogen and progesterone, and have low serum
testosterone levels, suggested that these female sex hormones participate in

decreasing ILC2 cytokine production. These discrepancies clearly indicate the
need to continue studying how sex hormones, especially estrogen and
progesterone, modulate the activity of ILC2s.
Neutrophils
Neutrophils are the most abundant leukocyte subset in circulation and are
significant players in defense against microbial pathogens and viral infections.
These cells can modulate the immune response by producing pro-
inflammatory cytokines, chemokines, and reactive oxygen species. Sex
hormones may modulate effector functions of neutrophils, as these cells
express estrogen and androgen receptors, and sex differences in the number
and function of neutrophils in humans have been reported.
High concentrations of estrogen and progesterone during pregnancy and
the luteal phase of the menstrual cycle correlate with an increase in the number
of neutrophils in the blood, suggesting these hormones as promoters of
neutrophil numbers. Moreover, some authors have demonstrated that both,
estrogen and progesterone contribute to the delay in neutrophil apoptosis by
decreasing expression of the pro-apoptotic protein caspase 3, which may
explain why neutrophils from healthy young women have improved
survival in vitro compared to those of healthy young men. Moreover, healthy
young adult men displayed significantly enhanced immature neutrophils
compared to age-matched healthy women.
Monocytes and Macrophages
Monocytes, and their mature form, the macrophages, are innate myeloid cells
and crucial modulators of the innate and adaptative immune responses.
Monocytes are released from the bone marrow in a CCR2-dependent manner
and circulate in the blood, from where they can migrate into inflamed tissues.
Monocytes produce inflammatory cytokines in response to danger signals, and
they can also take up cells and toxic molecules. Once in tissue, monocytes can
maturate into macrophages in inflammatory conditions. Macrophages are
tissue-resident phagocytic cells. Macrophages participate in maintaining
homeostasis in a steady state via the clearance of apoptotic cells and the
production of growth factors. However, they can easily recognize pathogens
and danger signals through their wide variety of PRRs and start the production
of inflammatory cytokines and chemokines.
Several authors have demonstrated that both monocyte and macrophages
are susceptible to modulation by sex hormones, as they express estrogen,
progesterone, and androgen receptors. Different concentrations of estrogen
can modulate monocyte and macrophage activity in different manners:
physiological (low concentration) of estrogen enhances the pro-inflammatory
phenotype of human and murine macrophages and monocytes (enhancing the
production of pro-inflammatory cytokines such as IL-1, IL-6, and TNF),
whereas high estrogen concentrations, such as those found during pregnancy
suppress their pro-inflammatory profile. In vitro exposure of mouse
macrophages to low estrogen levels has been observed to attenuate the LPS-
induced TNFα production and IL1 and IL6 gene expression. Moreover,
similar findings were obtained when exposing male monocytes to different
concentrations of estrogen. Estrogen also promotes monocytes to differentiate
into inflammatory dendritic cells with increased production of IFNα, increased
TLR7 and TLR9 signaling, and greater internalization and presentation of
antigen to naive T cells when stimulated with granulocyte–macrophage
colony-stimulating factor (GM-CSF). However, contradictory effects of
estradiol have also been reported, showing that low concentrations of estrogen
can enhance TNFα and IL-6 production in male PBMCs but not similar cells
from females. In macrophages, interesting differences have been described
between females and males. In general, female murine macrophages have
more efficient phagocytosis and higher levels of TLR2, TLR3, and TLR4
compared to male cells. In contrast, male macrophages seem to respond better
to TLR4 ligands, possibly explaining the greater susceptibility to endotoxic
shock in males. Therefore, estrogen is generally known to enhance pro-
inflammatory immune responses. On the other hand, progesterone, the other
important female sex hormone, is better known for its anti-inflammatory
effects. Macrophages exposed to progesterone are less active and produce less
TNF and IL-1b compared to untreated macrophages. Moreover,
progesterone facilitates the M2 polarization of macrophages, as indicated by
the enhanced expression of M2 markers and the reduced production of
inducible nitric oxide synthase (iNOS) and nitric oxide (NO).
In general, estrogen enhances the immune response while progesterone
has an overall negative regulatory function, attenuating, for example, the IL-6
and IL-12p40 production induced by LPS. Androgens, on the other hand, are
more likely negative modulators of inflammation. Studies using mouse cells
have demonstrated that in response to IL-4, bone marrow–derived
macrophages (BMDM) and alveolar macrophages from female mice display

greater expression of M2 genes in vitro and after allergen challenge in vivo.
These sex differences were mediated by estrogen receptor activation. In
humans, important sex differences have been described in both monocytes and
macrophages. CD14+ monocytes from women express higher levels of CCR5
compared to the same cells from men, which can correlate with enhanced
maturation and differentiation in female cells. Moreover, androgens modulate
macrophage biology. Testosterone reduces the expression of TLR4 in vitro,
while gonadectomy of male mice results in an increase of the expression of
this ligand and enhanced susceptibility to endotoxic shock.
Dendritic Cells
Dendritic cells are professional antigen-presenting cells representing the main

bridge between innate and adaptative immune response being the major
activators of T cells. Briefly, DCs are subdivided into three subtypes:
conventional DCs (cDCs), monocyte-derived DCs, and plasmacytoid DCs
(pDCs). DCs express estrogen receptors, but most of the evidence regarding
the role of estrogen in modulating DCs function has been obtained through in
vitro assays by stimulation bone marrow cells with GM-CSF, showing that the
presence of E2 in the culture media for differentiating DCs promotes the
development of CD11c+MHCII+ DCs. Notably, the promotion of CD11+ DC-
like cells mediated by E2 occurs even at very low concentrations, in the order
of 10−10–10−9 M range, which are the levels provided by the simple addition
of fetal calf serum (FCS) supplementation of the media, and that are
comparable with the physiological E2 concentrations in female mice during
diestrus and estrus. This process is mediated by the activation of ERα and not
by ERβ. Contrarily, androgens seem to have an overall inhibitory effect on
DCs. However, the regulatory effect of androgens on DCs is still unclear,
debating if it is direct or indirect because the expression of AR by DCs is not
consistently determined.
Sex and Sex Hormones in Innate Immune Cells

in Allergic Asthma
Asthma is a chronic disease of the lungs characterized by the inflammation of

the airways mediated by an aberrant type 2 immune response against
otherwise innocuous stimulus. Significant sex differences in incidence,

prevalence, and severity have been reported in asthma. Boys are more likely
to develop asthma during childhood than girls, with this pattern being reversed
after puberty when females present increasing number and severity than
males. This change in the incidence of asthma right after puberty suggests that
sex hormones play a pivotal role in affecting this disease.
In response to inhaled allergens such as those derived from pets, house
dust mites, and pollens, the exacerbated production of type 2 cytokines such
as IL-4, IL-5, and IL-13 initiate allergic lung inflammation. These cytokines
participate in the production of allergen-specific IgE production, the
recruitment of eosinophils, and in general, activating the innate cell in the lung
and further initiating the Th2 immune response characteristic in allergic
asthma. Importantly, female sex hormones, such as estrogen, have been related
to worsening asthma. At the same time, androgens seem to contribute to
maintaining a better lung function in men, and low testosterone levels in serum
correlate with moderate to severe asthma in men. Moreover,
dehydroepiandrosterone (DHEA), another androgen, has been found to be
present in low concentration in women with severe asthma, while DHEA
concentration are known to correlate with lung function.
Group 2 Innate Lymphoid Cells
ILC2s are the dominant innate lymphoid cell population in the lung, despite
being a scarce population. These cells have been recently reported to play a
critical role in asthma and allergic lung inflammation. ILC2s are an essential
source of IL-5 and IL-13 in the lung. ILC2s are decisive for initiating Th2-
mediated allergic lung inflammation by promoting the migration of lung
dendritic cells into the draining lymph node mediated by IL-13. Although
ILC2s are present in the healthy lung, these cells also produce important
amounts of IL-5 and IL-13 in the lung of asthmatic patients.
Immunosuppressive effects of androgens on ILC2s have been reported to
ameliorate allergic lung inflammation in mice: male mice are resistant to IL33-
induced asthma due to dampened ILC2 activation. At the same time, castration
abolishes these male-protective effects, resulting in a significant expansion of
ILC2s. Similar observations have been reported in asthmatic humans, where
circulating ILC2s were less abundant in men compared to women.
Neutrophils
Although neutrophils are not a key of allergic lung inflammation, some studies
indicate that asthmatic adults who are also obese have increased neutrophils
in sputum, which correlates with increased asthma severity and reduced
treatment response. This increase was only observed in females while no
difference in the number of neutrophils in sputum in obese compared with
nonobese males was found. Moreover, this increment was only observed in
reproductive-age women, with no older females presenting differences in the
neutrophils in sputum when comparing obese and non-obese participants,
suggesting sex hormones as mediators of the obese-asthma phenotype.
Patients with severe asthma may also have IL-17-mediated inflammation with
increased neutrophil infiltration. A study in mice using adoptive transfer of
ovalbumin (OVA)-specific Th17-mediated inflammation demonstrated that
OVA-specific Th17 cells from female mice caused increased neutrophilic
inflammation in recipient mice compared to cells from male mice. Indeed,
there is very little information regarding how sex mediates differences in
neutrophils in asthma, and further work is needed.
Besides monocytes and monocyte-derived macrophages, lung tissue-resident

macrophages are critical mediators in asthma. Lung macrophages are a unique
subset of macrophages that display substantial differences compared to other
tissue-resident macrophages. Resident alveolar macrophages are seeded
embryonically from the yolk sac and fetal liver monocytes and play an
essential role in lung homeostasis. On the other hand, recruited alveolar
macrophages derived from blood monocytes are the cells involved in the
pathogenic activity in asthma. The presence of M2 macrophages in the lungs
correlates with asthma severity. Importantly, there is a dominance of M2
macrophages in airways and lung tissue in asthmatic women. A recent report
highlighted important differences in monocytes and macrophages between
women and men and between healthy and asthmatic subjects. Blood
monocytes from healthy women have higher expression of CCR5, while
CCR5 was barely expressed in monocytes from healthy men.
However, a significant increase of this receptor was found in monocytes

from asthmatic male subjects. Contrary, the expression of another chemokine
receptor, CCR2, is decreased in monocytes from asthmatic subjects but only
significantly different in male cells. These disparities, along with essential
differences found in the expression of C, a subunit of the Type I IL-4 receptor,
between asthmatic women and men, could explain, at least in part, the greater
susceptibility of women to suffer more severe asthma than men. However,
more studies are needed to fully understand the sex differences in monocytes
and macrophages in asthmatic subjects.
Sex and Sex Hormones as Modulators of COPD
Chronic obstructive pulmonary disease (COPD) is a chronic disease of the

lungs characterized by the presence of obstruction ventilator trouble, which
can be described as a persistent limitation of airflow that is not fully reversible.
COPD is usually progressive and associated with exposure to noxious particles
or gases, being the most common cigarette smoking (related to more than 90%
of COPD in Westernized countries). Both innate and adaptative inflammatory
immune responses are present in COPD. The presence of alveolar
macrophages, neutrophils, T lymphocytes, DCs, and B cells is characteristic
in the peripheral airways and lung parenchyma, with higher numbers of B cells
and neutrophils correlating with increased severity.
Historically, COPD has been reported to affect more men than women,
and it has especially been perceived as a disease of elderly men. Notably, now
it is known that this perception is not accurate. The reason why older men used
to suffer more COPD was gender habits, with a higher prevalence decades ago
of smoking men than women. However, in the last decades, the smoking rates
have increased in women, as well as the number of COPD cases in this
demographic group exceeded that of men. Nevertheless, gender is not the only
component driving the differences in incidence and severity of COPD in
women and men. Now we know that women develop more severe COPD with
early-onset disease (<60 years) and have greater susceptibility to develop
COPD even with lower tobacco exposure. This evidence highlights the
importance of including women and men, and females and males in basic
research studies and clinical trials and analyzing results segregating by sex and
gender.
The number of macrophages in the different compartments of the lung is

increased during COPD as a result of enhanced blood monocyte recruitment
in response to chemokines such as CCL2 and CXC-chemokine ligand-1,
probably due to the high concentrations of these molecules in the sputum and
bronchoalveolar lavage fluid of patients with COPD. The activation profile of
the macrophages presents in COPD patients, unlike allergic asthma, is toward
an M1 profile. Although for some authors, there is a lack of information to
establish a predominant profile, suggesting an intermediate M1/M2
phenotype. Macrophages are indispensable participants in this disease: the
number of macrophages in the small airways correlates with the severity of
COPD.
Moreover, macrophages are an important source of inflammatory
mediators such as matrix metalloprotease-12 (MMP12). How sex and sex
hormones modulate monocyte and macrophage function in COPD is still a
question. It is known that ovariectomy of mice before the induction of smoke-
induced COPD model results in ameliorated disease. There are reports
suggesting that female macrophages are greater producers of inflammatory
mediators such as MMP12 in other diseases. Significant differences in the
expressions of certain proteins in alveolar macrophages from male and female
smokers suffering from COPD have been reported. Moreover, an impairment
in the lysosomal function that may result in macroautophagy inhibition in
alveolar macrophages has been hypothesized to contribute to airway
inflammation. Female sex hormones have also been described to be players in
this condition. Estrogen is involved in maintaining cell structures and in lung
elastic recoil that keeps airways open. The level of airway obstruction in post-
menopausal women who are not receiving hormone replacement therapy is
greater than in those who receive it. Furthermore, a potential anti-estrogenic
effect of cigarette smoke has been described and may contribute to impaired
lung function in smoking women. Female mice chronically exposed to smoke
present a more significant number of foamy macrophages in the lungs
compared to male mice, and ovariectomy abrogates this difference. It is known
that foamy macrophages are cells that can contain cigarette smoke-induced
lipid oxidation products and are associated with chronic inflammation.
Although important sex differences have been observed in COPD and
reports studying how monocytes and macrophages from females and males
have critical disparities, there is still work to be done to understand better how
sex and sex hormones affect this disease. Besides, it is unclear if androgens
could play a protective role during COPD, as men have lower severity
compared to women. Again, more profound studies in how sex and sex
hormones can impact the monocyte/macrophage and other cell type function
and activation profile during COPD are needed.
The Role of Other Innate Immune Cells in COPD
Several innate immune cell types besides monocytes and macrophages also
contribute to airway remodeling and COPD progression. In COPD, innate
immune cells can induce structural injury to the airways promoting the release
of damage-associated molecular patterns (DAMPs) that PRRs further
recognize. After this recognition, DAMPs can lead to the recruitment of
activated innate immune cells, facilitating COPD progression.
There is an increased influx of DCs that are recruited to the lungs in
COPD. The epithelial integrity is diminished in this disease, which leads to an
enhanced exposure of intraepithelial DCs to antigens, promoting their
migration toward draining lymph nodes where they activate naive B and T
cells. However, DC maturation is impaired after long-term exposure to
harmful stimulus, inhibiting their antigen-presenting capacity and inducing the
appearance of “non-functional DCs.” This is important as the number of
immature DCs is increased in the small airways; there is a greater release of
CCL3 and CXCL2 by immature DCs, promoting neutrophil recruitment.
Neutrophils are a key marker for the diagnosis of COPD progression. An
excessive neutrophilic infiltration to the lungs leads to the release of MMPs
and neutrophil elastase, which mediate a protease-antiprotease imbalance in
the lungs, enhancing tissue damage. The exacerbated presence and activation
of neutrophils in the lungs mainly affects the small airways by provoking the
dissolution of the alveolar wall, injury to the ciliated epithelium and
connective tissue matrix, causing mucus hypersecretion, and squamous
metaplasia in epithelial cells, which together leads to emphysema and fibrotic
repair in the small airways. Similar to neutrophils, natural killer T (NKT) also
participate in enhancing COPD severity. Activated NKT cells are an important
source of IFNγ and TNFα and enhancers of T and B cell function, facilitating
COPD progression.
Although there is evidence of how innate immune cells mediate COPD
progression and severity, participating in airway destruction and aberrant
repair, no studies focused on how the innate immune response participates in
mediating the sex differences observed in this disease. Moreover, there is also
an evident lack of information on how sex hormones impact the innate

immune response in COPD. Understanding the early stages of COPD
development, in which innate immune cells may have a determining role, is
necessary to develop better therapies against the remodeling process,
particularly for women who are more affected by this health condition.
Considering Sex and Sex Hormones

in Chronic Lung Inflammation
Studying chronic lung inflammation is challenging itself. Most human-based

reports are merely descriptive or correlative, and understanding the differences
between the immune response of healthy versus chronically ill subjects is
complicated. Obtaining BAL samples from ill persons is not an easy thing to
do, even when they can be already hospitalized and have intubating procedures
that facilitate access to sample obtention. Collecting the same type of samples
from healthy subjects is even more complicated. People do not always
appreciate the importance of thoroughly understanding lung biology under
homeostatic conditions and how this knowledge help to understand the
changes that occur during disease. Moreover, obtaining lung tissue samples is
even more complicated. Setting up the suitable protocols to be approved
requires the joint work of basic scientists and a number of health providers.
Working with mouse models also presents important caveats as not all human
disease models can be fully developed in animals. Results might have
significant variations depending on animal genetic background and housing
conditions (due to differences in the microbiome).
Even though literature highlights the influence of sex and sex hormones
on the incidence and severity of the inflammatory response in lung diseases,
controlling for these factors during basic research is difficult to address.
Variation of hormone levels during the menstrual cycle, consumption of
hormonal-based birth control medication, hormone, among other factors, are
complicated to predict when working with human subjects but can modify the
outcome of a variety of biological readings and push the research to notably
increase the number of participants in order to get more reliable results, which
is, as mentioned above, complicated itself.
Conclusion
This chapter highlights the importance of considering sex and sex hormones
when studying chronic lung inflammation. Chronic lung diseases affect
differently to women and men, and historically women have been an
underrepresented group, which has impacted the development of efficient
therapies for this demographic detrimentally. Moreover, sex hormones have
been observed to affect the innate immune responses to several lung diseases.
However, it has not been completely addressed, and this is something that
needs to be understood as sex hormones can have a beneficial or detrimental
role in disease. In general, male sex hormones have demonstrated a beneficial
anti-inflammatory role in diseases such as asthma and COPD. In contrast,
female sex hormones, particularly estrogen, displays both anti-inflammatory
and pro-inflammatory properties in lung diseases. However, there is still too
much research to be done in order to develop novel therapeutic approaches
that eventually lead to individualized and more efficient treatments.
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Chapter 3
Asthma beyond Adaptive Immunity:

Fighting Corticosteroid Resistance
Claudia Andrea Morales-Garay1,

Claudia Hallal-Calleros2
and Claudia A. Garay-Canales3,*
1InstitutoTecnológico y de Estudios Superiores de Monterrey,
Campus Ciudad de México, Ciudad de México, Mexico
2Facultad de Ciencias Agropecuarias, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos, Mexico

3Departamento de Inmunología, Instituto de Investigaciones Biomédicas,
Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Abstract
Asthma is a multifaceted heterogeneous syndrome characterized by

hyper-responsiveness of the respiratory tract, bronchial obstruction,
mucus overproduction, and airway remodeling. It is one of the most
rapidly growing disorders, which affects around one-third of the world’s
population. According to WHO statistics, around 461,000 deaths annualy
are attributed to severe disease annually, and most asthma-related deaths
occur in low and middle-income countries.
*
Corresponding Author’s E-mail: clausgaray@iibiomedicas.unam.mx.

ISBN: 978-1-68507-510-1
62 C. A. Morales-Garay, C. Hallal-Calleros and C. A. Garay-Canales
There are increasing reports of resistance to conventional therapies,

such as steroids or bronchodilators. Misdiagnosed or inadequate
treatment leads to poor quality of life and creates a burden on families,
societies, and countries.
Its heterogeneity is linked to its multifactorial nature, which leads to
various mechanistic inflammatory pathways. The common
misconception is that asthma is mainly set off by common allergens like
pollen or dust. Furthermore, asthma has been commonly classified into
two main types: allergic and nonallergic. Nonetheless, this classification
appears to be an oversimplification. Nowadays, asthma can be classified
in different endotypes, based on the source of the stimulus that triggers
the immunopathology; pollution particles, fungus or viral infections,
work environment, aspirin, etc. The pathogenesis can also be related to
the age of onset of the disease (childhood vs. late-onset asthma), obesity,
or even menstruation.
Traditionally, airway inflammation in asthma was considered
exclusively part of adaptive immunity. Nonetheless, the identification of
innate lymphoid cells, the difference in the pathogenesis according to the
presence of eosinophils vs. neutrophils, an increased presence of
monocytes or dendritic cells, the secretion of different cytokines, and
their potential role in atopic disorders significantly contributes to
determining the inflammatory response that characterizes the different
asthma phenotypes. In fact, the pathophysiology of asthma shares
common cascades and signaling molecules in both types of immunity.
Therefore, any immunological mechanism leading to asthma also
involves the activation of the innate immune system. In this chapter, we
address the innate immune mechanisms of different endotypes involved
in the diversity of phenotypes of asthma, the approaches for an accurate
diagnosis, and the current options for its proper management.
The deeper knowledge of the role of different cells of the innate
immune system, the mechanisms involved, and the identification of
asthma endotypes will lead to a better understanding of the
pathophysiology of this heterogeneous condition. Additionally,
biomarkers involved in the disease will provide a better diagnosis, which
will help physicians offer a safer, more precise, and more effective
treatment focused on targeted medicine.
Keywords: asthma therapy, inflammation, asthma endotypes, innate immune

response.
Asthma beyond Adaptive Immunity 63
Introduction
Asthma is the most common long-term, non-communicable disease (NCD) in

the world. It is frequently characterized by chronic airway inflammation,
accompanied by a history of intermittent respiratory symptoms like wheeze,
shortness of breath, chest tightness, and cough that vary over time in intensity
(Hammad and Lambrecht 2021). The clinical features of asthma revealed it to
be a heterologous disease, reflected by major differences in the patient age of
onset, associated risk factors, level of severity, comorbidity, and response to
treatment (Wu et al. 2019).
This condition affects patients of all ages, but mainly children, as it is the
most common chronic disease that affects them (WHO, 2019). Additionally,
it was ranked as the 16th leading cause of years living with disability and the
28th cause of burden disease. Ultimately, it is estimated that in 2019, asthma
caused 461,000 deaths; 21.6 million Disability-adjusted life year (DALYs),
and more than 300 million people worldwide suffer from this disease. This
condition affects people from different ages, race or ethnicity. Nonetheless,
ethnicity and socioeconomic status have a critical impact on prevalence,
morbidity, and mortality worldwide.
Furthermore, statistics from the Global Initiative for Asthma 2021 or
GINA report (Reddel 2021) show that asthma’s prevalence has increased over
the years, affecting 1-18% of the population in different countries, reaching
15 up to 20% in developed countries (Enilari and Sinha 2019). Moreover, it is
challenging to estimate regional and global scale prevalence, incidence, and
mortality rates due to the lack of a precise and globally accepted asthma
definition, a non-standardized diagnosis, and the diversification of methods
for gathering information.
Nonetheless, the global and yearly increase of disease indicators is
remarkable. According to The Global Health Metrics report in 2020, using
both sexes and age-standardized data from 2010 to 2019, stated a percentage
change of (cases millions) of 15.7% in global prevalence, 13% in global
incidence, 5.4% in global deaths, -1.9% in years of life lost (YLLs), of 15.4%
in years of healthy life lost due to disability (YLDs), and 5.6% in DALYs.
The second noteworthy remark retrieved from global data was the
difference in disease indicators between high-income and low and middle-
income countries. The World Health Survey disclosed that high-income
countries like Australia (21%), UK (18.15%), Sweden (20.18%), or The
Netherlands (15.32%) have the highest rates of clinical asthma prevalence.
Nevertheless, the death rates corresponding to asthma in these countries
remain low. On the contrary, some low- and middle-income countries like
South Africa (6.09%), Mexico (2.39%), or Guatemala (2.42%), which have
lower prevalence rates, suffer from more annual deaths (Global asthma report)
and DALYs (Global health metrics).
One possible reason for the increasing rates of asthma prevalence in high-
income countries could be pollution, westernization, and higher rates of
obesity and urbanization. Another plausible explanation may be the “hygiene
hypothesis”; in which reduced and later onset exposure to microbes and
infections translates into higher asthma incidence and prevalence. On the other
hand, in low and middle-income countries, the incidence is more likely
attributed to lifestyle and environmental causes than genetic variations
between populations. Also, misdiagnosis and under-treatment of asthma have
caused higher rates of preventable deaths and personal, social-economic, and
health burdens (Enilari and Sinha 2019).
Although asthma has low mortality, the morbidity prevalence is still
representative. The burden that asthma patients have due to visits to the
emergency room, hospitalization, tests for diagnosis, medication, and visits to
physicians are noteworthy. In the U.S. alone, asthma costs around $80 billion
annually between medical expenses and loss of productivity. Furthermore, the
costs of asthma go beyond the economic ones. Indirect costs of asthma
include; loss in productivity, missed school and work days, waiting times, and
forecast losses due to missing opportunities or working days. These expenses
increase with severity, misdiagnosis, or incorrect treatment. (Bahadori et al.
2009) (Dierick et al. 2020).
Differential Diagnosis
A clear asthma definition will provide patients better medical care and
will avoid more social and economic burdens. The landing of this definition
will lead to a standardized diagnosis and later on to a personalized and more
precise treatment that will effectively control the symptoms. Since there is not
a standardized asthma method of diagnosis, Table 1 is presented as a first
approach for physicians to perform a differential diagnosis and confirm
asthma or present an alternative diagnosis.
The acceptance of an asthma diagnosis should be based on the recognition

of a characteristic pattern of respiratory symptoms and an airway obstruction
described in the Global Initiative for Asthma 2021 or GINA report (Reddel
2021).
Furthermore, this diagnosis should rely on the identification of chronic
respiratory manifestations that are peculiar to asthma and not confuse them
with an acute alternative pathology. The patterns of asthma symptoms are a
result of chronic inflammation of the airways, which may trigger
overproduction of mucus, remodeling of the airway wall, and bronchial
hyperresponsiveness, which is smooth muscle’s reaction to nonspecific
stimuli (Hammad and Lambrecht 2021). It is important to establish the initial
diagnosis and document the symptoms before treatment options.
The identification of frequency and intensity of the symptoms drives the
diagnosis of asthma. It is important to recognize the pattern of symptoms, to
differentiate acute or chronic conditions other than asthma. If the patient
presents more than one feature, increasing the probability that the patient has
asthma, the physician should identify if the patient has a characteristic pattern
of respiratory symptoms like a wheeze, shortness of breath, cough, or any
other suspicious symptoms presented in Table 1. To support the diagnosis the
patients could experience more than one sign, that often worse at night or early
in the morning; different factors such as viral infections, allergies, pollution,
or even exercise could provoke a crisis.
On the other hand, some signs decrease the possibility to experience
asthma, like isolated cough, chronic production of sputum, shortness of breath
presented with dizziness, chest pain, or exercise-induced dyspnea. Table 1
shows some symptoms presented in asthma or an alternative diagnosis, the
features observed are clustered upon age, since manifestations vary in the
lifetime. A flowchart is presented in Figure 1, to be followed by the physician
to corroborate or deny an asthma diagnosis. It is relevant to remark that the
information of this diagram was retrieved from the GINA 2021 report, which
is a classical and accepted method to confirm or reject an asthma diagnosis.
Moreover, we also added sputum cytology complementing the steps for a
complete asthma diagnosis. This addition will provide evidence of
eosinophilic, neutrophilic, or mixed complex inflammation. In some cases, it
will provide information on a few inflammatory cells (paucigranulocytic)
(Moffatt et al. 2010). The mix between the recognition of symptoms and
sputum cytology will lead to a more personalized diagnosis, which will be
further discussed in this chapter.
Table 1. Differential diagnosis per group age (GINA, 2021)
Age (years) Common asthma symptoms Alternative Diagnoses

Any age Chronic cough Tuberculosis
Inspiratory wheezing (stridor)
Throat-clearing
Dyspnea
Hemoptysis
Blocked nose
Sweats and fever
Fatigue
6 to 11 Recurrent infections Chronic upper airway cough syndrome
Productive cough Inhaled foreign body
Sinusitis Bronchiectasis
Sudden onset of symptoms Primary ciliary dyskinesia
Excessive cough and mucus Congenital heart disease
production
Gastrointestinal symptoms Bronchopulmonary dysplasia
Cardiac murmurs Cystic fibrosis
12 to 39 Dizziness
Paresthesia Chronic upper airway cough syndrome
Sighing Inducible laryngeal obstruction
Productive cough Hyperventilation, dysfunctional breathing
Excessive cough and mucus Bronchiectasis
production
Cardiac murmurs Cystic fibrosis
Shortness of breath Congenital heart disease
Early emphysema Alpha-antitrypsin deficiency
Sudden onset of symptoms Inhaled foreign body
Cough and sputum Inducible laryngeal obstruction
40 or more Dyspnea on exertion Hyperventilation, dysfunctional breathing
Smoking or noxious exposure Chronic obstructive pulmonary disease
(COPD)
Productive cough Bronchiectasis
Recurrent infections Cardiac failure
Nocturnal symptoms Medication-related cough
Ankle edema Parenchymal lung disease
Non-productive cough Pulmonary embolism
Finger clubbing Central airway obstruction
Chest pain
Figure 1. Methodology to corroborate or reject an asthma diagnosis (GINA, 2021).
Intrinsic and Extrinsic Factors That Trigger Asthma
Asthma has been described as a single disease entity characterized by chronic

inflammation and categorized into either one of two groups: allergic and non-
allergic phenotypes (Khalaf et al. 2019). These categories differ from each
other by the reactivity of a patient to display symptoms in the presence of an
allergen or not. Nowadays, the Global Initiative for Asthma (GINA 2021) has
incorporated new ways of clustering the disease in different asthma
phenotypes. These asthma phenotypes include both previously mentioned
categories, late-onset asthma, asthma with fixed airflow limitation, and asthma
with obesity.
As a consequence of the limited categorization, the prescribed treatment
may be insufficient and oblivious to other factors that may influence the
severity and prognosis of the disease. Therefore, to propose a more effective
grouping of asthma, we have to discuss the different intrinsic and extrinsic
factors that may trigger it. Figure 2 displays various environmental, biological,
genetic, and even factors that have to do with the patient’s habits that could
trigger the apparition of asthma, and the presence of a hyperreactive
respiratory epithelium. It has been studied that infections caused by viruses,

bacteria, or fungal colonization may produce bronchoconstriction. In the case
of viruses, their infection causes damage to the respiratory epithelium, which
permits allergen penetration. On the other hand, fungal species like
Aspergillus fumigatus or Candida albicans produce proteases to the
respiratory epithelium that could lead to bronchiectasis and airflow
obstruction (Gans and Gavrilova 2020).
Figure 2. Intrinsic and extrinsic factors that trigger asthma. Inflammation starts with
the recognition of nonself structures from microorganisms or chemical compounds.
However, in asthmatic patients, hyperreactive respiratory epithelium can respond to
indirect factors, such as AINES, exercise, cold air, premenstrual hormones, etc.
Furthermore, susceptibility to present exacerbation of symptoms can be influenced
by obesity, polymorphisms, and female hormones.
Moreover, many environmental factors are considered risk factors with

the potential to set off asthma. Frequent and prolonged exposure to tobacco
smoke, pollution particles, allergens like pollen, and even cold air are known
to cause bronchoconstriction, and later on hyperreactive airway epithelium.
The patient’s habits may also play a role as risk factors of asthma; elite or
amateur athletes may present dehydration or increased ventilation that could

cause airway damage. Further, sex and more specific, sexual hormones in
women have also been identified as potential triggers of asthma. Premenstrual
hormones like estrogens and progesterone can cause bronchoconstriction in
females (Han et al. 2020). Additionally, obesity(Peters, Dixon, and Forno
2018), gastroesophageal reflux disease, or genetic polymorphisms may also
lead to airway obstruction.
The interaction of the host with the environment influences the
pathophysiology of asthma. Despite the idea that asthma is only triggered by
allergens, there is evidence showing that there are numerous factors causing
episodes of airway obstruction characteristic of asthma. Six groups of triggers
have been identified thematically associated with psychology, animal allergen,
pollen allergen, physical activity, infection, and air pollutants/irritants (Ritz et
al. 2006). However, there are “other factors” that influence the initiation and
outcome of the disease. Among these elements, there are polymorphisms for
IL-33, IL1RL1, IL8R1, HLA-DQ, SMAD3, IL2RB, and polymorphisms in
the locus of chromosome 17q21 including ZPBP2, GSDMB, and ORMDL3.
These genes disturb the epithelial barrier and innate and adaptive immune
response contribute to asthma (Moffatt et al. 2010). The airway smooth muscle
is hypercontractile, which is amplified by co-located mast cells and other
immune cells, this condition responds to disturbance like cold air, exercise,
etc.
Occupational asthma is induced by exposure to irritants or allergens: high
or low molecular weight agents. High molecular weight agents include
proteins, and cause immunological occupational asthma via an IgE mediated
mechanism. On the other hand, low molecular weight agents, essentially
chemical agents, have an IgE independent mechanism (Muñoz and Romero-
Mesones 2020). Among some chemicals related to asthma, there are
Endocrine-disrupting compounds (EDC). These compounds are complex
mixtures found in everyday products such as fragrances, cosmetics, and other
personal care products. Some of these compounds are parabens, phthalates,
bisphenol A (BPA), triclosan, ethanolamines, alkylphenols, glycol ethers,
cyclosiloxanes, etc. The overall effect observed was worsening pulmonary
function and airway inflammation in asthmatic patients (Dodson et al. 2012)
(Kim et al. 2018).
Sex differences found in asthma may be explained by changes in the level
of sex hormones during life. Women are more susceptible to asthma after
puberty, menstruation, menopause, hormonal contraceptives, and hormonal
replacement therapy (Yung, Fuseini, and Newcomb 2018) (Han et al. 2020).
Exercise-induced bronchoconstriction has been recognized since 1960 in

asthma patients, and it has a negative impact on the quality of life of athletes.
This type of asthma is commonly underdiagnosed but usually responds to
classical pharmacological treatment. The condition may even improve with a
warm-up before exercise (Aggarwal, Mulgirigama, and Berend 2018).
Aspirin and other drugs exacerbate nasal symptoms such as nasal
obstruction/congestion and rhinorrhea and may progress over time up to life-
threatening asthma (Philpott et al. 2018). Obesity represents an increased risk
for asthma patients, especially in menopause women; obese asthmatics present
more symptoms, frequent and severe exacerbations, reduced response to
several asthma medications, and decreased quality of life (Peters, Dixon, and
Forno 2018) (Han et al. 2020).
Clinical features along with proteomics, genomics, metabolomics, and
transcriptomic studies have elucidated that all these risk factors are directly
related to the innate immune response. The immunopathophysiology of
asthma involves both the activation of the innate and adaptive immune system,
which are responsible for airway inflammation, and later on
hyperresponsiveness and chronic problems. Subsequently, this chronic
inflammation leads to mucus overproduction, mucus plugging, and later on,
airway remodeling. This permanent structural remodeling is caused by the
activation of several Th helper cells (1, 2, and 17), and by the participation of
innate immune cells like eosinophils and neutrophils.
Overall, the diverse collection of risk factors shows the heterogeneity of
asthma. This statement shows the need for reclassification of this disease
based on its triggers and the molecular mechanisms that underlie them. Mainly
focusing on the molecular mechanisms that involve the innate immune
response. Therefore, in the following sections of this chapter, the authors will
unravel: the relationship of asthma with the innate immune system, the
triggered immune response by risk factors, the possibility of identifying innate
immune cells as biomarkers of the disease, and novel treatments of asthma.
Asthma Classification
Understanding the immunological mechanisms of asthma, and identifying the

key players that induce chronic airway inflammation can optimize
management and drive personalized treatment. First, it is critical to
acknowledge the major patient-specific differences in age of onset, associated
risk factors, and degrees of severity, comorbidity, and response to treatment
(Wu et al. 2019). Traditionally, asthma was only considered allergic (type 1)
or non-allergic (type 2). Now there are two major classifications, based on the
phenotype (the observable characteristics) or the endotypes (clinical features
of the disease and their underlying mechanism). The Global Initiative for
Asthma (GINA) has defined groups based on demographic, clinical and
pathophysiology asthma phenotypes: allergic, non-allergic, late-onset, asthma
with fixed airflow limitations, and asthma with obesity (Reddel 2021). An
endotype classification was proposed by Lötvall as a subtype of a condition
defined by a distinct pathophysiology mechanism. This classification is based
on clinical characteristics, biomarkers, lung physiology, genetics,
histopathology, epidemiology, and treatment response summarized in Table 1
(Lötvall et al. 2011) (Lambrecht and Hammad 2017).
In asthma, chronic inflammation is promoted by Type 2 cytokines such as
IL-4, IL-5, and IL-13, leading to eosinophilia in the airways, hyper-production
of mucus, bronchial hyperresponsiveness, hyper-secretion IgE and
susceptibility to exacerbation. Nevertheless, only less than half of patients
present this type-2 high endotype. The rest of the asthma population present
type-2 low endotype with different features; it is associated with obesity, the
presence of neutrophils and unresponsiveness to corticosteroids (Table 2)
(Hammad and Lambrecht 2021).
In type-2 high asthma, key features are the presence of high numbers of
eosinophils in the airways and iNOS in exhaled air. Additionally, in the airway
epithelium, there have been sights of goblet cell metaplasia and hyper-
production of MUC5AC. Upon allergic recognition by pattern recognition
receptors (PRRs), dendritic cells are recruited to epithelium and activate
memory Th2 cells, producing Th2 cytokines as mentioned above IL-4, IL-5
and IL-13. On the other hand, non-allergic type-2 high patients activate the
innate lymphocytes type 2 (ILC2) directly by epithelial cytokines such as IL-
33 and TLSP to produce IL-5 and IL-13. ILC2. An innate immunity cell shares
features with Th2 cells, such as the expression of cytokine receptors, and some
transcription factors (GATA3 and CRTH2).
IL-4 stimulates IgE synthesis and prepares endothelial cells for
extravasation of eosinophils inducing the expression of vascular cell adhesion
molecule (VCAM) and intercellular adhesion molecule (ICAM-1) through IL-
4R. Moreover, IL-5 promotes the development and activation of eosinophils,
which produce Charcot-Leyden proteins, and upon activation, degranulate
releasing toxic compounds such as Eosinophil peroxidase (EPO), eosinophil
cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). Altogether,
these compounds damage lung structural epithelium cells by activating innate
immune cells. IL-13 stimulates iNOS production, goblet cell metaplasia and
bronchial hyperreactivity (Figure 3).
Table 2. Type 2 asthma phenotypes (Type 2-high and Type 2-low)

In type-2 low asthma, neutrophils play a crucial role in inflammation.

Environmental stimuli such as pollutants, chemical compounds like cigarette
smoke or even microorganisms, activate the airway epithelium and alveolar
macrophages, producing pro-inflammatory cytokines, which include IL-1,
IL-6, and epithelium produce CXCL8, a potent neutrophil attractant. This
cytokine microenvironment activates Th1 and TH17 cells, which in turn
recruit and activate neutrophils releasing neutrophil elastase,
myeloperoxidase, or reactive oxygen species (ROS), which provoke
epithelium damage increasing mucus production and exacerbated
inflammation (Figure 3).
Figure 3. Type 2-high asthma’s main features are high production of IL-4, IL-5, and
IL-13, recruitment of eosinophils and mast cells in lung epithelium, and exacerbated
mucus production. Type 2-low asthma is associated with obesity, the presence of
neutrophils, and unresponsiveness to corticosteroids.
Role of Innate Immune System in Asthma
The characteristic airway inflammation of asthma has been commonly

associated with the adaptive immune system. And the role of the innate
immune system has been frequently overlooked and positioned as secondary.
The pathogenesis of asthma is strongly influenced by environmental agents as
shown in figure 2. The pathogenesis leads to exacerbate allergen-induced type
2 immune responses leading to airway narrowing, blood eosinophilia, mucus

overproduction, and increased levels of serum IgE antibodies. The innate
immune system has evolved to process environmental stimuli, shaping the
adaptive immune response and playing a key role as both an inductor or
protector regulator of the disease.
During asthma onset, genetic studies have shown that polymorphism in
classical innate immunity genes influences the development of the disease.
Among these genes, there are toll-like receptors (TLRs), nucleotide-binding
oligomerization domain-containing proteins (NODs), and CD14 (Vercelli
2008). Later, additional innate immunity genes associated with asthma were
identified, such as alarmin interleukin 33 (IL-33), IL-33 receptor interleukin-
1 receptor like1 (IL1R1)/ST2, thymic stromal lymphopoietin (TSLP), and the
ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) (Ober and Nicolae
2011). The alarmins IL-33, TSLP, and IL-25 can promote type 2 immune
response, demonstrated by elevated expression of IL-25 and its receptor found
in patients with allergic asthma (Tang et al. 2014), meanwhile TSLP
expression was increased in patients with airway obstruction, where anti-
TSLP antibodies reduced bronchoconstriction and levels of eosinophils in
patients with mild, moderate-severe asthma (Gauvreau et al. 2014).
Monocyte-derived dendritic cells and LPS-stimulated monocytes, human
bronchial epithelial, and smooth muscle cells express IL-33. Upon stimulation
with IL-33, type-2 innate lymphoid cells (ILC2) are major producers of Th2
cytokines like IL-13 and IL-5 (Halim et al. 2016) (Van Dyken et al. 2014).
Activation of innate pathways by allergens with protease activity or chitin
(major polysaccharide found in arthropod exoskeletons and fungal cell walls)
induces lung inflammation, the release of IL-33, and TSLP, which stimulate
ILC2 to produce IL-5 and IL-13 driving the induction of systemic Th2
response (Chang et al. 2011). Furthermore, after birth, immune cells such as
ILC2, eosinophils, basophils, and mast cells were found in an IL-33-dependent
manner (de Kleer et al. 2016).
Role of Innate Immunity as Protector of Asthma
The main reason for the increase of prevalence in asthma worldwide is not
only due to growing promoting environmental factors such as pollution, food
intake, physical activity and others, but the decrease in asthma-protective
innate stimuli such as farm effect or hygiene hypothesis. Epidemiologic
studies have clearly shown the connection between early-life exposure to
traditional farms and decreased prevalence of allergic diseases, including

asthma (Riedler et al. 2001) (Ege et al. 2011). Specific exposure includes
contact with livestock (cattle, pigs, and poultry), animal feed (hay, grain, and
straw), consumption of raw milk, high endotoxin levels, environmental
microbes, and physical proximity to traditional farms that affect asthma
protection (Pivniouk et al. 2020).
Figure 4. Role of innate immunity in asthma. Left, Innate immune response driving
asthma progression: many allergens and air pollutants trigger epithelial cell
production of Th2 cytokines through activation of pattern recognition receptors
(PRRs), such as Toll-like receptors (TLRs) activating Dendritic cells (DC), which in
turn migrate to lymphoid nodes to promote Th2 development. Also, innate immune
cells are activated and recruited to the airways, where they produce mediators that
contribute to airway inflammation. Right, Innate immune response driving asthma
protection: upon activation with “farm factors” there is an augmented expression of
TLR4, TLR2 and CD14; increasing levels of T regulatory cells and the production of
regulatory mediators.
Toll-like receptors (TLR) play a key role in this protection since they
become the link between the host’s innate immune system and the
environmental microbiome. These receptors are transmembrane or
intracellular proteins expressed on innate immune cells in mucosal lining, both
in lungs and gut, and detect a broad range of pathogen-associated molecular
patterns (PAMPs), through these pattern recognition receptors (PRRs).
Exposure to a myriad of environmental microbial diversity, as exposed in
farms, appears to prime a child’s immune response up-regulating TLR4, TLR2

and CD14 (co-receptor for LPS detection), increasing the levels of regulatory
T cells (Tregs) (Schaub et al. 2009), high expression of interleukin-1 receptor-
associated kinase (IRAK-4) and receptor-interacting serine/threonine kinase
(RIPK)-1 and augmented expression of regulatory molecules such as
transforming growth factor (TGF-), IL-10, suppressor of cytokine signaling
(SOCS-4) and IRAK-2 (Frei et al. 2014). The perinatal effect was detected in
cord blood from farm children where higher expression of TLR7 and TLR8,
and increased levels of IFN- and TNF- were found (Loss et al. 2012). These
pieces of evidence suggest that exposure to diverse microbial environments
up-regulates asthma protective innate immune components (Figure 4).
Immunological Therapy for Asthma Management
The primary goal of asthma treatment is focused on controlling the symptoms

and underlying inflammation to avoid future disease exacerbations. Although
asthma has low mortality, physicians are now aware that remodeling the
airway walls can lead to chronic or even fatal airflow limitation, especially in
severe asthmatic patients. Standard anti-inflammatory (corticosteroids and
bronchodilator medications) are the gold standard treatment options for
asthma, but they do not consider the heterogeneity of this syndrome.
These treatments are effective for the majority of patients, but an
important percentage of asthmatic patients do not reach clinical and functional
control even using high doses of the standard drugs (Khalaf et al. 2019). More
personalized and precision therapeutic approaches based on immunological
therapeutic targets, depending on asthma endotypes, suitable for non-
controlled or severe asthmatic patients, are being researched. Precision
medicine may contribute to a better quality of life for patients.
Alternative treatments of severe asthma must be considered since the
increase of Inhaled CorticoSteroids (ICS) usually provides no significant
improvement for the patient and increases the risk of adverse effects and
increased complexity of the pathology. Allergen immunotherapy changes the
course of asthma, decreasing symptoms for years after cessation of therapy. It
is known that when an allergen is exposed to the body, epithelial cells activate
dendritic cells, which present the allergen antigen to naïve T cells to cause an
IgE-mediated Th2 response. The allergen immunotherapy proposed
mechanism is a shift of Th2 to a Th1 immune response, since repeatedly
exposition of antigens stimulate a Th1 response of dendritic cells and a
blockade of Th2 response. Allergen immunotherapy is indicated only in

patients with the confirmed allergic disease (Epstein and Nyenhuis 2019).
Furthermore, several anti-IgE, anti-cytokines or anti-cytokines receptor
antibodies have been studied. GINA 2021 found enough evidence for a
recommendation as an add-on treatment for several antibodies used as
immunotherapy.
Anti-IgE Antibodies
Omalizumab is a recombinant monoclonal that binds to IgE and blocks the

interaction of IgE with IgE receptors on mast cells, avoiding its downstream
action. Following it decreases airway inflammation by increasing eosinophil
apoptosis, decreasing IgE receptor expression on basophils and on mast cells,
and subsequently decreasing the release of mediators of inflammation. Also,
this monoclonal antibody inhibits T cell maturation inducing a reduced
production of IgE. The clinical indication for Omalizumab is an add-on
subcutaneous injection for patients older than six years with moderate or
severe allergic asthma uncontrolled with ICS-formoterol or medium-dose
ICS-LABA maintenance plus Short-Acting Beta2-Agonists (SABA) as
needed (GINA 2021). There is limited data on omalizumab’s effectiveness in
non-allergic patients. Studies targeting IgE with different monoclonal
antibodies such as lumiliximab, quilizumab, ligelizumab have not shown
enough safety or efficacy pattern (Gans and Gavrilova 2020).
Anti-Interleukin 5 (Anti-IL-5) Monoclonal Antibodies
Mepolizumab and Reslizumab are other therapeutic options for severe

eosinophilic asthmatic patients. They are monoclonal antibodies targeting
circulating IL-5, as this cytokine has a critical role in determining and
maintaining the eosinophilic inflammation in the Type 2 phenotype of asthma.
Mepolizumab induces a decreased proliferation, maturation, and survival of
eosinophils, prevents asthma exacerbation, and allows the reduction of the use
of oral corticosteroids with subsequent diminution of their adverse effects
(Canonica et al. 2019). Mepolizumab clinical indication is subcutaneous
administration of 100 mg every four weeks, for severe eosinophilic asthmatic
patients with evidence of blood eosinophils greater than 300/mcl (Varricchi et
al. 2017). GINA 2021 indicates an add-on subcutaneous administration of
Mepolizumab for patients older than six years, or intravenous Reslizumab for
patients older than eighteen, in both cases for patients with severe eosinophilic
asthma uncontrolled with ICS-formoterol or medium-dose ICS_LABA
maintenance plus SABA as needed.
Anti-Interleukin 5 Receptor (Anti-IL5R) Monoclonal Antibody
Benralizumab binds directly to the alpha subunit of the IL-5 receptor to block
IL-5 from binding to its receptor, then blocking the IL-5 action. Benralizumab
induces an antibody-dependent cell-mediated cytotoxicity mechanism in
natural killer cells against eosinophil and basophil cells. It is indicated
intravenously with an adjustable dose (from 100 to 575 mg) (Nair et al. 2017)
diminishing the exacerbations risk of asthma. Gina 2021 indicates it
subcutaneously as an add-on therapy for patients older than 12 years with
severe eosinophilic asthma uncontrolled with ICS-formoterol or medium-dose
ICS-LABA maintenance plus SABA as needed.
Different Monoclonal Antibodies Commercially

Available or at Clinical Trials
Dupilumab targets the IL-4 receptor alpha chain that is a subunit shared with
the IL-13 receptor inhibiting the binding of these pro-inflammatory cytokines
to their respective receptors. It is indicated subcutaneously for atopic
dermatitis and as an add-on therapy in moderate or severe asthma, for patients
older than twelve years with an eosinophilic phenotype or those patients
dependent on oral corticosteroids (GINA 2021), and for patients with chronic
rhinosinusitis with nasal polyposis (FDA 2019). Dupilumab induces a
reduction of exacerbation rates, improvement of lung function and favorable
tolerability with glucocorticoid withdrawal (Rabe et al. 2018), and it is a
promising treatment for patients with concomitant asthma and rhinosinusitis
with nasal polyposis.
Additionally, several trials have been performed looking for anti-
cytokines antibodies for asthma control, but their commercial use is still not
approved (reviewed in Gans and Gavrilova 2020, Khalaf et al. 2019).
Anakinra is an antibody that binds to the IL-1 type 1 receptor to prevent IL-
1 and IL-1 from binding to their receptor. It is effective in reducing
neutrophilic airway inflammation. Lebrikizumab and Tralokinumab block IL-
13, a Th2 cytokine; they have not shown a significant decrease in exacerbation
rates. Secukinumab is an antibody against IL-17A and Brodalumab against IL-
17R; neutrophilic asthma is driven by a predominant Th17 response, but none
of these antibodies are available as an effective treatment. ABM125 has been
hypothesized as a treatment for asthma in mice by targeting IL-25.
ANB020, an anti IL-33, inhibits airway hyperresponsiveness and
inflammation in a house dust mite model of asthma. Tezepelumab binds the
alarmin TSLP and blocks its interaction with its receptor, diminishing the
recruitment of antigen presenting cells to mature cells of the adaptive
immunity; it suppresses type 2 inflammation, decreases blood eosinophils, IgE
and FeNO levels, decreases exacerbation episodes. Fevipiprant is a reversible,
but selective competitive antagonist of CRTH2, a prostaglandin receptor that
perpetuates chemotaxis of Th2 cells, and is commonly expressed on the
surface membrane of many immune cells; it induces reduction of exacerbation
rate and lung function improvement, while AZD1981 antibody has not been
shown efficacy.
IL-9 is believed to play a role in asthma, but a humanized anti-IL-9
monoclonal antibody in patients with moderate to severe asthma failed to
show efficacy. CRTh2 is a prostaglandin receptor that maintains chemotaxis
of Th2 cells; anti-CRTh2 AZD1981 antibody has not been effective in treating
asthma, but there has been some efficacy for Fevipiprant anti-CRTh2. Anti‐
TSLP antibodies reduced allergen‐induced bronchoconstriction and number
of blood and sputum eosinophils in patients with mild allergic asthma, and
decreased asthma exacerbation rates in adults with moderate to severe asthma.
Conclusion
Asthma is a heterogeneous, multifactorial disease that has been extensively

studied and characterized but still represents major public and global health
issues. In asthma pathogenesis, inflammation is crucial, and the primary goal
of treatment is to achieve the control of symptoms and the underlying
inflammation to avoid future disease exacerbations. Moreover, not all asthma
patients have reversible airway obstruction, some present persistent airway
obstruction caused by airway-wall remodeling and mucus plugging, which is
a major concern among physicians (Dunican et al. 2018).
The morbidity prevalence of asthma affects a large number of people
worldwide and represents an economic and humanitarian burden. Moreover,
statistics show an underestimation because of the ambiguity of an asthma
definition and the resulting underdiagnosis. Consequently, a global diagnosis

acknowledging the different intrinsic and extrinsic factors will provide a more
precise diagnosis. Clinical features of the patient are important, but deeper
conditions must be analyzed upon the severity of the symptoms and the
response to classical therapies.
The study of inflammatory mechanisms involved in asthma pathology has
been traditionally associated with the adaptive immune system since there is
considerable production of Th2 cytokines. Later, the identification of cells
from the innate immune system and the initiation mechanisms of asthma has
driven attention to this system and has led to extensive studies to confirm this
hypothesis. Moreover, the dual role of innate immunity is fascinating. Apart
from having an inducer role, the innate immune system also has a protective
character. For instance, at the beginning of life, the contact with products from
different microorganisms drives immunity to a regulatory pathway, which
increases the number of Tregs and the expression of some TLR diminishing
allergies. These mechanisms provide protection against diseases like asthma.
Contrary, at asthma’s initiation, innate immune cells like ILC2 activate and
release cytokines and molecules that recruit eosinophils or neutrophils
depending on the extrinsic factors, which provoke inflammation of the airways
and furthers asthma’s development.
The use of new “-omics” technologies like proteomics, metabolomics,
transcriptomics, etc.; could indicate the origin of the exacerbation factor and
could support the diagnosis, and the understanding of different pathways
involved. Nonetheless, these expensive studies are not the only way to achieve
a proper characterization of the mechanisms of inflammation in each asthma
patient. For example, sputum cytology can provide useful information
regarding the recruitment of cells like eosinophils vs. neutrophils or the
presence of other innate immune cells leading to a more precise diagnosis and
treatment.
Once we have a proper diagnosis, the next step is to provide the patient
the most accurate therapy to avoid unwanted side effects. In mild asthma,
symptoms can be controlled with the use of corticosteroids and bronchodilator
medications. For athletes with exercise-induced asthma, a previous warm-up
routine is recommended to avoid bronchoconstriction. The challenge is those
severe asthma patients; their condition might lead to multiple visits to
heathcare institutions causing economic and social burdens to life-threatening
conditions.
To provide adequate therapy for severe asthma patients, physicians must
consider the use of different immunotherapies, while identifying the patient’s
phenotype and endotype. Monoclonal antibody therapy is getting more

attention, and FDA and other regulatory agencies approve new antibodies
every year. For severe asthma patients with uncontrolled symptoms, the use
of monoclonal antibodies seems the right way to proceed. Although the cost
of these therapies seems elevated, the reduction in exacerbations and
hospitalization costs, as well as the indirect costs regarding loss of working
days or unpaid activities, should be considered (Bagnasco et al. 2021). The
most important factor into account is providing the best quality of life for
asthma patients.
Note
During the global pandemic of COVID-19, infectious disease caused by

severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), asthma
patients were a major concern due to susceptibility and severity of the disease.
However, several studies worldwide have not shown the expected prevalence
of asthmatic patients. Certain factors of type 2 immune response, such as type
2 cytokines including IL-4, IL-13, and accumulation of eosinophils, might
provide potential protective effects against COVID-19. Moreover,
conventional therapy for asthma such as inhaled corticosteroids, allergen or
anti-cytokines immunotherapy, or anti-IgE monoclonal antibodies, might also
reduce the risk of asthmatic from suffering infection of the virus through
alleviating inflammation or enhancing antiviral defense (Liu, Zhi, and Ying
2020).
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Section 2. Innate Immunity
and Non-Classic Immunomodulators
Chapter 4
Arthritis Rheumatoid Onset

and Development: The Gaze of TLRS
Laura del Carmen Sánchez García*

Departamento de Ciencias de la Farmacia,
División de Ciencias de la Salud, Universidad de Quintana Roo,
Chetumal, Quintana Roo
Abstract
Rheumatoid arthritis (RA) development as a chronic inflammatory

disease involves the early activation of innate immunity. RA patient
monocytes strongly express TLR2, TLR4, TLR5, TLR7, and TLR8
induced by their exogenous and endogenous ligands in synovial tissue
that activated the initial production of inflammatory cytokines,
chemokines, and destruction of the cartilage and bones by destructive
enzymes. Fibroblast hyperplasia is an anticipatory process during the
development of RA, followed by the persistence of inflammatory
cytokine that recruits cells by enhancing the expression of a broad range
of inflammatory chemokines and TLRs. Moreover, macrophages and
monocytes contribute significantly to the release of cytokines such TNF-
alpha, IL1, and GM-SCF and the production of proteases collagenase,
gelatinase B, stromelysin, and leucocyte elastase that contribute to the
accelerated reabsorption bone process. Besides, some pieces of evidence
indicated that miRNAs act as key immunoregulators of TLRs and their
multiple components of the signaling pathways, including signaling
proteins, transcription factors, regulatory molecules, and cytokines.
*
Corresponding Author’s E-mail: laurasan@uqroo.edu.mx.

ISBN: 978-1-68507-510-1
90 Laura del Carmen Sánchez García
Thus, innate immunity control of RA is beyond what was expected before

and opens an extended therapeutic option that can control the pathology
at very early steps. This chapter reviews the innate central actors such as
TLRs that act as a trigger to develop non-clinical manifestations of RA.
Keywords: TLRS, rheumatoid arthritis, fibroblast, miRNAs, signaling

cascade MAPK
Introduction
Rheumatoid arthritis (RA) is an autoimmune arthropathy associated with

articular damage disease characterized by chronic inflammation and its
comorbidities, particularly in the cardiovascular system. Some characteristics
such as heterogeneity, variability in clinical presentation, and the pathogenic
mechanism mounted by the individuals lead to AR as a disease that requires
early diagnosis and a pharmacotherapeutic treatment that controls the loop
inflammation established by the activation of innate and adaptive immunity.
Genetic and non-genetic factors determine the complexity of AR and trigger
the disease in genetically susceptible individuals according to the
environmental factors that influence their physiological condition. Due to the
complexity and diversity of genetic and non-genetic factors, determining the
etiology of RA has become an enormous task, especially to accord the
classification criteria of RA and the characterization of new autoantibodies to
facilitate an early diagnosis. As AR is non-curable, patients do not reach
remission and there is an increase in disability and socioeconomic decline, it
is critical to establish biomarkers to use in the early detection of AR, identify
the innate immunity factors involved in the onset of AR, and determine the
biological therapies that can control those innate receptors that are involved in
the establishment and persistence of the inflammatory condition. In addition,
it is necessary to identify the trigger mechanism induced by environmental
factors to prevent or mitigate the onset and development of AR in genetically
susceptible patients. Furthermore, in patients without a genetic risk but who
are in a risk environment, the generation of early attention programs can
contribute to prevention.
Thus, this chapter tries to guide the reader towards being involved in the
main issues of the innate immunity involved in the comprehension of the onset
and establishment of crosstalk between inflammatory cells and the signaling
Arthritis Rheumatoid Onset and Development 91
pathways activated throughout TLR after ligand pathogens and microbiome

antigens.
Innate Immunity
The innate immune response is the first mechanism for host defense.
Moreover, its activation tries to prevent infection and attack the invading
pathogens (Flaherty, 2012) while protecting the host from various toxins and
infectious agents such as parasites, fungi, bacteria, and viruses (Janeway,
2002). Innate immune protection has two steps: first, identifying pathogens
and abnormal tissues and cells, and second, by orchestrating humoral and cell
effectors to neutralize and eliminate the identified targets (Flaherty, 2012).
Innate immunity response includes a non-specific mechanism that acts within
minutes to hours and includes different components, including physical
factors, anatomical barriers, and epithelial and phagocytic cells that prevent
microorganisms from entering the host (Chaplin, 2003). A variety of epithelial
cells produce antimicrobial peptides to eliminate pathogens. Furthermore, the
pathogen presence enhanced the action of inflammatory cytokines such as
interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) produced by
several innate cells (Chaplin, 2003; Delves and Roitt, 2000).
Elements of the Innate Immune System
Innate immunity consists of three components: physical barriers, cells, and

soluble factors: skin, mucosal surfaces, cellular components, chemotactic
factors, and receptors. The skin is the outermost organ of the body, and
constantly struggles with external pathogens (Kabashima et al. 2019). Due to
the continuous exposure to chemicals and pathogens, several immune cells are
skin residents, inducing homeostasis upon inflammatory challenges
(Kabashima et al. 2019). The structures of the skin are the epidermis and the
dermis. The stratum corneum is the outermost layer of the epidermis.
Likewise, it is composed of dead keratinocytes and intercellular lipids in the
hair follicles and sweat ducts. Langerhans cells, γδT cells, and resident
memory T cells are between keratinocytes (Kabashima et al. 2019). In
addition, there are different immune cells in the dermis, such as dermal DC,
macrophages, mast cells, innate lymphoid cells, and γδT cells (ILCs) (Tong et
al. 2015).
The mucosal immune system comprises a variety of epithelial cells among

the gastrointestinal and respiratory tracts. Innate immunity protects the
mucosal tracts by effectively recognizing and eliminating pathogens (Perez-
Lopez et al. 2016; Thaiss et al. 2016). Although many cell types are involved
in the innate response, dendritic cells, macrophages, and epithelial cells
initiate the specific pathogen recognition throughout the receptors (PRRs)
such as TLRs, CLRs, RLRs, and NLRs which, once activated, mediate the
immune response, and regulate mucosal homeostasis or contribute to the
development of inflammation related to various diseases. (Thaiss et al. 2016;
Akira et al. 2006; Gilbert et al. 2016).
Innate immunity includes various antimicrobial molecules and enzymatic
compounds such as lysozyme, which affects microbial growth, as well as
hydrochloric acid and proteins like pancreatin and peptidase in the
gastrointestinal tract. Additionally, the growth regulators of the human biome
and pathogenic microbiome are involved: fatty acids, bile acids, fibronectin,
lactoferrin, and transferrin (Kumagai and Akira, 2010; Zasloff, 2002), as well
as a wide variety of opsonins: MBL, CRP, C3b complement factors, and
fibrinogen (can sense microorganisms). This opsonins stimulated
phagocytosis, favoring the control of the infection (Al-Azad et al. 2016). The
cellular components play a vital role in developing the inflammatory process
and the management of the pathogens, and include phagocytic cells, epithelial
cells, endothelial cells, mast cells, natural killers, innate lymphoid, and
platelets (Al-Azad et al. 2016).
Synoviocytes and Fibroblast
The freely moving diarthrodial joints with a synovial cavity provided the
structure and support mobility. The synovium encapsulates the joint and
lubricates the surfaces, providing nutrients to the cartilage. The joint lining is
a delicate membrane divided into two anatomical and functional
compartments, the intimal lining and the sublining layer (Bartok and Firestein,
2010). The former is a superficial layer in contact with the intra-articular
cavity and produces the lubricious synovial fluid. It is organized generally in
two or three cells deep in a loose association, including two or three types of
cells like Type A or macrophage-like synovial cells and Type B or fibroblast-
like synoviocytes. The Type A, macrophage-like synovial cells in the intimal
lining consist of monocyte-macrophage lineage with little capacity to
proliferate (Edwards and Willoughby, 1982). The type B synoviocytes or FLS
are mesenchymal cells that display many characteristics of fibroblasts with

some unique properties. Uridine diphosphoglucose dehydrogenase (UDPGD)
is preferentially expressed by the intimal lining fibroblasts and reflects the
ability to synthesize hyaluronan, an essential constituent of synovial fluid and
extracellular matrix (ECM) (Firestein., 1996). Moreover, fibroblasts present
in the intimal lining secrete lubricin. Type B synoviocytes express type IV and
V collagens, vimentin, CD90 (Thy-1), intercellular adhesion molecule 1
(ICAM-1), and CD55 (decay-accelerating factor) (Szekanecz et al. 1994,
Morales-Ducret et al. 1992). The sublining layer comprises cellular
components such as fibroblasts and macrophages organized loosely along the
blood vessel network (Filer, 20013).
Macrophages
Macrophages and monocytes are critical effector cells of innate immunity and
possess a variety of pattern recognition receptors such as Toll-like Receptors
(TLRs), Nod-like receptors (NLRs), RIG-I like receptors (RLRs), and C-type
Lectin receptors (CRLs) (Sweet and Bokil, 2010). This receptor group is either
expressed at the cell surface or intracellularly, enabling the cell to sense
intracellular or extracellular pathogens. The receptor signaling produced an
immediate response such as phagocytosis, clearance, oxidative burst, cytokine
secretion, adhesion, and several morphological changes. In addition, gene
regulation is involved in several biological functions like survival/death,
inflammatory response, leukocyte recruitment, anti-microbial response,
pathogen destruction, antigen presentation, and adaptive immunity activation
(Sweet and Bokil, 2010; Williams and Ridley, 2000; Doherty et al. 1989).
Macrophages are a diversified group of cells that acquired a phenotype
according to the specificity of the local microenvironment, resulting in cells
that produced an answer depending on the pathogen and the signaling
molecules produced by innate and adaptive cells and tissue damage (Kumagai
et al. 2008). The plasticity and heterogeneity of these cells separate
macrophage activation into either classical or alternative (Sweet and Bokil,
2010; Taylor et al. 2005).
The polarization of classically activated or inflammatory macrophages
(named M1) is by cytokines like tumor necrosis factor (TNF-α), interferon
(IFN)γ, bacterial lipopolysaccharide (LPS) and the TLRs activation
(Abdulkhaleq et al. 2018). In addition, M1 shows higher anti-microbial
capacity due to ROS generation, the release of pro-inflammatory cytokines
(Interleukin (IL)-1β, TNF-α, IL-6), and cytotoxicity via the phagocytosis of

microorganisms and necrotic cells (Yang et al. 2013). Additionally, M1
upregulates TNF-alpha and inducible nitric oxide synthase during infections
(iNOS) (Mosser et al. 2008; Chow et al. 2011).
Non-classical monocytes are named M2, and their phenotype includes a
patrolling and anti-inflammatory cell. M2 has a close relationship with
basophils and mast cells because IL-4 (Martinez and Gordon, 2014) can
mediate its differentiation. In M2, transforming growth factor (TGF-beta),
IL5, IL-10, and Stat-3 are elevated, linking the inflammatory actions and tissue
repair (Lv et al. 2017; Wang and Kubes, 2016) M2 macrophages respond to
different cytokines and chemokines. Activation by IL-4 or IL-3 polarized the
M2a phenotype. Immune complexes (ICs) combined with IL1-beta or LPS
activate M2b, and IL-10, TGF-beta, or glucocorticoids polarized the M2c
macrophage phenotype (Laria et al. 2016).
TLR Function
Innate immunity is the fundamental defense mechanism against infections;

this response is a fast and non-specific immune response (Wei-Chiang and
Stan, 2005). The cellular, vascular, and humoral activation is followed by
developing inflammatory conditions that include several pathways involved
in developing this mechanism, a migration of white blood cells such as
monocytes, neutrophils, eosinophils and basophils, and fluids to the site of the
insult. In addition, signal molecules such as histamine, serotonin,
prostaglandins, leukotrienes, and nitrogen-derived free radicals accompany
the process (Abdulkhaleq, 2014).
Inflammation is mediated by germline-encoded pattern recognition
receptors (PRRs), recognizing pathogen-associated molecular patterns
(PAMPs). PRR activation initiates a series of signaling programs that execute
the first host defensive responses mounting the inflammation (Janeway, 1989).
Toll-like receptors (TLRs) are type I transmembrane proteins that contain
leucine-rich repeats which mediate the recognition of PAMPs, and the
cytosolic Toll-IL-1 receptor (TIR) mediated the downstream signaling
pathways (TIR) (Beutler, 2009; Akira et al. 2006). TLRs are the first PRRs
characterized and widely characterized in PAMP recognition and activation.
To date, ten functional TLRs have been identified in humans (Akira et al.
2006). Each TLR detects specific PAPMs derived from bacteria, parasites,
viruses, mycobacteria, fungi, and parasites. These include lipoproteins
(recognized by TLR1, TLR2, and TLR6), double-stranded (ds) RNA (TLR3),

lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), single-stranded (ss) RNA
(TLR7 and TLR8), and DNA (TLR9) (Akira et al. 2006). Furthermore, TRL
recognition initiates downstream signaling events that produce type I
inflammatory cytokines, chemokines, and antimicrobial peptides. (Kawai and
Akira, 2010). Those events mainly cause the recruitment or induction of
cytokine genes and neutrophils, and the induction of macrophages. Besides,
TLR signaling induces adaptive immunity by leading to the maturation of
dendritic cells (Kawai and Akira, 2011).
TLRs Cellular Location and Signaling
TLRs such as TLR1, TLR2, TLR4, TLR5, and TLR6 are membrane
glycoproteins. Furthermore, TLR3, TLR7, TLR8, and TLR9 are expressed
within intracellular vesicles and recognized nucleic acids. The recognition of
several PAMPS for TLRs and coreceptors are sufficiently relaxed to permit
the accommodation of numerous isoforms of antigens (Beutler et al. 2006;
Hoebe et al. 2004). Also, TLRs do not discriminate between host and
microbial molecules, and TLRs activated an inflammatory immune response
(Kawai and Akira, 2011).
Following TLR recognition of the respective PAMPs, a signaling pathway
is activated, producing a specific response. TLRs depend on a particular or
unique combination of TIR domain-containing adaptor proteins such as
MyD88, TIRAP, TRIF, TRAM, IKK-α, osteoponina, and IRF7 (Kawai etal.,
2004; Saitoh et al. 2011). Several studies support the fact that MyD88 is
utilized by all TRLs (except TLR3) (Kawai and Akira, 2010). The activation
of MyD88 produced the phosphorylation of several MAP kinases and the
translocation of the transcription factor NF-KB to the nuclei to initiate the
synthesis of inflammatory cytokines. TIRAP, a member of the TIR family, is
an adaptor molecule that recruits MyD88 which is used in TLR2 and TLR4.
Besides, TRAM functions as a bridge between TLR4 and TRIF (Kawai and
Akira, 2011).
There are differences between TLR signaling cascade activation and the
adaptor proteins involved. TLR2, TLR1, and TLR6 are cell membrane
receptors, and activate signaling pathways through MyD88 and TIRAP
recruited to phagosomes during phagocytosis to induce the synthesis of
inflammatory cytokines (Kawai and Akira, 2011). Also, TLR4 recruits four
adaptor proteins and activates two specific signaling pathways: the MyD88-
dependent and TRIF-dependent pathways (Kawai and Akira, 2010). TLR4

recruits TIRAP and MyD88, while PIP2 serves as a bridge for interactions
between MyD88 and TIRAP. MyD88 recruits IRAKS, TRAF6, and the TAK1
complex leading to the early-phase activation of MAP kinases and NFκB57.
Already, TLR4 is endocytosed and delivered to intracellular vesicles to form
a complex with TRAM and TRIF leading to the recruitment of TRAF3 and
the protein kinases Ikki and TBK1, which catalyze the phosphorylation of
IRF3 resulting in the synthesis of interferon (IFN) type 1 (Husebye et al. 2010;
Barton and Kagan, 2009).
The TLR4 TRIF-dependent pathway recruits TRAF6 and TAK1 to
activate MAP-kinases and NFKB. In addition, TLR4 recruitment depends on
the small GTPases Rab11a-positive recycling endosomes, and RAB11
regulates TLR4 mobilization from recycling endosomes to phagosomes, thus
activating the TRI-dependent type I IFN induction pathway (Husebye et al.
2010). Thus, for a robust response to TLR4 activation, a MyD88 and TRIF-
dependent pathway is necessary to induce the release of inflammatory
cytokines (Palsson-McDermott et al. 2010). (For more information about the
trafficking of TRLS, see Figure 1. TLR Trafficking and Signaling Kawai and
Akira, 2011).
Trafficking of Intracellular TLRS
TLR translocation among intracellular compartments requires several

chaperones proteins; for example, Gp96 or Grp94, a paralog of the Hsp90
family present in ER, act as chaperones for TLRs, various integrins, and IgGs
(Randow and Seed, 2001; Yag et al. 2007a). The UNC93B1 protein interacts
directly with the TLR3, TLR7, TLR9, and TLR13 transmembrane domains
and mediates their translocation from the ER to the endolysosomes (Kim et al.
2008; Brinkmann et al. 2007). Some evidence indicates a rivalry between
TLR9 and TLR7 associated with UNC93B1 and the more robust association
with TLR9, favoring its translocation and resulting in more robust signaling
through TLR9 and weaker TLR7 responses (Fukui et al. 2009; Wang et al.
2006). In brief, UNC93B1 is a necessary chaperone protein for intracellular
TLR responses, and it defines the efficiency of each TLR to move from the
ER to the endolysosomes where they encounter and respond to their respective
ligands (Wang et al. 2006). The endosome acidification is a critical process
for disassembling the pathogen to the TLRs (TLR3, TLR7, and TLR9) and
processing the TLRs themselves. For example, the TLR9 ectodomain, upon
reaching the endolysosomal compartment, is proteolytically cleaved by

proteases including various cathepsins and asparagine endopeptidase. The
proteases seem to depend on the cell type (Park et al. 2008; Sepulveda et al.
2009; Fukui et al. 2009). Additionally, cleavage is required to recruit MyD88
and the initiation of the signaling pathways. Trafficking and Processing of
Intracellular TLRs Blasius Al, Beutler B). (Blasius and Beutler, 2010).
Arthritis Rheumatoid Generalities
Rheumatoid arthritis (RA) is an autoimmune disease typified by the chronic

inflammation of joints and the production of IgG antibodies such as
rheumatoid factor (RF) and anti-citrullinated proteins antibodies (ACPAs).
RA is a complex disease that involves environmental and genetic factors (60%
of risk), susceptibility genes (HLA-DRB1), and epigenetic modifications, as
well as non-genetic risk factors (40%), smoking, microbiota, female sex,
western diet, and ethnic characteristics (Smolen et al. 2018) (Figure 1
Development and progression of RA. Smolen et al. 2018).
Genetic Risk
The major histocompatibility complex (MHC) molecules show a robust

association with RA, and may contain a shared epitope, which recognizes a
specific amino acid motif commonly encoded by some alleles of HLA-antigen
D related with the DR locus HLA-DBR1*01 and HLA-DBR1*04 (Gregersen
et al. 1987). However, studies suggest that genetic RA alleles are
heterogeneous across ethnic groups. Many alleles that associate weakly with
RA interact with other genes and with the environment. The effect of this
interaction must be modest or not in several alleles that are considering the
risk for RA (Steinbrocker et al. 1949). Furthermore, genetic differences
between ACPA-positive and ACPA-negative RA revealed the presence of
variants such as HLA-DRB1, PTPN22, BLK, ANKRD55, and IL6ST
independently of RA seropositivity (Smolen et al. 2018). Meanwhile, AFF3,
CD28, and TNFAIP3 are only found in seropositive patients. Thus, even
though patients with different racial backgrounds share the HLA region as a
significant genetic risk locus, there is no consensus about the alleles and genes
used as general RA markers of patients. (Smolen at al., 2018, Hey-Soon et al.
2009).
Epigenetic Factors
Nine clusters of DNA methylation with differential patterns in the HLA in RA

patients indicate that HLA variants provide a mechanism by which
environmental factors can induce changes in cellular function (Liu et al. 2013).
Furthermore, the different patterns of DNA methylation and transcription
described by fibroblast-like synoviocytes (FLS) in different joints could
explain the symmetry of RA and the severity in some joints but not in others
(Frank-Bertoncelj et al. 2017).
No Genetic Risk
Gender
The overall risk of developing an inflammatory autoimmune rheumatic

disease is mostly unknown. In the USA, the risk for women is 8.4%, equivalent
to 1 in 12 women. For men, the risk percentage is 5.1%, which means 1 in 20
men can develop RA (Crowson et al. 2011). The overall prevalence of RA is
age- and gender-specific; for women, more than 2% of 65 years old are prone
to developed AR1 (Crowson et al. 2011). It is well accepted that the higher
prevalence of AR for females seems to be associated with hormonal control
of the immune system. Evidence indicates that temporal remission is
associated with pregnancy period time (Alpizar-Rodriguez et al. 2017) The
residual lifetime risk of rheumatic disease at the age of 50 is 7% for women
and 4.1% for men (Crowson et al. 2011). Even though the percentages reported
for AR are lower than those reported for common diseases such as dementia
or hip fracture, the lifetime risk and primary inflammatory autoimmune
rheumatic diseases must be greater than generally reported (Crowson et al.
2011).
In Smoking
RA trigger factors such as tobacco smoking remain controversial regarding

the association with antibodies from ACPA-positive individuals. However,
some results indicated that smoking conditions increase the levels of pro-
inflammatory cytokines, and the anti-CCP-positive RA patients with a current
smoker status present higher levels of disease activity associated with higher
levels of inflammatory serum cytokines. Conversely, patients with a
discontinuous smoker habit have lower inflammatory cytokines and lower
ACPA titers5. (Källberg et al. 2011; Sokolove et al. 2016).
Contribution of the Microbiome and Another Pathogen to RA
Freire de Carvalho et al. 2009 report that microbial agents are critical
environmental contributors to the development of autoimmunity. The
mechanism involved in the development of autoimmune disease included
epitope spreading, which refers to the exaggerated local activation of ag-
presenting cells due to an inflammatory state. In addition, the bystander effect
is characterized by the generation of expansion of auto-reactive T cells. Also,
the biome seems to have an essential role in the development of AR disease.
Some rare bacteria species seem dominant during the early and late
establishment of AR. Recent studies showed two novel autoantigens isolated
from HLA-DR molecules, with significant sequence homology at the peptide
level with Actinobateria, Prevotella and other gut bacteria species peptides,
as well as parvovirus B19 and Chikungunya (Scher et al. 2013; Planta et al.
2017; Naciute etal., 2016; Gasque et al. 2016).
RA Pathogenesis
As mentioned before, genetic and non-genetic factors orchestrate the

development of AR; one of the crucial processes to operate is post-
translational modifications (citrullination, acetylation, carbamylation)
(Makrygiannakis et al. 2008) For example, cigarette smoke can act on cells in
mucosal sites favoring the post-translational conversion of several amino acids
from arginine to citrulline. Proteins involved in this process are intracellular
and matrix proteins such as histones, fibronectin, collagens, fibrinogen,
enolase, and vimentin. Additionally, the microbiome may also induce
citrullination. For example, P. gingivalis, a bacteria present in periodontal
disease, expresses peptidyl arginine deaminase that promotes ACPA. Also, A.
actinomycetemcomitans produces a toxin that increases the influx of calcium
and the citrullination of peptides in neutrophils (Dissick et al. 2010; Chen et
al. 2016).
The citrullinated peptides bind to MHC protein heterodimers to present

the shared epitope to T cells in the next step. Besides, once T cells are
activated, they stimulate B cells to synthesize various antibodies that
recognize self-proteins, including rheumatoid factor (targeting IgGs) and
ACPAs (targeting citrullinated proteins) (Holers et al. 2013).
Once autoantigens are shown to belong to self-epitopes activated T and B
cells, the autoantibodies are produced. They circulate in the serum, favor
immune complex formation and activation of the microvascular system, and
synovitis is mounted. Synovitis may develop in three ways. The first event is
the development of vasculitis throughout the deposition of the circulating
preformed immune complex along the basement membrane of synovial
postcapillary venules. The second is related to the preformed immune complex
of autoantibodies settled directly to the synovium. Thirdly, autoantibodies
now enter the synovial space and bind directly to specific antigens in the
cartilage. The influx of mononuclear cells feeds synovitis, neutrophils, CD4 T
cells, and early stromal cell activation. There is high expression in the synovial
intimal lining of matrix-degrading enzymes (such as matrix
metalloproteinases (MMPs), type 2 collagen (particularly in oxidized form),
glucose-6-phosphate isomerase, proteoglycans, and nuclear antigens.
Additionally, autoantibodies (ACPAs, RF) recognize immunoglobulins and
other autoantigens that expand the pathways, whereby autoantibodies likely
contribute to expand and maintain pathogenesis. (Muller and Radic, 2005;
Trouw et al., 2013; Steiner, 2007; Arend and Firestein 2012) (Figure 1.
Proposed mechanism of initiation of RA. Steiner, 2007).
Innate Immunity of RA Synoviocytes and Fibroblasts
During the onset and development of RA, the joint goes through modifications
such as dramatic hyperplasia of the lining layer, occasionally reaching 10-15
cells in depth. The articular borders of the lining layers are rich in fibroblasts
and osteoclasts, which form a mass of pannus tissue invading adjacent
articular cartilage, and the subchondral bone (Filer, 2013) Also, the sublining
layer expands and recruits inflammatory cells, including macrophages, T cells,
B cells, and plasma cells; sometimes, T and B cells form complex aggregates
(Takemura et al. 2001). Fibroblast hyperplasia seems to relate to two possible
events. First, some evidence suggests the inhibition of proapoptotic pathways
and factors like PTEN, SEN-P1, and microRNA-34A, and second, the large-
scale proliferation and a significant increase in pro-survival factors including
FLIP, SUMO-1, and the overactivity of the Ras, Myc, and NF-kB pathways
(Korb et al. 2009). Another critical event related to RA damage and
persistence is the increase in the size of the synovial fibroblast population and
eroding phenotype, fibroblast enhanced secretion of matrix metalloproteinases
and cathepsins.
Additionally, some evidence indicates that fibroblasts can acquire a
migratory phenotype (Lefèvre et al. 2009). Moreover, fibroblast-secreted
RANK-ligand, which promotes osteoclast differentiation and activation
favoring bone erosion (Shigeyama et al. 2000), and DKK-1, which inhibits
anabolic osteoclast function, preventing the repair of bone erosion (Diarra et
al. 2007). Following the persistence of inflammation, fibroblasts recruit cells
by enhancing the expression of a broad range of inflammatory chemokines,
including CXCL8, CCL2, CCL5 and CXCL10, CXCL5, and CXCL1 (Koch
et al. 1995; Koch et al. 1991; Patel et al. 2001). Furthermore, fibroblasts
enhance BAFF- and APRIL-mediated B-cell survival and function in the RA
synovium (Bomnardieri et al. 2011). Another outstanding role for fibroblasts
is the retention of cells in the inflamed joint that would otherwise traffic to
lymph nodes through CXCL12, CXCL13, and CCL21 (Bomnardieri et al.
2011; Bucley et al. 2000). (For more information about the role of fibroblasts
in RA, see Figure 2. The persistent RA fibroblast phenotype. Synovial
fibroblasts interact with multiple cell types in the RA synovium to maintain
inflammation and continued joint destruction (Filer, 2013).
In rheumatoid arthritis, the synovial membrane contains a surface layer of

HLA-DR+CD14+ and CD68 macrophages distributed in lymphoid aggregates
or diffuse infiltrates (Janeway and Medzhitov, 2002).
Macrophage abundance correlates with joint pain and the general
condition of inflammation. When these cells are present at the destruction site
(cartilage-pannus and bone-pannus junction), they contribute significantly to
the production of inflammatory cytokines such TNF-alpha, IL1 and GM-SCF,
as well as the production of proteases collagenase, gelatinase B, stromelysin,
and leucocyte elastase (Ahrens et al. 1996). A piece of evidence indicated an
amplifier role of macrophages rather than as primary effectors. Also, the
activation of circulating monocytes produced the spontaneous production of
prostaglandins E2, prostanoids, and cytokines (Sesster et al. 1999; Mastroeni
et al. 2000; Garcìa et al. 2000). Additionally, a series of events related to the
release of the degrading enzyme gelatinase B, and superoxide dismutase,

increased integrin expression, and monocyte adhesiveness (MacMicking et al.
1997; Jesh et al. 1997), producing a series of events that amplify the
inflammatory process.
Macrophages like synoviocytes produce a variety of inflammatory
cytokines (like IL-1, IL-, tumor necrosis factor (TNF-alpha). Fibroblast-like
synoviocytes (FLS) mainly produce an incredible amount of MMPs,
prostaglandins, and leukotrienes (Bartok and Firestein, 2010). Over time, FLS
acquired an invasive and migratory phenotype which contributed to cartilage
damage and the propagation of disease because of its migratory potential
(Lefèvre et al. 2009; Philippe et al. 2013).
A second cell wave associated with AR is adaptive immune activation.
CD4+ memory T cells migrate to the synovial sublining. T cells cluster either
as diffuse infiltrate or form ectopic germinal centers in which B cells
proliferate and differentiate, producing ACPA autoantibodies (Chemin et al.
2019).
Cartilage and bone damage are due to macrophages, neutrophils, and mast
cell infiltration via the release of cytokines, collagenase, and stromelysins
(Paul et al. 2000).
Bone erosion is mainly controlled by the activation of osteoclasts
(resorbing bone cells), the receptor activator of nuclear factor-kB ligand
(RANKL) produced by T cells, and the inflammatory cytokines (TNF, IL-6,
IL-1) produced by macrophages and FLS (Panagopoulos and Lambrou, 2007).
In addition, ACPAs play a pivotal role in leading the osteoclast to mature;
once activated, they initiate bone damage (Harre et al. 2012).
Cytokines for the Innate Response
Cytokines involved in RA are central to the development and permanence of

pathogenesis. Cytokines contribute to AR pathology in compartments, namely
innate, adaptive, and stromal. Cellular components produce the cytokines
involved in the development of RA. Infiltrating and resident populations of
macrophages, mast cells, natural killer (NK) cells, neutrophils, FLS, and
innate lymphoid cells certainly contribute to the production of cytokines
(Krishnamurthy et al, 2019; McInnes et al. 2016). Cytokines produced by
synovium cells such as FLS, APCs, macrophages, T, and B cells create a
crosstalk communication system that influences the action and activation
among transversal cells. Pleiotropic activity, paracrine and autocrine actions
of the cytokines, and the persistence of the adaptive immune cell responses
perpetuate inflammation and cartilage-bone destruction. Inflammatory
cytokine production in AR pathogenesis is involved in the articular destruction
in the joints, and autoimmunity, the T helper (TH1) and/or TH17 cell
activation phenotypes in association with synovial the interleukin-15 (IL-15),
IL-1, IL-6, transforming growth factor-β (TGFβ), IL-12 and IL-23. IL-17 is
produced by macrophages. The synovial produce IL-6, IL-10, B-cell
activating factor (BAFF), and a proliferation-inducing ligand (APRIL) that
lead to B-cell differentiation and expansion. The production of cytokines like
TNF, IL-1, IL-6, IL-15, and IL-18. Form Macrophage-derived synovial tissue.
And the lead to osteoclast maturation and activation of the Nuclear factor-κB
ligand (RANKL) and TNF, IL-17, and IL-1. (McInnes and Schett, 2007).
Besides the model called “pathologic compartments” proposed by McInnes et
al. 2015 considered a holistic RA tissue response. This proposal considered
the integrative role of the inflammatory cytokines among the different
compartments in the RA pathobiology during the early and the establishment
phase. Innate cytokines act together with Genetic and non-genetic factors
(Figure 1).
(For more information related to the cytokine network and functions, see
McInnes et al. 2016 and McInnes and Schett, 2007).
Figure 1. (Continued)
Figure 1. Cytokines interaction and actions subserve during the arthritis process. I)
Cytokines are deeply involved in the development of RA and drive the early immune
differentiation, mediated the transition to chronicity, and favor the maintenance and
the loss of remission all along with the discrete and activation/differentiation phases
and during the loss of tolerance in preclinical or early arthritis. Furthermore,
cytokines defining the endotype with a discrete response capability (A vs. B) and the
immune activation to mediate maintenance or loss of remission (C vs. D). II. An
intricated crosstalk is mounted among the innate immune, mediated by inflammatory
cytokine in the synovia. And it is closely related to the inflammatory cytokine
control mounted by adaptive immunity as well. Cell components of the inflamed
rheumatoid synovial membrane included adaptive and innate components such as
dendritic cells (DCs), T cells, B cells, and macrophages that uphold the cytokines
dysregulation. And drive the activation of the effector cells, including neutrophils,
mast cells, endothelial cells, and synovial fibroblast. Only key cytokines are shown
for simplicity. Bidirectional arrows represent a relationship between cells that is
influenced by the cytokines listed. APRIL, a proliferation-induced ligand, BAFF, B-
cell activation factor, bGFG, basic fibroblast growth factor, CCL21, CC-chemokine
ligand 21, CXCL3, CXC-chemokine ligand 13, CXC-chemokine ligand 13; FCyR,
Fc receptor for IgG, IFN, interferon; IL, interleukin; LTB, lymphotoxin-B, M-CSF,
macrophage colony-stimulating factor; PAR2, protease-activated receptor 2;
RANKL, receptor activator of nuclear factor-kB (RANK) ligand; TGF, transforming
growth factor; TH, T helper; TLR, Toll-like receptor, TNF, tumour-necrosis factor;
VEGF, vascular endothelial growth factor. Figure were adapted from McInnes 1B,
Buckley CD, Isaacs JD. 2016. “Cytokines in rheumatoid arthritis - shaping the
immunological landscape.” Nature Review Rheumatology, no. 12(1):63-8. doi:
10.1038/nrrheum 2015.171 and McInnes 1B, Schett G. 2007. “Cytokines in the
pathogenesis of rheumatoid arthritis.” Nature Review Immunology, no. 7(6):429-42.
doi: 10.1038/nri2094.
TLR and Autoimmunity
Two processes avoid autoimmunity via the recognition of self-DNA by TLRs:

a) the intracellular compartment trafficking, which is crucial to distinguish the
self from foreign nucleic acids; and b) the physical separation of self and non-
self-DNA permits self to be distinguished from invader DNA (Blasius and
Beutler, 2010). DNA remains isolated in the mitochondria and nuclei, and
extracellular and endosomal DNases and RNases hydrolyze DNA to prevent
the improper activation of the innate immune system; in this manner, cells and
their endogenous TLR ligands are silent eliminated (Blasius and Beutler,
2010).
The innate recognition of self-DNA is initiated when intracellular TRLs
or other cytoplasmic nucleic acid sensors are activated. Several pieces of
evidence indicate that TLR7 and TLR9 produce antibodies of cytoplasmic and
nuclear DNA/RNA antigens in systemic lupus erythematosus (SLE)-like
disease (Marshak-Rothstein, 2004). The nucleic acids that escape degradation
during programmed cell death produced an inappropriate encounter between
host nucleic acid sensors and self-host nucleic acids (Blasius and Beutler,
2010). Some evidence indicates that the dysfunction of endonucleases
promotes the development of autoimmunity. Undigested self-DNA within
apoptotic bodies due to dysfunction of the Flap endonuclease 1 (FEN1) is
associated with chronic inflammation and autoimmunity (Zheng et al. 2007).
The mutation of Trex1 endonuclease causes SLE, familial chilblain lupus,
inflammatory myocarditis, and Aicardi-Goutieres syndrome. Trex1 mediates
DNA degradation during granzyme A-mediated cell death (Lee-Kirsh et al.
2007a; Lee-Kirsh et al. 2007b). The dysfunction of lysosomal DNase II
produces the inadequate degradation of DNA and results in various
autoimmune syndromes, for instance, arthritis and anemia (Kawane et al.
2006; Yoshida et al. 2005).
TLRS and AR
The primary events of the onset of AR occur outside the joint at the mucosal
compartments such as the mouth, pulmonary system, and gut. Antigen-
presenting cells (APCs) lead to the primary immunization of autoantigens in
the secondary lymph nodes. TLR activation plays a pivotal role in the synovial
compartment to amplify the crosstalk signal produced by autoantigens and the
APCs, and T and B cells; after the activation of TLRs, resident and recruited
cells produce a high number of inflammatory cytokines. In addition, the

autoreactive T cells expand together with the local detection of autoantigens
such as FR and ACPA. Those conditions persist for a long time, leading to
bone and cartilage damage, FLS hyperplasia, and inflammatory cell
recruitment (Larionova et al. 2019). Inappropriate TLR activation is related to
the chronic inflammatory state. Several events supported dysfunctional TLR
activation: the alteration of the microbiome and the abnormal response to the
infection of pathogens, the presence of abnormal TRL-ligands in the synovial
serum of AR patients, and the overexpression of TLR molecules. The
overproduction of inflammatory cytokines is due to activation of the TLR
signaling pathway. Also, the hyper-TLR activation is due to genetic variants
of the patients and the changes produced by epigenetic factors (Larionova et
al. 2019).
TLR Expression in Inflammatory Cells in the Synovium
AR patient monocytes strongly express TLR2, TLR4, TLR5, TLR7, and

TLR8; TLR ligation is induced by their exogenous and endogenous ligands in
synovial tissue, with the fluid activating the production of inflammatory
cytokines (Goch and Midwood, 2012; Seibl et al. 2003).
TLR2 is responsive to microbial ligands, and M-CSF and IL-10 may
induce TLR2 expression in monocytes and synovial macrophages. Besides,
endogenous TRL ligands such as HsP60 and FcγRIIIA-ligand small immune
complexes control the TNF-alpha production of monocytes present in the AR
(Iwahashi et al. 2004). This interplay favors TLR2 expression and the
synthesis of TNF-alpha. (Elshabraw et al. 2017 highlighted that ligation of
TLR2, TLR4, TLR5, and TLR7 of myeloid cells by their natural ligands can
remodel naïve cells present in the synovium into pro-inflammatory
macrophages. Additionally, TLR5 and TNF-α pathways have a strong positive
feedback regulation in attracting the circulating monocytes and further
remodeling the newly recruited cells into mature osteoclasts. And TLR5 and
TNF-α amplifying the polarization of M1 in the synovium compartment (Kim
et al. 2014; Him et al. 2013). Moreover, fibroblasts and macrophages make
from 1-3 to 10-15 lining layers in the inflamed joint (Noss, 2008). Fibroblasts
in hyperplastic RA overgrow the underlying cartilage surface and invade
cartilage and bone to promote joint damage. The fibroblasts of synovial RA
tissue express TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 (Elshabrawy et
al. 2015; Szekanecz et al. 2010). Also, the inflammatory cell crosstalk favors
pleiotropic and synergistic interactions between the cytokines produced.

TLR2 ligation in synovial tissue fibroblasts enhanced the secretion of pro-
inflammatory cytokines (IL-6, CCL8), adhesion molecules (ICAM-1), matrix
metalloproteinases (MMPs 1/3/13), and proangiogenic factors (VEGF, IL-8,
CXCL2) (Makrygiannakis et al. 2008; Dissick et al. 2010; Chen et al. 2016).
Hypoxia activates and potentiates the expression of transcription factors
TLR2, TLR3, and TLR9 for the synthesis of IL-6 and IL-8 (Holers et al. 2013).
TLRs (TLR2, TLR3, TLR4) express fibroblast activated nuclear factor-kB
(RANKL), MMP1, and MMP2 (Hu et al. 2012; Arleevskaya et al. 2020).
Several pieces of evidence indicated that TLRs promote osteoclast-
mediated bone resorption associated with inflammatory conditions during RA.
TLRs also activated homeostatic mechanisms that suppress osteoclastogenesis
and can limit the extent of pathologic bone erosion associated with infection
and inflammation. TLR ligands suppress osteoclastogenesis by inhibiting the
expression of the receptor activator NFκB (RANK) and producing precursor
cells that are unresponsive to the effects of RANKL. M-SCF signaling inhibits
the expression of RANK. TLRs inhibited M-CSF signaling by rapidly down-
regulating cell surface expression of the M-CSF receptor c-Fms via a matrix
metalloprotease- and MAPK-dependent mechanism. Moreover, TLRs
collaborated with IFN-γ to inhibit the expression of RANK and the CSF1R
gene that encodes c-Fms and synergistically inhibits osteoclastogenesis (Ji et
al. 2009). Also, TLR5 activation by flagellin in a dose-dependent manner and
via TNF production promotes osteoclast maturation and monocyte infiltration.
Thus, potent feedback between TLR and TNF is mounted to inflamed joints
and remodel the newly infiltrated cells into mature osteoclasts in mouse
myeloid cells (Kim et al. 2014; Chamberlain et al. 2012).
Learning TLR Role in Mice Model
In mice models, autoimmune arthritis can be reproduced in IL-1 receptor

antagonist (Ra)-deficient mice due to excessive IL-1R/TLR signaling. The
microbiome plays an essential role in developing arthritis since germen-free
mice do not develop the illness in L-1 receptor antagonist (Ra)-deficient mice.
Knockdown mice in IL-1Ra and TLR4 had a significantly lower capacity to
produce IL-17. These findings suggest a role for TLR4 in mucosa for the
expansion of TH17 cells and the development of autoimmune disease in mice
(Rogier et al. 2017).
The microbiome plays a vital role in the development of RA; several

studies have demonstrated the control of TLR-ligand on the synthesis of
several cytokines and chemokines. For example, TLR4 antagonist (Bartonella
quintana LPS) suppressed the severity of experimental arthritis and
diminished the expression of IL-1 (Abdollahi-Roodsaz et al. 2007). On the
other hand, the antigen of Streptococco (Streptococcal cell wall-SCW)
induced arthritis in mouse models. When TLR2-/- mice challenge SCW, a
significant decrease in the levels of CCL3, CCL5, and CXCL1 is produced.
These chemokines are responsible for the infiltration of inflammatory cells
into the arthritic joint (Joosten et al. 2003). Also, in the chronic model of SCW,
TLR4 deficiency resulted in impaired osteoclast formation and reduction in
TH-17, inducing cytokines such as IL-1, IL-6, and IL-23. Besides, TLR
signaling might additionally activate MyD88 and perpetuate joint
inflammation (Rogier et al. 2017).
Given that IL-1 and TLR signaling pathways converge on MyD88, Choe
et al. hypothesized that MyD88 activation could occur via either IL-1R or TLR
stimulation; the results are bone erosion and joint inflammation. The
transgenic K/BxN model of spontaneous arthritis and the disruption of IL-1R
signaling by anti–IL-1R antibody treatment diminished the extent of
spontaneous disease. Furthermore, the co-administration of the TLR-4 ligand,
LPS, and K/BxN serum induced paw swelling in IL-1R-/-, but not MyD88-/-
mice (Choe et al. 2003).
Angiogenesis is a premature event and critical in RA. It promotes
leukocyte infiltration and pannus formation, thereby perpetuating chronic
inflammation (Szekanecz and Koch, 2009; Szekanecz et al. 2009). Synovial
tissue fibroblasts and myeloid cells trigger angiogenesis indirectly by TLR2
and TLR4 ligation via the production of potent proangiogenic factors such as
TNF-alpha, VEGF, IL-8 (Grote et al. 2011; Cho et al. 2007). TLR2 induced
angiogenesis and cell invasion due to the secretion of Ang-2 from RA synovial
explants and its binding to Tie2 expressed on synovial sublining endothelial
cells (Saber et al. 2011). Moreover, TLR5 ligation induced endothelial
chemotaxis through activation of the PI3K/AKT1 pathway; TLR5 agonists
enhance CIA disease severity by promoting TH-17 cell differentiation as well
as joint neovascularization. Also, TLR5 ligation mediates RA angiogenesis
throughout and the differentiation of TH-17 cells and production of joint IL-
17 (Kim et al. 2016).
Results of the effects of the lentiviral vector-mediated delivery of TLR7
short hairpin RNA gene (Lt. shTLR7) on collagen-induced arthritis (CIA)
demonstrated a reduction in joint micro-vessel density and VEGF release from
fibroblasts. Besides, CIA alleviated in TLR7-/- mice due to the suppression of

the IL-17 response and the elevation of joint Tregs. In addition, the K/BxN
serum transfer arthritis model reduced the joint inflammation in TLR7-/-
compared to wild-type mice, in part due to the reduced serum levels of IL-1ß,
CXCL1, CXCL10, and CCL3 (Chen et al. 2012). In addition, the TLR7
ligand miR-let-7b incorporated exosomes, promoting M1 macrophage
differentiation, which secrete high levels of TNF, IL-1, IL-6, CCL2, CCL5,
express the M1 marker iNOS and produce an increase in lining thickness. The
local injection of miR-let-7b induced arthritic joint inflammation by recruiting
myeloid cells from the circulation into the joint. Joint swelling blunted in
TLR7-deficient mice which received an ectopic injection of adenovirus (Ad)-
Let7b or Ad-Ctl, signifying that the ligation of miR-Let7b to TLR7 is
responsible for arthritis induction (Kim and Chen, 2016).
Data discussing RA preclinical models indicated that the activation of
TLR4, TLR5, and TLR7 orchestrate joint inflammation and bone destruction.
However, the ligation of TLR2, TLR3, and TLR9 may have dual effects on
disease manifestations depending on the experimental arthritis model
employed (Elshabrawy et al. 2017).
Role of the Aberrant Expression of Micrornas (MIRNAS)
miRNAs are single-stranded, non-coding, endogenously generated, and the

phyla are evolutionarily conserved. Mature microRNAs are small, non-
coding, naturally occurring RNA molecules with a length of 18-22 nucleotides
(Ruvkun, 2001) miRNA genes constituted about 1-2% of the whole genome.
Therefore, they can potentially regulate 30% of all protein-coding genes (Ha
and Kim, 2014). Also, more than 60% of human genes comprise at least one
miRNA binding site and are present mainly within the junk DNA, the
intergenic regions, and introns of the protein-coding genes (Furer et al. 2010;
Sujitha and Rasool, 2017).
MicroRNAs are pivotal regulators of the gene expression that bind across
the 3ÚTR of target messenger RNA through sequence complementarity,
thereby downregulating gene expression through translational repression,
mRNA cleavage, and deadenylation. Most miRNAs are negative regulators of
the expression of target genes and act by repressing the translation or via the
direct separation of mRNAs, and participate in several cell physiological
processes such as growth, apoptosis, and cell differentiation (Sujitha and
Rasool, 2017).
Until now, the mechanistic details of miRNA biogenesis and gene

silencing are still unclear. However, in general, the biogenesis of human
miRNA consists of a two-step process, in which Drosha and dicer ribonuclease
III endonuclease are involved. The biogenesis takes place first in the nucleus;
the polymerase transcribed the miRNA gene, engendered the pri-miRNA and
a miRNA duplex, and released the pre-miRNA (Sujitha and Rasool, 2017).
The process held in the nucleus includes miRNA genes which are
transcribed by RNA polymerase II to generate a long primary transcriptome
named the pri-miRNA which is processed by the multiprotein complex
(Microprocessor) that comprises the RNA-binding protein DGCR8 (DiGeorge
Syndrome Critical Region 8) and RNAase II type endonuclease Drosha (or
RN3). DCGR8 recognizes the substrate, while the Drosha enzyme catalyzes
the conversion of pri-miRNA to precursor pre-miRNA (Churov et al. 2015).
The nucleocytoplasmic transporter factor exportin-5 assembly the pre-miRNA
and RNA-GTP (preventing nuclear degradation and cytoplasmic
translocation). The following process included the translocation of the pre-
miRNA and the production of a double-strand miRNA with 3´-overhanging
ends. The process is led by the multi-domain protein (RNase III Dicer) that
functions as dicer. pre-miRNA is composed of a guide (or active) RNA
sequence and the passenger RNA. The active strand selectively is incorporated
into a ribonucleoprotein (RNP) complex named RNA-induced silencing
complex (RISCs). Meanwhile, the passenger is usually released and rapidly
degraded at the final step of maturation (Ha and Kim, 2014). (For more
information about miRNA biogenesis, see Figure 1. The canonical pathway of
miRNA biogenesis Churov et al. 2015).
Rheumatoid Arthritis, TLRS and MiRNAs
Taganov et al. 2006 mentioned miRNA-146 for the first time and
demonstrated that this miRNA increased expression in response to LPS while
regulating the signaling of TLRs by TNF receptor-associated factor 6 and
Interleukin 1 receptor-associated kinase. Furthermore, other studies indicated
that miRNA-146 increased its expression by proinflammatory cytokines and
could be an essential modulator of the function and differentiation of innate
and adaptive immunity (Tganov et al. 2006; O’Connell et al. 2010). MiR-146
is one of the most studied miRNAs in RA. miR-146 increased expression
levels in AR patients in synovial fluid, synovial tissue, PBMC, and whole
blood. Some evidence indicated that miRNAs act as key immunoregulators of
TLRs and their multiple components of the signaling pathways. Those

included signaling proteins, transcription factors, regulatory molecules, and
cytokines (Banerjee et al. 2021).
(For more information about the TLRs control by miRNAs, see Banerjee
et al.142. Table 1. The regulatory role of miRNA in TLR signaling pathway in
Inflammatory diseases).
TLR Signaling Cascade Mapk and Mirna Control
The MAPK signaling framework is complex, with constant crosstalk and

many interacting pathways that have switch-like activations of regulatory
factors and fine-tuning. Nevertheless, MAPK pathways converge in the
amplification of crucial molecules involved in cell proliferation, survival, and
growth (Braicu et al. 2019). The MAPK signaling cascade outlines one or
more growth factors with their specific growth factor receptors. The growth
factors bind to membrane glycoproteins of the receptor tyrosine kinase (RTK)
family and activate the signal transduction cascade through cytosolic
intermediates to finally start the transcriptional/translational regulation of
effector genes (Cargnello, 2011).
After the growth factor ligand activated the signaling pathway, the first
line cytosolic intermediates RAS superfamily of GTPases (HRAS, KRAS,
NRAS) activated MAPK phosphorylation (Johnson, 2011). Then, with the
help of the EGFR-associated nucleotide exchange factor Son of Sevenless 1
(SOS1), GTPases were activated (Matallanas et al. 2011). SOS and RAF limit
the active form of RAS, such as the downstream effector of RAS, and it is
dependent on the interaction with an activated RAS. The RAF family includes
several variants (such as BRAF, ARAF, CRAF). They consist of
serine/threonine kinases responsible for the pathway progression by activating
MEK (MAP kinase-ERK kinase) and ERK1/2 (Extracellular signal-regulated
kinases). Both enzymes are involved in processes such as differentiation, cell
survival, and proliferation (Matallanas et al. 2011; McCain, 2013).
The order of MAPK cascade activation is the following:
1. MAPKKK (Mitogen-activated protein kinase kinase kinases,

represented by RAF and its variants)
2. MAPK kinase (MAPKK: MEK1/2/3/4/5/6/7) is involved in cell
survival, proliferation, and differentiation, depending on the
phosphorylated targets of MEK.
3. MAPK includes three main classical MAPKs with different isoforms

ERKs (with ERK1 and ERK2 isoforms), JNKs (c-Jun N-terminal
kinases, with JNK1, JNK2, and JNK3 isoforms), and p38 MAPKs
(with p38α, p38β, p38γ, and p38δ isoforms) (24–26). ERK1/2 is
involved in cell survival, proliferation, and differentiation, depending
on the phosphorylated targets of ERK1/2 (Braicu et al. 2019).
(For details about the MAPK signaling pathway, see Figure 1. Parallel
outline of several physiological roles of the TGF_/p38, mitogen-activated
protein kinase (MAPK), and P13k/AKT/mTOR signaling pathways (Braicu et
al. 2019)).
Mitogen-activated protein kinases (MAPKs) are highly conserved
serine/threonine protein kinases activated mainly by extracellular stimuli such
as cytokines, TLRs, neurotransmitters, and oxidative stress. MAPKs are key
TLR activation pathways that contributed notably to the inflammatory
condition in RA (Schett et al. 2000). In arthritis, rheumatoid MAPK
(p38MAP) abnormally increases the phosphorylated levels of this enzyme.
This event is accompanied by increased ERK and JNK signaling molecules in
synovial-like fibroblasts and macrophages. TLR activation depending on
MAPK signaling pathways in synovial like fibroblast induces MMPs,
hypoxia-inducible factor-1a (HIF-1a), TGF—ß, and VEGF. Another critical
element of the TRL-MAPK signaling pathway is JNK, which is involved in
the induction of MMP expression in synovial like fibroblasts and macrophages
derived from RA (Tganov et al. 2006). Some studies of the use of the selective
inhibition of p38MAPK demonstrated the effective suppression of joint
destruction and TNF-a release in multiple RA disease models. Moreover, a
JNK selective inhibitor showed excellent protection against bone degradation
in the adjuvant-induced arthritic rat model, which is mainly attributed to the
inhibition of the JNK-mediated activation of AP-1, collagenase-3, and MMP
expression (Ha et al. 2006).
Increasing evidence about the regulation of miRNA of several molecules
of the TLR signaling pathway; for example, miR-146a suppresses the
expression of TRAF-6 and IRAK-1.
Consequently, it inhibits the production of crucial adaptor molecules
downstream of Toll-like and cytokine receptors in RA144. miR-155 regulates
the overall NFκB and MAPK signaling using both positive and negative
effectors. In addition, it mediates the release of major pro-inflammatory
cytokines and negatively regulates the inflammatory pathways pf TLR/IL-1R,
as well as targeting TAB-2, which inhibited the activity of RAK-1 (Tili et al.
2007; Ceppi etal., 2009).
miR-26a negatively regulated the expression of TLR3 and reduced the
development of AR in rats152. Moreover, miR19a/b targeted the TLR-2 mRNA
and downregulated the release of IL-6 and MMP-3 in the fibroblast-like
synoviocyte in RA patients (Philippe et al. 2012).
Additionally, synovial fibroblasts express miR-451, and miR-451
treatment significantly decreased the cell proliferation fibroblast ability. Also,
p30MapK reduced significantly after MiR-451 treatment. Also, MiR-451
transfected synovial fibroblasts secreted considerably lower levels of IL-
1beta, TNF-alpha, and IL-6. Those results support the fact that miR451
reduces synovial fibroblast proliferation and can down-regulate p38MAPK
protein expression (Wang et al. 2015). Different experiments showed a lower
expression of miR-573 in the synovial tissues of patients of RA, and this
decrease might contribute to the abnormal expression of TXNDC5
(thioredoxin domain containing 5). Also, the silencing of TXNDC5 abrogates
the pro-invasive effect of MiR-573 inhibitor treatment, indicating the essential
mediation of TXNDC5 to the effect of MiR-573 on the invasion of rheumatoid
arthritis synoviocyte-like fibroblasts (RASFs). Likewise, MiR-573 regulates
the expression of the significant metalloprotease MM3 involved during the
invasion and damage of the cartilage in AR. In addition, miR-573 negatively
regulates the synthesis of IL-6, COX, and TNF-α. Also, TLR2 is a direct target
of MiR-573, which silences TLR2, but not TXNDC5; miR-573 transfection
duplicates its expression. Furthermore, the strikingly lower values for IL-6,
TNF-α, and IL-1β, support the existence of a TLR-2-dependent manner during
miR-573, which functioned to negatively regulate inflammation in RA. The
evidence demonstrated that miR-573 acts as a protective regulator of
inflammation in synovial tissue in AR by switching TCBDC5 and TLR2
(Wang et al. 2016).
The fibroblast-like synoviocytes stimulated with LPS (lipopoly-
saccharide-TRL2 ligand), and bacteria lipoproteins (TLR4 ligand) showed a
significant down-modulation of miR-19, miR20, and miR-17s92 clusters.
Besides, miR-19a and b negatively regulate the TLR2 expression, thereby
reducing the inflammatory response induced by bacterial lipoproteins in
fibroblast-like synoviocytes, which is characterized by the secretion of IL-6
and matrix metalloproteinases (MMP-3) (Philippe et al. 2012).
Conclusion
Rheumatoid Arthritis (RA) is a multifactorial illness that compromises the

patient due to the clinical diagnostic and pharmacotherapeutic treatment
complexity. Considering the genetic and non-genetic factors associated with
the etiology of RA, its prevalence may grow rapidly and constantly for the
next ten years. Genetic predisposition in patients displays considerable
variability in the Human Leukocyte Antigen (HLA), including ethnicity,
which could determine the expression of the Major Histocompatibility
Complex (MHC). The RA complexity increases when we look at the TLRs,
which are TLR signaling pathway polymorphisms that can control and
perpetuate the inflammatory process by activating the immense variability of
these critical regulators together.
Additionally, environmental elements contributed about 40%
susceptibility to developing AR; thinking about the enormous variability,
heterogeneity, and numbers of these factors and their influence on the
individual, genetic AR has become a Pandora’s box. Furthermore, the
diversity of clinical manifestations, biomarkers, antigens involved, previous
infections, the biome, the polymorphism, and the environmental factors
obligate the scientific community to recognize the diversity of many molecular
and cellular processes that initiate and orchestrate the development of RA.
Regarding the pharmacotherapeutic options, it is urgently and
fundamentally needed to base the design of the drug considering the signaling
pathways of TRL-ligands and the miRNA regulation. Then, those treatments
can offer the patients individual, personalized, and adequate attention. This
contributes to diminishing adverse reactions, increasing patient safety, and
hopefully, a lifelong treatment that avoids reactivation and contributes to
remission.
Another essential topic to mention is race and the particular genetic and
environmental factors involved in the onset and development of RA. Paying
attention to the specific environmental factors and the molecular mechanism,
the activation of innate and adaptive immunity should be a determinant in the
race treatment. It is well established that genetic factors are dissimilar,
depending on race; particular factors such as diet and environmental pollution
grade will impact on the commencement and evolution of RA.
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Chapter 5
Modulation of the Innate Immune System

by Extracellular Vesicles
César Díaz-Godínez1, Alejandra Garduño-Nieto2,

Raúl Bobes-Ruíz1 and Julio César Carrero1,
1Departmento de Inmunología, Instituto de Investigaciones Biomédicas,
UNAM, Ciudad de México, Mexico
2Escuela de Dietética y Nutrición-ISSSTE,
Ciudad de México, Mexico
Abstract
Extracellular vesicles (EVs) include a wide range of structures delimited

by a lipid bilayer that are released into the extracellular space by virtually
any cell studied. EVs are classified according to their subcellular origin
and diameter into exosomes, microvesicles, apoptotic bodies and other
uncharacterized EVs. EVs are key structures in intercellular
communication allowing the exchange of numerous molecules that
constitute their cargo, including soluble and membrane proteins,
peptides, carbohydrates, lipids, DNA, and diverse RNA types. When EVs
reach a target cell, they are usually internalized by endocytosis,
phagocytosis, or by fusion with the acceptor membrane to deliver their
cargo into the cytosol, thereby inducing changes in the recipient cell. In
the case of innate immune cells, EVs can influence many processes,
including maturation, activation, migration, cytokine and chemokine
release, antigen presentation and effector functions. The effects can result

Corresponding Author’s E-mail: carrero@unam.mx.

ISBN: 978-1-68507-510-1
130 C. Díaz-Godínez, A. Garduño-Nieto, R. Bobes-Ruíz et al.
in either activation or suppression of their function depending on the

source of EVs and the context in which they are produced. During a
pathological process, host cell EVs tend to promote inflammatory
responses that activate the innate and adaptive immunity to control the
disease. In contrast, EVs released by pathogens during infection diseases
or EVs from tumor cells during cancer tend to exert an important
immunosuppressive role on innate immune cells that prevent the
resolution of the infection or favor the development of tumors and
metastatic niches, respectively. On the other hand, during autoimmune
diseases, EVs tend to promote the development and maintenance of
inflammation. However, due to their immunomodulatory effects, EVs are
promising tools for the treatment of many diseases in humans and
animals. Thus, immunotherapy based on EVs is a field that is gaining
popularity recently and it is envisioned that it may have a great impact
on processes such as transplantation and the treatment of acute
inflammation. Therefore, EVs play an important role in the maintenance
of innate immune homeostasis and their study will make an important
contribution to understanding the intricate communication of innate
immune cells in health and disease.
Keywords: Extracellular vesicles, exosomes, microvesicles,innate immunity,

infectious diseases, autoimmune diseases, immunotherapy, cancer
Introduction
The presence of rounded membranous bodies inside cells has been an

important aspect to consider during the characterization of different cell types,
including a great variety of structures such as lysosomes, granules, vacuoles,
among others (Cowland and Borregaard, 2016; Ballabio and Bonifacino,
2020; Shitan and Yazaki, 2020). Nevertheless, it was the discovery of the
ability of cells to release membranous bodies, also named extracellular
vesicles (EVs), which marked a before and after in cell biology, by allowing
us to understand how cell to cell communication occurs carrying information
with the capability to influence the behavior of other cells. In 1967, Wolf
reported the obtention of a fraction from platelet-free plasma by high-speed
centrifugation, which he called platelet-dust. It was rich in lipids, but its
composition was different from that of chylomicrons, platelets, and
erythrocytes. In addition, the fraction possessed Platelet Factor 3 activity and
was thought to be derived from the osmophilic granules of platelets. Platelet-
dust was further characterized and identified as vesicles derived from platelets
Modulation of the Innate Immune System … 131
possessing ATP-ase activity and contractile proteins (Crawford, 1971). The

author hypothesized that these EVs could play an important role during the in
vivo coagulation process. During the 1970s, it was found that an increase in
intracellular calcium concentration (using the ionophore A23187) induced the
transformation of erythrocytes into echinocytes, a red blood cell with
abnormal membrane, with the subsequent release of EVs from their
microvillus-like structures (Figure 1A) (Allan and Michell, 1975; Allan et al.,
1976). These EVs consisted of spherical bodies about 100 nm in diameter with
a membrane lipid composition different from that of erythrocytes, being
enriched in 1,2-diacylglerol and reduced in phosphatidylcholine (Allan et al.,
1976). Subsequently, EVs release was reported to be a normal process during
aging of erythrocytes stored for transfusions and was related to the
transformation of erythrocytes into spherocytes (Rumsby et al., 1977). Their
characterization showed changes in the membrane lipid composition and
absence of some members of the spectrin proteins with respect to the original
cell (White et al., 1979).
The first functional studies of cell derived EVs were carried out during
the reticulocyte maturation process. The experiments of Pan and Johnstone
(1983) showed that the transferrin receptor was eliminated from the
reticulocyte membrane in vesicles released to the extracellular space, which
would explain the lack of this receptor on the surface of erythrocytes.
Moreover, the transferrin receptor was recycled in the reticulocytes and
released to the extracellular space through the fusion of multivesicular bodies
(MVBs) with the plasma membrane (Harding et al., 1983). The mechanism
was further elucidated by demonstrating that the MVBs, with a diameter of
approximately 1-1.5 μm and containing vesicles of approximately 50 nm
whose surface carries the transferrin receptor, finally fused with the plasma
membrane, releasing the EVs outside the cell (Figure 1B) (Pan et al., 1985).
From these first descriptions, EVs were also soon identified in bacteria
but no function was attributed to them (Pope and Wyss, 1970). Specifically,
EVs from Azotobacter vinelandii were only considered the result of the
membrane compaction during the encystment of this microorganism. Overall,
EVs have also been identified in fungi, protozoa, helminths, and even in virus-
infected cells, ruling out that these are generated as secondary products in
cellular processes (Coackley et al., 2017; Kosanović et al., 2019; de Toledo
Martins et al., 2019; de Souza and Barrias, 2020; Caobi et al., 2020). On the
contrary, EVs have been attributed important roles in intercellular
communication, in the interaction of the host with pathogens during infections,
and in immune regulation, among others (Mathieu et al., 2019; Chen et al.,
2019; Wu et al., 2019).
Figure 1. First descriptions of extracellular vesicles formation. (A) In the 1970s, Allan et al.
described that calcium influx, caused by the ionophore A23187, induced the release of spherical
bodies from microvillus-like structure during transformation of erythrocytes into echinocytes. (B)
In 1985 during the study of the transferrin receptor, Pan et al. described that multivesicular
bodies (MVB) present in reticulocytes fuses with plasma membrane releasing small EVs
to the extracellular space.
Maintenance of homeostasis has also been linked to the production of EVs

as their release is related to feedback signals in different organs (Stahl and
Raposo, 2019). An example is the increase in lipogenesis and arrest of lipid
oxidation in adipocytes after detection of EVs released by hepatocytes when
a lipid overload is detected in liver, which allows a remodeling of adipose
tissue (Zhao et al., 2020). In nervous system, the choroid plexus epithelium
releases EVs during systemic inflammation that can reach the brain
parenchyma and being taken up by astrocytes and microglia, triggering the
upregulation of genes related to inflammation (Balusu et al., 2016).

Furthermore, the release of annexin A1-loaded EVs by epithelial cells in the
gut has been reported to provide the necessary signals to initiate the repair
process after injury (Leoni et al., 2015).
As mediators of intercellular communication, EVs have also been related
to pathological processes. Several authors have reported that EVs released by
tumor cells allow the creation of premetastatic niches in different organs such
as lungs and liver through the promotion of angiogenesis, fibroblast activation
and the recruitment of immune cells (Costa-Silva et al., 2015; Liu et al., 2016;
Zeng et al., 2018; Kong et al., 2019). EVs have also been linked to the
appearance of different eye diseases including diabetic retinopathy, glaucoma,
and autoimmune uveitis (Liu et al., 2020). Finally, EVs play a preponderant
role in triggering the inflammatory response by the activation of most immune
cells, which if not properly regulated can lead to tissue damage (Li et al., 2019;
Jiang et al., 2020).
In this chapter, we explore the role of EVs in the innate immune system
under steady state conditions, infectious diseases, cancer, and autoimmune
diseases. Finally, we focus on the possibilities of using EVs as immunotherapy
for different pathological processes.
Extracellular Vesicles: An Overview
EVs define a heterogeneous set of bodies delimited by a phospholipid bilayer

that are released by cells, which differ mainly in their secretion mechanism,
although there are also important differences in size and in their cargo (Chiang
and Chen, 2019; Doyle and Wang, 2019). EVs are produced by practically any
cell during the normal life cycle and do not require a specific stimulus for their
release; however, certain stimuli have been observed that increase their
production (Arraud et al., 2014; Panteleev et al., 2017; Palviainen et al., 2020).
From a functional point of view, EVs act as an important cell-to cell
communication pathway, since once released, they can be internalized by
other cells (target cells) triggering a biological function (Shifrin et al., 2013;
Maacha et al., 2019; Lizarraga-Valderrama and Sheridan; 2021). Although
they have been widely studied under pathological conditions, the different
types of EVs fulfill very important functions in the steady state, being a source
of molecules with the ability to induce cell differentiation, modification of the
microenvironment, cell activation and adhesion, among many other functions
(Quesenberry et al., 2015; Stronati et al., 2019; Oggero et al., 2019; Bagi et
al., 2019).
Figure 2. Main characteristics of microvesicles, exosomes and apoptotic bodies.

Microvesicles (30 - 1,000 nm) are formed as result of plasma membrane protrusion leaded by
cytoskeleton rearrangement culming in membrane fission and MV release. Exosomes (30 - 100 nm)
are formed inside MVBs during endocytic trafficking; finally, MVB fuses with plasma membrane
shedding exosomes out of the cells. Apoptotic bodies (500 – 5,000 nm) are formed during
disassemble of cells caused by apoptosis program; these EVs even contain entire organelles. Cargos
differ between three types of EVs, however, these include nucleic acids, soluble and membrane
proteins, as well as lipids.
Noteworthy, the composition and cargo of EVs can vary, even if they
come from the same cell, since the detection of external stimuli by the cell can
modify the molecules and the amount of them that are incorporated into the
vesicles (Groot Kormelink et al., 2016; Kolonics et al., 2020). In this sense,
EVs released by a cell may have different functions depending on the
biological context in which they are secreted, which makes them a mechanism
with exceptional plasticity (Holm et al., 2018; Fowler, 2019). In addition, the
uptake of EVs is not a random phenomenon, since the molecules present on
their surface can direct them in a specific way towards the target cells
(Mulcahy et al., 2014; Jurgielewicz et al., 2020). In the next section we will
review the features, biogenesis, and cargo of the three well-defined groups of
EVs: microvesicles, exosomes, and apoptotic bodies (Figure 2).
Microvesicles
Microvesicles (MVs) are a heterogeneous group of membranous bodies

released by blebbing from the cytoplasmic membrane (Allan and Michell,
1975; Tricarico et al., 2016). In the literature, these have also received other
names such as microparticles (MPs), ectosomes (EcMs) or oncosomes (OcMs)
(Freyssinet, 2003; Barteneva et al., 2013; Sadallah et al., 2011; Surmanet al.,
2017; Ciardiello et al., 2019; Di Vizio et al., 2009). MVs have a size range of
30-1000 nm (inclusively 2000 nm), making them the largest EVs together with
apoptotic bodies (ApoBDs) (Minciacchi et al., 2015; Willms et al., 2018; Ståhl
et al., 2019). Dynamics of MVs formation obeys a mechanism that is not fully
characterized; however, redistribution of membrane lipids as well as
rearrangements of the cytoskeleton are required for release of nascent MVs by
fission (Frey et al., 2021).
During the formation of MVs, phosphatidylserine (PS) is exposed in the
surface by the activity of flippases and floppases, which are responsible for
redistributing this lipid to the outer membrane (Liu et al., 2009; Morel et al.,
2010; Fujii et al., 2015; Hankins et al., 2015). This change starts the bleb
formation in the cytoplasmic membrane, where cholesterol-rich lipid rafts
serve as a platform for this process (Liu et al., 2006; Wei et al., 2018). The
calcium influx also appears to play an important role in the process as it
increases MVs formation and release to the extracellular space (Pasquet et al.,
2996; Taylor et al., 2020). The release of MVs is led by the contraction of the
cytoskeleton through the activation of a cascade of signals. ADP-ribosylation
factor 6 (ARF6)-GTP activates phospholipase D to recruit the extracellular
signal-regulated kinase (ERK) to the membrane where it phosphorylates the
myosin light-chain kinase (MLCK) (Muralidharan-Chari et al., 2009; Clancy
et al., 2019). Finally, the phosphorylation of the myosin light chain by MLCK
induces a contraction of the cytoskeleton that conclude with the release of
MVs.
Trafficking of cargo into MVs is a regulated event since not just any
molecule will be included in these. The mechanism of sorting to MVs is not
completely understood, but it could be regulated by some of the typical MVs
proteins such as ARF6, CD40 or flotillin-2. However, due to the diversity of
molecules that are included in MVs, it is likely that multiple pathways are
involved in sorting the cargo (Clancy et al., 2019; Saliba et al., 2019).
Moreover, the great diversity of cargos in MVs depend, in some instances, on
the progenitor cell (Nguyen et al., 2016; Menck et al., 2017: Fraser et al.,
2019). Nevertheless, MVs are characterized by the constant presence of
proteins GP96, actin-4, and mitofilin, which are absent or reduced in exosomes
(Kowal et al., 2016). On the other hand, MVs also appear to be enriched in
nucleic acids including DNA and a variety of RNAs, including messenger
RNA (mRNA), ribosomal RNA (rRNA), micro-RNA (miRNA), long non-
coding RNA (lncRNA), and small interference RNA (siRNA) (Waldenström
et al., 2012; Elsemüller et al., 2019; Aliotta et al., 2010; Rani et al., 2017;
Kogure et al., 2013; Zhang et al., 2014).
Exosomes
Unlike MVs, exosomes are a more homogeneous group of EVs with well-
defined characteristics. They originate in the endosomal system rather than
being direct evaginations of the cytoplasmic membrane (Hessvik and Llorente,
2018; Zhang et al., 2019; Tschuschke et al., 2020). Exosomes are the smallest
EVs with a size range of approximately 30-100 nm (Paulaitis et al., 2018; Patel
et al., 2019). The mechanism of exosome formation has been widely
characterized and its function has been related to various physiological
processes in health and disease conditions (Hornung et al., 2020; Tutanov et
al., 2020). As mentioned above, the first description of exosome release was
observed during reticulocyte maturation, when MVBs fused with the
cytoplasmic membrane releasing small vesicles into the extracellular space
(Harding et al., 1983).
The biogenesis of exosomes is directly related to endosomal trafficking,
which is in turn one of the main mechanisms for recycling molecules in a cell
(Gruenberg, 2001; Grant and Donaldson, 2009; O’Sullivan and Lindsay,
2020). In this regard, cells internalize the components that will be recycled
through clathrin-mediated endocytosis, caveolae-mediated endocytosis, or
clathrin-independent endocytosis (Mousavi et al., 2004; Mayor and Pagano,
2007; Kiss and Botos, 2009). Subsequently, an invagination of the membrane
is formed giving rise to the early endosome, which can be recycled by being
sent back to the membrane, sent to the trans-Golgi network, or sent to
lysosomes for degradation (Brown et al., 2007; Elkin et al., 2016). Some
endosomes mature by lumen acidification given rise to late endosomes where
exosomes will form through invaginations that result in MVBs (Huotari and
Helenius, 2011; Scott et al., 2015). Numerous molecules are loaded into the
intraluminal vesicles that form within MVBs, but their nature will be discussed
later.
The formation of the intraluminal vesicles within the MVBs involves two
fundamental steps: enrichment with CD9 and CD63 tetraspanins at the
membrane site where invagination will take place (Andreuand Yáñez-Mó,
2014; Hurwitz et al., 2017; Böker et al., 2018), followed by the recruitment of
endosomal sorting complexes required for transport (ESCRTs). First, ESCRT
0 is recruited by phosphatidylinositol 3-phosphate and ubiquitinated
molecules present outside the endosomal membrane, which in turn recruits
ESCRT I and II, both responsible for forming the invagination in the late
endosome membrane, and the process is completed with the recruitment of
ESCRT III (Tamai et al., 2010; Colombo et al., 2013; Alonso Y Adell et al.,
2016). Recruitment of ESCRT III occurs through the intervention of
programmed cell death 6-interacting protein (ALIX), which is responsible for
also binding tumor susceptibility gene 101 protein (TSG101), subcomponent
of ESCRT I, and charged multivesicular body protein 4a (CHMP4A),
subcomponent of ESCRT III (Baietti et al., 2012). During this process, the
cargo is loaded into the vesicles (Wei et al., 2021). Fusion of MVBs with the
plasma membrane occurs through different pathways involving proteins of
Rab GTPase family (RAB11, RAB35, RAB27A/B) and proteins of SNARE
family (vSNARE and tSNARE) (Savina et al., 2005; Hsu et al., 2010;
Ostrowski et al., 2010). Finally, the MVBs membrane fuses with the plasma
membrane releasing exosomes to the outside.
Exosomes are characterized by having certain membrane proteins and
lipids in their composition, as well as different soluble molecules inside them.
Lipids associated with the exosomal membrane include PS, sphingomyelin,
cholesterol, and ceramides (Haraszti et al., 2016; Skotland et al., 2017;
Skotland et al., 2019). Proteins that can be found in the exosomal membrane
include the tetraspanin family (CD9, CD63, CD81 and CD82), major
histocompatibility complex (MHC)-I, MHC-II, as well as other proteins
related to vesicular trafficking (Admyre et al., 2003; Andreuand Yáñez-Mó,
2014; Schey et al., 2015). On the other hand, exosome lumen is characterized
by the presence of proteins such as actin, TSG101, heat shock protein 70
(HSP70) and ALIX (Greening et al., 2015), as well as a wide variety of nucleic
acids including mitochondrial DNA (mtDNA), single-stranded DNA
(ssDNA), double-stranded DNA (dsDNA), mRNA, miRNA, siRNA,
mitochondrial RNA (mtRNA), circular RNA (circRNA), lncRNA or transfer
RNA (tRNA) (Takahashi et al., 2017; Spada et al., 2020; Sansone et al., 2017;
Izumi et al., 2015; Li et al., 2018; Wang et al., 2019; Baglio et al., 2015). In
addition, other groups of proteins such as enzymes, RNA-binding proteins,
cytoskeletal proteins, HSP and proteins specific from the cell that produced
the exosomes have also been found (Prunotto et al., 2013; Kalra et al., 2013;
Wu and Li, 2018).
Apoptotic Bodies
ApoBDs originate from cells in the last stages of apoptosis. They are the
largest EVs with a size range of 500 nm to >5 μm (Xu et al., 2019; Kakarla et
al., 2020). ApoBDs are formed by the disassembly of apoptotic cells and
contain nuclear fragments, organelles such as mitochondria, as well as soluble
molecules (Battistelli and Falcieri, 2020). ApoBDs functions are less diverse
compared to MVs and exosomes, however, they have been related to the
induction of cell proliferation, which makes them messengers of tissue repair
after tissue death events (Li et al., 2020).
Apoptotic cells expose PS as a “eat me” signal, reason for which it is
common to find PS on the outer surface of the ApoBDs (Fadok et al., 1998;
Mariño and Kroemer, 2013). As PS is recognized by Annexin V, this is used
as a marker for ApoBDs (Genderen et al., 2008; Liu et al., 2009). Other
proteins used for the identification of these EVs are thrombospondin and the
complement fraction C3b, which interact with oxidized molecules on the
surface of ApoBDs (Mevorach et al., 1998; Krispin et al., 2006). Noteworthy,
some mRNAs and miRNAs have been found within ApoBDs, so it has been
suggested that they may also establish communication through these
molecules (Li et al., 2020).
Effects of EVs on Innate Immunity during Steady State
Despite the pathological role that EVs have on the immune responses such as
the maintenance of inflammation, the recruitment of cell in cancer, or the
induction of tissue damage, they have also been related to the normal
functioning of immune cells. Here, we will review some of the roles that EVs
have in non-pathological conditions.
Relation of EVs with Monocytes and Macrophages

during Steady State
Monocytes are bone marrow-derived mononuclear cells found in the blood

stream and tissues. Although they were originally considered as an
intermediate state in macrophage differentiation, it is now known that these
are functional cells and have different roles in both infection resolution and
tissue repair (Ginhoux and Jung, 2014). In addition, as is well-known, classic
monocytes can differentiate into monocyte-derived macrophages and
monocyte-derived dendritic cells (mDCs) (Kapellos et al., 2019). On the other
hand, non-classical monocytes participate in phagocytosis of antibody- or
complement-opsonized antigens, as well as in antiviral defense (Sampath et
al., 2018).
On the other hand, macrophages are immune cells present in tissues as
resident cells and, together with neutrophils, are the first cells to interact with
pathogenic microorganisms during infections (Silva and Correira-Neves,
2012). Macrophages are phagocytic cells that possess different strategies to
kill microorganisms, including hydrolytic enzymes, nitric oxide (NO),
reactive oxygen species (ROS) and antimicrobial peptides, among others.
Moreover, macrophages are also antigen presenting cell since they express
MHC-II and costimulatory ligands (Hirayama et al., 2018). Macrophages are
cells with great plasticity that, under different stimuli, change from a basal
phenotype called M0 to other phenotypes with an increased response. M1
macrophages become proinflammatory producing cytokines such as
interleukin (IL)-12, IL-6, IL-1β, tumor necrosis factor (TNF)-α, or monocyte
chemoattractant protein (MCP)-1 and increase their pathogen-killing
potential. But in the same way, if the immune response is not controlled, M1
macrophages are also associated with tissue damage (Yunna et al., 2020).
Conversely, M2 macrophages are characterized by the production of the anti-
inflammatory cytokines transforming growth factor (TGF)-β as well as IL-10
and by their role in tissue repair; nevertheless, they are also associated with
the survival and prevalence of pathogens (Martinez and Gordon, 2014). Here
are some interesting cases regarding the effect of EVs on monocytes and
macrophages in non-pathological conditions.
The interaction between monocytes and endothelial cells is regulated by

a series of complex chemical signals resulting, in some cases, in the
recruitment of these innate cells to certain tissues during inflammation
(Gerhardt and Ley, 2015). Nevertheless, in some biological contexts, this
cellular interaction results in pathologies, such as atherosclerosis, in which
monocytes are recruited by endothelial cells due to an increase in the
expression of adhesion molecules, resulting in chronic inflammation (Mestas
and Ley, 2008). Interestingly, many EVs (mainly MVs) of different origins
have been detected in blood from healthy donors (Fendl et al., 2016). In this
sense, it has been reported that EVs released by endothelial cells suppress
monocytes activation against stimuli such as lipopolysaccharide (LPS), which
are polarized by the suppression of pro-inflammatory genes and the activation
of genes related to immunomodulatory activity such as IL-10, TGF-β and
macrophage mannose receptor (MRC)-1 (Njock, et al., 2015). This
communication through EVs between endothelial cells and monocytes could
be a strategy to regulate the activation of monocytes in the bloodstream to
avoid exacerbated inflammatory responses that generate endothelial damage
(Figure 3A).
As mentioned above, some monocyte subsets differentiate into
macrophages during migration from bloodstream to tissues (Italiani and
Boraschi, 2014). This process is regulated by different factors such as the
cytokines macrophage colony stimulating factor (M-CSF), granulocyte
macrophage colony-stimulating factor (GM-CSF), or IL-4, as well as proteins
of the extracellular matrix (Jacob et al., 2002; Sander et al., 2017). It has been
described that macrophage-derived MVs induce the differentiation of
monocytes into monocyte-derived macrophages as these EVs contain
miRNAs (especially miR-223) that are taken up by the target cells after fusion
(Ismail et al., 2013). Accordingly, inhibition of miR-223 resulted in a
reduction in the differentiation rate of monocytes into macrophages. This
opens a field of study regarding the role that resident macrophages play in the
monocyte differentiation during migration (Figure 3B).
Hair growth is a process in which the hair follicle undergoes a series of
cyclical changes: regression phase (catagen), resting phase (telogen) and
growth phase (anagen) (Chen et al., 2020). The hair follicle maintains a
microenvironment that allows the activation and arrest of hair follicular stem
cells (HFSC), regulating hair growth through their active division or entering
a quiescent state. One of the signaling pathways involved in this cycle is the
Wnt/β-catenin pathway, which promotes the activation and division of HFSCs
(Choi et al., 2013). Perifollicular macrophages have been linked to the hair
regeneration process, mainly because these cells express Wnt, which allows
HFSCs to enter in anagen (Castellana et al., 2014). Macrophage-derived EVs
contain the Wnt protein (Wnt3a and Wnt7b) mainly on their surface, and their
interaction with papillary dermal cells (PDC) stimulate their proliferation,
migration and expression of markers related with survival and proliferation.
Interaction between EVs and PDCs occurred firstly by attachment to the
membrane, followed by the EVs internalization into the cells (Rajendran et al.,
2020). Therefore, EVs restored the hair growth in a mice model and increased
de quantity of hair follicles in these animals, highlighting the importance of
macrophage-derived EVs in maintaining hair follicle homeostasis and hair
regeneration.
Apoptosis is defined as a form of programed cell death that occurs in
normal conditions to eliminate damaged cells through the intrinsic (by
intracellular sensors) and extrinsic (by immune cells) pathways (D'Arcy,
2019). This form of cell death is important to homeostasis in the body as it
allows the elimination of aged and unfunctional cells. Furthermore, it helps to
maintain a constant population of cells in the organs, with a balance between
cell proliferation and death (Elmore, 2017). Tissue resident macrophages are
important in the establishment of this balance as they clear apoptotic cells
(Gordon and Plüddemann, 2018). The clearance of apoptotic cells could
trigger inflammation mainly by the recognition of DNA via toll-like receptor
(TLR), however, specialized macrophages perform this function without
releasing pro-inflammatory mediators (Roberts et al., 2017). In agreement
with the above, apoptotic cells release a large quantity of EVs that promote
the in vitro and in vivo production of TGF-β by macrophages, which could
explain why inflammation is not primed during apoptotic cell clearance (Chen
et al., 2019). Additionally, the production of TGF-β in macrophages was
triggered by the detection of PS on the surface of EVs, activating the
transcription factor forkhead box O3(FOXO3) (Figure 3C).
Relation of EVs with Neutrophils during Steady State
Neutrophils, the most abundant leukocytes in the bloodstream of mammals,

are characterized by having a multilobed nucleus and abundant cytoplasmic
granules (Davis et al., 2008). These cells are rapidly recruited to infection sites
in periods of minutes to hours and represent the first line of defense against
pathogenic microorganisms during acute inflammation (Wright et al., 2010).
Neutrophils have different strategies to combat microorganisms including
phagocytosis, degranulation, ROS generation and the release of neutrophil

extracellular traps (NETs) (Rosales, 2018). They have many molecules with
antimicrobial activity including enzymes: neutrophil elastase (NE), cathepsin
G (CG), proteinase 3, myeloperoxidase (MPO), azurocidin (AZU), lysozyme
(LYZ); antimicrobial peptides (cathelicidins and defensins); and oxidant
agents such as hydrogen peroxide, superoxide, hypochlorous acid and
peroxynitrite (Sheshachalam et al., 2014; El-Benna et al., 2016). Mutations
that affect the normal functioning of neutrophils increase the susceptibility of
individuals to infections, emphasizing their protective function as part of the
host´s innate immunity (Lakshman and Finn, 2001).
In vitro characterization of EVs derived from neutrophils has focused on
the MVs released by these leukocytes (microparticles and ectosomes). To
obtain them, some stimuli are used that favor their release, such as bacteria,
LPS, N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-myristate
acetate (PMA) (Duarte et al., 2012; Dalli et al., 2013; Gasser et al., 2003;
Gasser and Schifferli, 2005; Finkielsztein et al., 2018), therefore, these cannot
be considered as produced in a steady state. However, unstimulated
neutrophils from equines release exosomes (diameter 30-80 nm) and MVs
(Vargas et al., 2016). A detailed characterization of these exosomes revealed
the presence of 246 proteins whose functions were related to: adhesion
molecules (tetraspanins, CD90, CD44 and integrins); immune functions
(LYZ, tissue factor, NME proteins, S100); protein protection and assembly
(chaperones); cytoskeleton and membrane trafficking, among others.
Interestingly, functional studies on neutrophil-derived exosomes have
shown a differential role for neutrophil-derived EVs depending on whether
they come from unstimulated or activated cells. Thus, exosomes derived from
unstimulated neutrophils were unable to promote the proliferation of airway
smooth muscle cells, unlike exosomes derived from LPS-stimulated
neutrophils. Another study found that MVs spontaneously released by
unstimulated human neutrophils lack antibacterial activity and present a lower
amount of proteins derived from cytoplasmic granules such as lactoferrin
(related to microbicidal activity) (Lorincz et al., 2015). In contrast, EVs
released by neutrophils stimulated with opsonized bacteria or zymosan-coated
particles showed a greater antibacterial activity linked to a greater quantity
(almost double) of antimicrobial proteins in their cargo (Lorincz et al., 2015;
Timar et al., 2012). A similar effect was identified using EVs spontaneously
released by unstimulated human neutrophils (sEVs) with respect to EVs from
Aspergillus fumigatus-infected neutrophils (afEVs), observing that sEVs have
no impact on fungal growth, whereas afEVs limit the development of A.
fumigatus (Shopova et al., 2020). Like the previous study, a higher proportion
of proteins with antimicrobial activity was also detected in the cargo of afEVs
respect to sEVs, containing enzymes such as NE, CG, MPO, AZU and
defensin-1. Taken together, these data suggest that the inability of
unstimulated neutrophil-derived EVs to kill microorganisms would be a
mechanism to prevent damage as neutrophil-antimicrobial proteins can also
damage host cells (Wang, 2018). Therefore, EVs loaded with the anti-
microbial enzymes will only be produced in response to infection (Figure 3D).
The paradigm that links neutrophils with inflammatory responses (Mortaz
et al., 2018) could be broken thanks to the study of EVs. Noteworthy, EVs
produced by unstimulated neutrophils significantly reduced ROS and IL-8
secretion in PMA- stimulated neutrophils, but promoted coagulation
(Kolonics et al., 2020). In contrast, EVs from neutrophils stimulated with
zymosan-coated particles or fMLP promoted a pro-inflammatory state in
unstimulated neutrophils characterized by increase ROS and IL-8 production,
CD11b/CD18 and CD35 expression and augmented phagocytosis (Kolonics
et al., 2020; Amjadi et al., 2021). This immunomodulatory effect of
neutrophil-derived EVs is not only restricted to the neutrophils themselves.
Neutrophil-derived EcMs and MVs treated with fMLP or TNF-α, respectively,
prevented the pro-inflammatory state in zymosan- and LPS-treated
macrophage, reducing the expression of cytokines such as TNF-α, IL-1β, IL -
6, IL8, IL-10 or IL-12, and promoting the production of the anti-inflammatory
cytokine TGF-β (Eken et al., 2010; Rhys et al., 2018). These observations
place the neutrophil as a cell capable of avoiding inflammation during the
steady state and regulating the inflammatory process once it has started
(Figure 3E).
Relation of EVs with Natural Killer Cells (NKs)

during Steady State
NK cells originate from the lymphoid lineage in the bone marrow and are
mononuclear cells classified as innate lymphoid cells (Abel et al., 2018). The
importance of NK cells is related to their ability to kill cancer cells and cells
infected by certain viruses (Wu et al., 2020).
Figure 3. Effect of EVs on innate immune cells. (A) Endothelial cells release EVs to diminish
monocyte activation through downregulation of pro-inflammatory cytokines and upregulation of
anti-inflammatory cytokines. (B) Macrophage-derived EVs induce monocyte differentiation to
macrophages due to transfer of miRNA (miR-223). (C) Apoptotic bodies prevent inflammation
during clearance of death cells thanks to phosphatidylserine-dependent TGF-β synthesis. (D) EVs
from unstimulated neutrophils are uncapable to kill pathogens as they are diminished in antimicrobial
protein NE, CG or MPO; on the other hand, EVs from stimulated neutrophil are enriched in
antimicrobial proteins and possess microbicidal effect. (E) Unstimulated neutrophils release EVs
with immunomodulatory effect on PMA-stimulated neutrophils downregulating IL-8 and ROS
production but promoting coagulation. (F) NK cells-derived EVs contain cytotoxic molecules such as
FasL, perforin and TNF-α that induce death of cancer cells.
NKs have two important mechanisms to fulfill their function: 1) they

detect a reduction in MHC-I expression thanks to the presence of receptors
that detect autologous MHC-I; or 2) they induce target cell death by antibody
dependent cytotoxicity. In both cases, once the NK detects the target cell, it
induces its death by activating death receptors (members of the TNF family)
or by releasing lytic enzymes such as perforin, granzymes or granulysin
(Topham and Hewitt, 2009; Paul and Lal, 2017). The importance of NK cells
in the maintenance of homeostasis has been related to their protective role
against cancer (immunosurveillance). Thus, mutations that affect the normal
development of these leukocytes increase the susceptibility of the organism to
develop malignant processes (Moon and Powis, 2019). Additionally, NKs
release chemokines and cytokines that actively participate in the inflammatory
process, mainly macrophage inflammatory protein (MIP)-1α, MIP-1β, TNF-α
and interferon (IFN)-γ (Fauriat et al., 2010).
Non-stimulated human NKs release exosomes under normal culture
conditions, even in the absence of IL-2 (cytokine normally used to activate
these leukocytes). Those exosomes are characterized by the presence of
cytotoxic proteins Fas Ligand (FasL; membrane and soluble forms) and
perforin, and interestingly, exhibited cytotoxic activity against different cancer
cell lines (Jurkat, K562, DAUDI, SKBR3 and 501mel) (Lugini et al., 2012).
Similar results have also been obtained using exosomes from the unstimulated
NK-92 cell line. Like the human exosomes, the exosomes of the cell line
contained perforin, FasL and TNF-α and showed cytotoxic activity against
melanoma, gastric carcinoma, and colon cancer cells (Zhu et al., 2017). EVs
from resting NKs have been related to the ability of these cells to prevent the
proliferation of pancreatic cancer cells as they contained increased amounts of
miR-3607-3p, a miRNA that suppress proliferation, migration, and invasion
of pancreatic cancer cells (Sun et al., 2019). No increase in the protein content
and cytotoxic activity has been observed in exosomes derived from stimulated
NKs (principally with IL-12), suggesting that priming is not necessary to
generate biological active exosomes (Neviani et al., 2019; Wu et al., 2019;
Federichi et al., 2020; Choi et al., 2020). Finally, NKs-derived exosomes from
the plasma of healthy donors also have cytotoxic activity against cancer cells
but not against mononuclear plasma cells (Lugini et al., 2012), suggesting that
these particles could act as vigilantes in the blood circulation with the ability
to discriminate between malignant and healthy cells (Figure 3F).
Relation of EVs with Mast Cells during Steady State
Mast cells are myeloid cells that leave bone marrow as immature precursors,
traveling through the blood until they become established in epithelial and
mucosal tissues (Dahlin and Hallgren, 2015). They possess dense
cytoplasmatic granules full of pharmacologically active molecules (histamine,
heparan sulfate, heparin) and proteases (tryptase, chymase, carboxypeptidase
A3) (Wernersson and Pejler, 2014). In addition, when they are activated
release inflammatory mediators such as leukotrienes, prostaglandins, and
cytokines (IL-6, TNF-α and IL-13) (Krystel-Whittemore et al., 2015). Mast
cells are mainly activated by cross-linking of membrane-bound IgE as they
express receptors for IgE antibodies (FcϵRI) on their surface (Galli, 2016),
which makes them important protagonists of inflammatory processes by
favoring the recruitment and activation of other cells. However, mast cells
contribute to pathology in allergic processes as they are sensitized with the
IgE produced against allergens (Amin, 2012).
The human mast cell line HMC-1 release exosomes in the absence of
inflammatory stimuli which are enriched with RNA (mainly mRNA and
miRNA), differentially expressed between the exosomes and the original cells
(Ekström et al., 2012). A functional study of these exosomes showed that they
are taken up by other mast cells and CD34+ hematopoietic precursors,
identifying cargo RNAs in the target cells cytoplasm. Unstimulated murine
mast cells also release EVs that contain lysosomal enzymes, non-specific
proteases, cytokines, and in general, protein profiles associated with glycolytic
processes, cell adhesion, chemotaxis, and degranulation, among others (Liang
et al., 2019). Like human EVs, murine mast cell EVs were also rich in RNA
(lncRNA and miRNA). As seen with EVs from other innate cells, EVs derived
from murine mast cells showed variations in size and composition between
resting cells and IgE-stimulated cells. Thus, while the EVs released by mast
cells at rest are larger and contain more phosphatidic acid, the EVs from
stimulated cells contain more phosphatidylinositol (Kormelink et al. 2016).
The fact that mast cell EVs are enriched in RNA suggests that they can
reprogram gene expression in target cells. This has been explored in alveolar
epithelial cells, where the incorporation of mast EVs induced a mesenchymal
phenotype by promoting active cell division and morphological changes due
to activation of different phosphorylation cascades (Yin et al., 2020). This
suggests an important role for mast cells in epithelial renewal, even in the
absence of damage.
Relation of EVs with Eosinophils during Steady State
Eosinophils are polymorphonuclear cells with abundant cytoplasmic granules

and a bilobed nucleus, representing 0-3% of blood leucocytes (Ramirez et al.,
2018). Eosinophils are poorly phagocytic but play a very important defense
role against helminth infections (Huang and Appleton, 2016). Eosinophil
granules contain different enzymes that help them fulfill this function, such as
the major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-
derived neurotoxin (EDN), and the ribonuclease eosinophil cationic protein
(ECP) (Rosenberg et al., 2013). These cells express FcϵRI, so they can exert
antibody-dependent cytotoxicity (Stone et al., 2012). However, this
characteristic also links them to a pathological role in allergic reactions,
releasing different chemical mediators that promote inflammation (Matucci et
al., 2018).
Unstimulated human eosinophils release MVs ranging 20 to 1000 nm in
diameter characterized by the expression of CD9 and presence of PS
(Akuthota et al., 2016). They also release exosomes in the absence of stimuli
whose proteomic analysis revealed the presence of 80 proteins including ECP,
MBP and EPO. The presence of other proteins associated mainly with cell
morphology, metabolism and immune response was also reported (Mazzeo et
al., 2014). It is noteworthy that eosinophil exosomes showed effects when
cultured with the eosinophils themselves, increasing the generation of ROS
(but not nitrite); however, they did not favor migration and adhesion (Cañas
et al., 2016). These data suggest that exosomes released by unstimulated
eosinophils could prime the cells to a more active ROS-protective phenotype,
but at the same time prevent their infiltration into the tissues during the steady
state.
Effects of EVs on Innate Immunity during Infectious Diseases
The history of successful infectious diseases is one of a continuous process of

adaptation in which the host suffers as little damage as possible while the
pathogen persists long enough to spread. This co-adaptation process requires
the transfer of information between both protagonists, a process that is now
known to be mediated by EVs. Once the pathogen enters the host environment,
the effect derived from its EVs is in principle that of the activation of the host's
innate immune system, mainly mediated by TLR ligands.
In turn, the EVs derived from the host´s innate cells that carry pro- and
anti-inflammatory preformed mediators and nuclei acids amplify the immune
response by interacting with other non-infected cells to facilitate bystander
activation of the innate immunity as well as activating complement and
coagulation, contributing significantly to the initial control of the infectious
load (Robbins and Morelli, 2014; reviewed in Karasu et al., 2018).
Figure 4. Release of EVs by infectious agents (upper half) and their corresponding reported effects on
macrophages (lower half). Within the EVs, the components usually identified for each of the infectious
agents are shown. In yellow: Gram-positive and negative bacteria release EVs outside the cell that are
recognized by TLRs and resulting in the activation of the NLRP3 inflammasome and the production of pro-
inflammatory cytokines and type I interferons. Inhibit transcription by NFκB. Bacterial and infected
macrophages EVs can inhibit the release of pro-inflammatory cytokines, prevent complement activation by
hiding bacterial components, and degrade NETs by carrying DNAses. In green: Leishmania, Trypanosoma
and Plasmodium parasites release EVs outside and inside the cell since in their life cycle they are extra and
intracellular. Its EVs can directly inhibit the inflammasome and NFκB signaling. In the case of
Plasmodium, EVs favor the expression of adhesion molecules ICAM and VCAM, which is associated with
the pathology, as well as activate a signaling pathway dependent on STING and NFkB that leads to the
release of a large amounts of pro and anti-inflammatory cytokines. EVs from adherent trichomonas can
transfer their ability to adhesion-deficient trichomonas. The EVs of infected macrophages can inhibit NO,
the expression of costimulatory molecules and inhibit the expression of pro-inflammatory cytokines by
PGE2. For their part, the helminth EVs (drawn in the lower part) exert mainly anti-inflammatory and
regulatory effects by inhibiting iNOS, the expression of pro-inflammatory cytokines and co-stimulatory
molecules, while favoring the differentiation towards alternately activated macrophages. In red: viruses can
use the secretion system of EVs mediated by ESCRT-Rab GTPAse to facilitate their dissemination, being
able to carry fully assembled particles inside, as is the case of the AIDS virus (Trojan horse hypothesis), or
carry all the coding genetic material, as is the case of hepatitis viruses. In addition, this allows them to
evade the immune system by avoiding recognition by complement and antibodies, as well as protection
against enzymes. Recently, COVID-19 patients have been reported to have circulating EVs that carry tissue
factor, which could be related to thrombosis. EVs released by infected macrophages can carry pre-formed
cytokines that recruit innate cells, or they can place neighboring uninfected cells into an anti-viral state
through induction of type I interferons and activation of virus cytosolic sensors. At the same time, EVs can
inhibit antigenic presentation and inflammation of the inflammasome, downregulating the immune
response. In all cases, EVs cargo contains small non-coding RNAs that induce epigenetic changes in the cell
(central nucleus).
Therefore, pathogens produce EVs with molecules that can modulate or

suppress the immune system, favoring the establishment of infection. The
outcome of the infection will depend on the combination of a multitude of
factors, among which the quality of the immune response mounted by the host
and the pathogenicity/virulence of the infectious agent are critical. Details on
EVs from innate immune cells and their role in activating the adaptive
immunity can be consulted in the review by Groot Kormelink et al. (2018).
It is now accepted that all cells in nature are capable of producing EVs. In
the case of infectious agents, the release of EVs has been demonstrated in
bacteria, fungi, and protozoan and helminth parasites. In the case of viruses, it
is the cells they infect and where they reproduce that produce EVs that
incorporate viral molecules that can activate or modulate the host´s innate
immune response. More interestingly, viruses can manipulate the EVs of
infected cells to, using them like a Trojan horse, carry viral particles and aid
in their dissemination (Kadiu et al., 2012).
The next section compiles the known effects that EVs derived from
different pathogens have on the innate immunity of the host, and the
exploitation of cells of innate immunity by viruses. A resume regarding the
effects of EVs from pathogens on macrophages is shown in Figure 4.
Bacterial EVs
EVs release from bacteria was first reported in the Gram-negative Escherichia
coli in the 60´s and in the Gram-positive Neisseria gonorrhoeae and Borrelia
burgdorferi in the late 80´s (Bishop and Work, 1965; Dorward, et al., 1989;
Garon et al., 1989). EVs from Gram-negative bacteria have been extensively
studied and is known that they are formed by budding from the outer
membrane (known as OMVs), thereby carrying components such as LPS,
lipoproteins and molecules of the periplasmic space. In addition, OMVs cargo
also include nucleic acids, toxins, adhesins and immunomodulatory molecules
(Schwechheimer and Kuehn, 2015; Brown et al., 2015). In contrast, EVs from
Gram-positive bacteria (known as membrane vesicles, MeVs) have been
scarcely studied as they lack outer membrane but instead have a thick
peptidoglycan cell wall outside of the cell membrane (OMVs and MeVs are
indistinctly called in this chapter as bacterial EVs). The exact mechanism by
which EVs are formed in Gram-positive bacteria is still unknown, but it has
been hypothesized that they may be formed by turgor pressure after release
from the plasma membrane, by the debilitating effect on the cell-wall of
modifying enzymes released with the EVs, or by tubulin-mediated passing

through protein channels (Brown et al., 2015).
Bacterial EVs may deliver virulence factors, resistance elements against
anti-microbial and immunomodulatory molecules, into mammalian cells by
endocytosis or macropinocytosis at long-distance, therefore contributing to
pathogenesis during the infection (Ketsy et al., 2004; Bomberger, et al., 2009;
O'Donoghue et al., 2016; Turner et al., 2018). In addition, the pathogen-
associated molecular patterns (PAMPs) present in the EVs cargo (LPS,
lipoproteins, DNA, RNA, toxins) bind to pattern-recognition receptors (PRRs)
such as membrane and cytoplasmic TLRs or cytosolic NODs triggering
signaling pathways that activate the effector mechanism of innate immune
cells (Kaparakis et al., 2010). In this sense, transfer of chromosomal DNA
from EVs of Pseudomonas aeruginosa into eukaryotic cells, including the
nucleus, has been demonstrated by polymerase chain reaction (PCR),
including the observation that most of the DNA of these EVs was carried in
the membrane of these structures (Bitto et al., 2017). Target immune cells
produce cytokines and chemokines that amplify the innate response, and if the
bacterial infection is not controlled in the short term, activate adaptive
immunity (Kaparakis et al., 2010; review in Groot Kormelink et al., 2018). To
circumvent the innate response, bacterial EVs can also transfer molecules that
have a detrimental effect, downregulating the host´s immunity.
Effects of Bacterial EVs on Host’s Innate Immunity

As mentioned above, the toxin and porins are virulence factors usually found
inside the bacterial EVs. In addition to cause damage to target cells, these
molecules are potent inhibitors or stimulators of the innate immune response.
Thus, it has been shown that toxins delivered by EVs from pathogenic Gram-
negative bacteria such as uropathogenic E. coli, N. gonorrhoeae and P.
aeruginosa inhibit the mitochondrial activity in macrophages that result in
BAK-dependent mitochondrial apoptosis (Deo, et al., 2020). In contrast, EVs
from the oral Gram-negative bacteria Fusobacterium nucleatum carrying the
porin FomA membrane protein were unable to cause any harmful effect on
human intestinal epithelial cells, rather triggering innate immunity by porin
TLR2-dependent NF-κB activation (Martin-Gallausiaux et al., 2020).
Examples of opposite effects of EVs porins from Gram-positive bacteria
have also been reported. EVs from the respiratory pathogen Streptococcus
pneumoniae, enriched in the pore-forming toxin pneumolysine, were found to
induce pro-inflammatory cytokines in the A549 cell line from a human lung
epithelial carcinoma and in human DCs. In contrast, pneumolysine-enriched
EVs was also found that activate the complement membrane-attacking

complex (MAC) in the extracellular environment, decreasing the killing of
encapsulated pneumococci by opsonophagocytosis and contributing to
immune evasion (Codemo et al., 2018).
Interestingly, there are examples of immunomodulation by toxins of EVs
derived from cells infected with bacteria, rather than EVs from bacteria per se.
Thus, EVs derived from eukaryotic cells carrying bacterial pore-forming
toxins were phagocytosed by human macrophages and polarize them to the
new macrophage phenotype CD14+ MHCIIlowCD86low, which enhances the
response to ligands of Gram-positive bacteria but inhibits it against ligands of
Gram-negative bacteria. Noteworthy, liposomes loaded with the toxins
showed the same polarizing effect on macrophages (Köffel et al., 2018),
suggesting that some components of the EVs can drive the differentiation of
immune cells favoring the response to some type of bacteria but inhibiting the
response to others. Another example was reported for EVs released from
Mycobacterium tuberculosis-infected macrophages, which were shown that
induce type I interferon release in target cells by a RIG-I/MAVS/TBK1/IRF3
RNA sensing pathway and promote the killing of mycobacteria by inducing a
RIG-I/MAVS-dependent maturation of the phagosomes, encouraging the use
of mycobacterial EVs as a new treatment (Cheng and Schorey, 2019).
Perhaps the most notorious component of EVs from Gram-negative
bacteria is the LPS as determined in biochemical studies and proteomic
analysis. LPS delivery by bacterial EVs into the cytosol of mouse
macrophages cause activation of caspase 11 (Vanaja et al., 2016), which in
turn activates the NLRP3 inflammasome by the non-canonical pathway,
contributing to the activation of the innate immunity (Santos et al., 2018) and
conferring in vivo protection against infections by bacteria (Aachoui et al.,
2013). However, caspase 11 activation can also cause gasdermin-D-dependent
piroptosis and lethality in an endotoxemic mouse model (Hagar et al., 2013),
suggesting that LPS delivery can also cause suppression of the innate
response. Therefore, the sensor ability of caspase-11 for LPS seems to be the
factor that determined the cellular fate and provide means to control the LPS
toxicity (Santos et al., 2018). Activation of the caspase-5-dependent
noncanonical inflammasome in human monocytes by TLRs has also been
reported for EVs from P. aeruginosa (Bitto et al., 2018). They also showed
that the physical form of LPS and the route by which is delivered into the cells
differentially activate the inflammasome by caspase-4 or 5.
Regarding the nucleic acids, activation of innate immunity by EVs from
the Gram-positive Clostridium perfringens that contained DNA encoding
perfringolysin O, 16S rRNA and α-toxin, determined by PCR, has been

reported (Jiang et al., 2014). In this case, the EVs were shown to be
internalized by RAW264.7 macrophages inducing the release of IL-1, TNF-α
and granulocyte colony-stimulating factor (G-CSF). More recently, it has been
shown that the action of bacterial EVs on host target cells also includes
epigenetic alterations. Such is the case of P. aeruginosa EVs that have been
reported to modify DNA methylation in human lung macrophages, potentially
resulting in an altered innate immune response (Kyung et al., 2020).
Other more general studies included proteomic analysis of purified EVs
and/or comparison between EVs from different conditions. Proteomic analysis
of EVs from the Gram-negative Klebsiella pneumoniae cultured in vitro
identified 159 proteins and it was shown that they were able to activate the
innate immunity by inducing the expression of proinflammatory mediators
such as IL-1β and IL-8 in epithelial cells (Lee et al., 2012). On the other hand,
evaluation of the EVs content from three Staphylococcus aureus strains
(methicillin-resistant, methicillin-sensitive and a laboratory-strain) showed
that they were different in number, morphology, and composition, with
variation in PAMPS as nucleic acids, peptidoglycan, and proteins. They also
showed that the EVs were degraded by autophagy in epithelial cells and that
PAMPS were recognized by epithelial PRRs as TLR 2, 7, 8, 9 and NOD2,
inducing the secretion of proinflammatory cytokines and chemokines (Bitto et
al., 2021). Strong induction of IFN-γ by NK cells was found when these cells
were exposed to EVs released by Chlamydia psittaci, a response that restrict
the bacterial growth contributing to protection (Radomski et al., 2019).
An example of evasion of cellular innate immunity is that of EVs from S.
pneumoniae providing DNases, such as TatD, to degrade DNA extracellular
traps from neutrophils. Consequently, a tatD-deficient strain of pneumococci
was less prone to cause lung damage in a murine sepsis model, which was
associated with a lower bacterial load in the tissue (Jhelum et al., 2018). EVs
from some pathogenic bacteria can also contribute importantly to the
pathology aggravating the infectious process. Such is the case of EVs from
Helicobacter pylori, one of the first reported bacterial EVs, which promoted
chronic pathology in human gastric epithelial cells by inducing the
exacerbated release of IL-8 (Ismail et al., 2003). More recently, it was shown
the same for EVs from S. aureus, which were capable of worsening atopic
dermatitis-type skin lesions in mice by exacerbating the local recruitment of
eosinophils and the inflammatory response (Hong et al., 2011). The process
was dependent on, on one hand, the induction of Th2-biased mediators such
as thymic stromal lymphopoietin, MIP-1α and eotaxin, and, on the other hand,
on the signaling by TLR2 and NOD2 since the knock-down of these molecules
using miRNAs reduced the expression of the IL-8 gene stimulated by the EVs
(Hong et al., 2011; Jun et al., 2016).
There are also studies of bacterial-derived EVs with potential benefits to
host physiology. Such is the case of the intestinal microbiota, which plays a
pivotal role in the maturation of the immune system and in maintaining human
intestinal homeostasis (Shen et al., 2012). The process is mediated by the
intercommunication between commensal and probiotic bacteria with the
intestinal epithelium. EVs released by commensal ECOR12 and probiotic
Nissle 1917 E. coli has been shown that trigger innate responses trough NOD1
activation, driving the NF-κB-dependent expression of IL-6 and IL-8 (Cañas
et al., 2018). On the other hand, a very important role for the host EVs in
modulating the innate response induced by Gram-positive probiotic bacteria
of the genera Lactobacillus and Bifidobacterium was found when serum EVs
were added to cellular cultures co-incubated with these bacteria. Serum EVs
enhanced the TLR2/1 and TLR4 responses while suppressed the TLR2/6
responses, favoring the innate response induced by Lactobacillus species and
inhibiting the response by Bifidobacterium species (van Bergenhenegouwen
et al., 2014). In the same work, serum EVs were also shown that promote
phagocytosis by dendritic cells (DCs) by modifying their interaction with the
bacteria. Inasmuch as EVs are released by all tissues and are found in
practically all body fluids, it is to be expected that their presence at infection
sites and its association to the microbe contributes to modulating the innate
response against pathogens, playing a very important role in the outcome of
the infection.
Parasitic EVs
The term parasite is applied to protozoa, helminths and ectoparasites (ticks

and others) that live at the expense of animals they infect. They are responsible
for the highest rates of morbidity and mortality from infectious organisms in
poor countries, infecting an estimated 30% of the world population. Their life
cycles are often complex including definitive, intermediaries, and reservoir
hosts. Parasitic infections are characterized by their chronicity due to the weak
host´s natural immunity and sophisticated immune evasion mechanisms.
Parasites produce EVs with variation in their cargo protein depending on the
developmental stage and environmental conditions, which usually carried
products that can suppress the host's innate immune response or favor their
capacity for colonization and invasion of target cells. On the other hand, EVs
offer a novel target to develop new alternatives for the diagnosis and the
development of therapeutic vaccines against infectious diseases.
Effects of Protozoa EVs on Host’s Innate Immunity

Protozoa are single-celled eukaryotic organisms that undergo developmental
changes during their life cycle, resulting in various functional stages such as
replicative trophozoites and amastigotes, non-replicative trypomastigotes and
dormant cysts. Some live outside the cells where it reproduces by binary
fission as the enteric parasites Entamoeba histolytica and Giardia lamblia,
while others are intracellular where they can reproduce asexually as
Leishmania spp., or sexually as Plasmodium spp.
EVs from Leishmania Parasites

Leishmania was the first protozoan parasites in which EV production was
described. In that study, EVs from both Leishmania donovani and Leishmania
major promastigotes was shown to be upregulated by stressful stimuli that the
parasite faces within the host (temperature and pH) and that those EVs are
taken up by THP-1 macrophages where induce the expression of IL-8
(Silverman et al., 2010a). However, the same group also showed that L.
donovani promastigote-derived EVs reduce the INFγ-mediated induction of
IL-8 and TNF-α secretion while increase the IL-10 release in infected human
monocytes, also impairing the ability of DCs´ antigen presentation to T
lymphocytes (Silverman et al., 2010b). In the same work, they also showed
that mice treated with L. major and L. donovani EVs prior to the infection
resulted in higher loads of parasites, probably due to the immunosuppressive
properties of the parasite EVs. Pro-inflammatory property of EVs from
Leishmania amazonensis has also been reported (Cronemberger-Andrade et
al., 2014), suggesting that the source of the EVs, i.e., parasite specie, the life
cycle stage and the immune target cell evaluated, are determining factors of
the effects observed. Regarding to the species, a recent study comparing the
ability of EVs from three different Leishmania species to activate the innate
immunity showed that, in contrast to EVs from Leishmania infantum and
Leishmania braziliensis that were unable to trigger a pro-inflammatory
response, EVs from Leishmania amazonensis were immunomodulatory by
eliciting the production of IL-6, IL-10, TNF-α and NO via NF-κB-dependent
TLR2 and TLR4 signaling (Nogueira et al., 2020). Regarding to EVs from the
target cells, in contrast to the EVs from infected-macrophages mentioned
above that suppressed the innate response, EVs from Leishmania-infected
DCs were seen to be immune stimulatory being proposed as a vaccine strategy

(Schnitzer et al., 2010).
The surface protease GP63 is a virulent factor found at higher levels in
exosomes from stationary or metacyclic promastigotes (stage at host´s blood)
than logarithmic promastigotes (stage at vector) of L. infantum, suggesting
adaptation of the parasite to the environment (Marshall et al., 2018). The
modulation of the innate immune response by Leishmania parasites can also
be mediated by EVs released by infected macrophages that carry parasite
molecules with immunosuppressive properties. Leishmania-infected
macrophages release GP-63-containing EVs that downregulate the innate
inflammatory response by modulating macrophage intracellular signaling and
transcription, including IL-1β production via inhibition of the NLRP3
inflammasome (Hassani et al., 2014). Noteworthy in the same work, EVs from
wild type and GP63 knockout (KO) parasites were shown that recruited more
neutrophils and macrophages in a mouse model than the wild type parasites,
suggesting that EVs from the parasite, independent of GP63, can be by itself
more prone to elicit an innate response than the parasite (Hassani et al., 2014).
In contrast, EVs from HSP100 KO parasites were more pro-inflammatory than
wild type EVs and parasites that induced regulatory responses, suggesting that
the presence of HSP100 altered the EV cargo making them more
immunosuppressive (Silverman et al., 2010b). In the same order of ideas,
elongation factor (EF)-1α-containing EVs from L. mexicana modulate the
immune response by interfering with the INF-γ signaling and inhibiting
macrophage production of TNF-α and NO (Silverman and Reiner, 2011).
Another study has shown that EVs from L. major exacerbated the footpad
lesions in mice compared to mice injected with the parasite alone (Atayde et
al., 2015). The pathology was associated with the activation of an IL-23-
dependent Th17 response that favored the recruitment of neutrophils.
In addition to GP63 and HSPs, the EVs from L. donovani and L.
braziliensis also contain non-coding small RNAs that could regulate the
expression of genes in the target cells (Lambertz et al., 2015), including
phosphatases such as the L. major phosphatase LmPRL-1, which has been
associated with the survival of the parasite inside macrophages (Leitherer et
al., 2017), and molecules associated with parasite invasiveness to mammalian
cells, such as the plasminogen binding protein enolase and the small
myristolated protein SMP-1 found in EVs derived from L. mexicana (Figuera
et al., 2013). Moreover, about 50 known virulent factors were recently
identified by proteomic analysis in EVs from logarithmic and stationary
phases of L. infantum chagasi, being higher in abundance in the log phase

(Forrest et al., 2020).
EVs from Trypanosoma Parasites

Trypanosoma brucei produce EVs from membrane nanotubes formed by
budding and extension of its flagellar membrane. Recognition of T. brucei
EVs by target cells is mediated by parasite proteins exposed on the surface of
the EVs, allowing the transfer of virulence factors such as flagellar proteins
and the serum resistance-associated protein (SRA), the latter being critical for
human infectivity due to its ability to inhibit the trypanosome lytic factor that
usually kill the parasites in primates (Szempruch et al., 2016). Thus, SRA-
contained EVs transfer the ability to resist to the lytic factor to non-human
trypanosomes allowing them to infect and destroy erythrocytes causing
anemia.
In the case of Trypanosoma cruzi, the EVs are produced from MVBs
(exosomes) and by shedding from the surface membrane (EcMs). These EVs
were formerly reported that contain mucin proteins (Goncalves et al., 1991),
and after, by proteomic analysis, that also contain glycoproteins such as the
major surface gp85, proteases such as cruzipain, cytoskeleton proteins, non-
coding small RNAs (with different proportion of tRNA/rRNA in
trypomastigotes vs epimastigotes EVs), antigens and proteins involved in
metabolism, signaling, nucleic acid binding and parasite survival; those EVs
cause differentiation in other trypanosome parasites and changes in the
cytoskeleton, extracellular matrix, and even, epigenetic changes in host cells
that result in modification of the immune response pathways (Bayer-Santos et
al., 2013; García-Silva et al., 2014). Injecting T. cruzi EVs into naive mice
makes them highly susceptible to infection, a process that appears to be related
to the production of IL-4 and IL-10 as monoclonal antibodies against both
cytokines restored survival (Trocoli Torrecilhas et al., 2009). Similar in vivo
results were obtained more recently, including the downregulation of the NO
production and the activation marker F4/80 in peritoneal macrophages (Lovo-
Martins et al., 2018). They also showed that in vitro pre-treatment of bone-
marrow derived macrophages with EVs from T. cruzi prior to infection
resulted in a significant increase of internalized parasites compared to
untreated cells (Lovo-Martins et al., 2018). In contrast, EVs can also confer
protection against the infection as T. cruzi EVs enriched in surface membrane
proteins (TcSMP), or secreted by the parasites, were shown that cause
calcium-dependent inhibition of the parasitophorous vacuole they form to gain
entry to the cytoplasm, reducing invasiveness of metacyclic trypomastigotes
to host cells and conferring protection to uninfected cells adjacent to the site
of high burden of parasites (Martins et al., 2015).
T. cruzi invades cells inducing the release of host´s EVs that are involved
in the evasion of the innate response. EVs from infected THP-1 cells has been
shown that bind to complement C3 convertase contributing to the parasite
complement evasion and to the invasiveness by phagocytosis, and therefore,
increasing the parasitemia (Cestari et al., 2012). Another potential evasion
mechanism of the innate immunity response is based on the reported capacity
of EVs from T. cruzi strain Y to induce formation of lipid bodies and
prostaglandin E2 release by macrophages, both contributing to immune
modulation (Lovo-Martins et al., 2018). More recently, it was reported that T.
cruzi EVs and EVs from immune and non-immune infected cells induce a
strong PARP1/cGAS-dependent release of pro-inflammatory cytokines IL-1β,
IL-6 and TNF-α in bone-marrow derived macrophages (Choudhuri and Garg,
2020). Recognition of oxidized DNA by TLR9 seems to synergize with
PARP1 to activate NF-κB and control the expression of the cytokines. This
exacerbated inflammation induced by the injection of EVs was also shown
that aggravate the chronic pathology in CD mice, showing the contribution of
the parasite EVs in Chagas disease (Choudhuri and Garg, 2020).
In summary, the above studies suggest that both parasite and host-derived
EVs are important in promoting parasite differentiation, pathology of the
disease or contributing to the evasion of host´s immune responses by
trypanosome parasites.
EVs from Plasmodium Parasites

EVs from Plasmodium falciparum was first reported that transfer proteins to
infected red blood cells (RBC) in a G-protein dependent manner, including
virulence factors Plasmodium falciparum erythrocyte membrane protein 1
(PfEMP1) and PfEMP3, contributing to the pathogenesis of malaria (Taraschi,
2003). In this sense, increase of TNF-α levels and sensitivity to this cytokine
by upregulation of the VCAM-1 adhesion molecule in endothelial cells has
been correlated with the levels of EVs circulation in blood of patients with
cerebral malaria, suggesting a role of malarial EVs in this pathology (Wassmer
et al., 2011; Sahu et al., 2013). In agreement, the treatment of patients with
antimalarial drugs result in a decrease of circulating EVs in plasma
(Nantakomol et al., 2011). Moreover, they also showed that EVs release
increase upon maturation of the parasites and that their cargo changes during
the differentiation of the parasite (Nantakomol et al., 2011). Noteworthy, an
increase of 10 times in the shedding of EVs from infected RBCs compared to
uninfected RBCs has been reported (Mantel et al., 2013), production that has
been recently related to the parasite ESCRT-III machinery activation by the
action of PfBro1 and PfVps32/PfVps60 proteins (Avalos-Padilla et al., 2021),
contributing to the disease pathogenesis. Even, EVs from infected RBCs
transfer miRNAs and proteins of the RNA-induced silencing complex (RISC)
that can regulate expression in target cells, mimicking what happens with the
infection by the parasite (Wang et al., 2017), as well as they can also transfer
drug resistance to susceptible parasites through episomal DNA (Regev-
Rudzki et al., 2013). Recently, the modulatory activities of EVs from malaria
patients has been demonstrated. Thus, EVs isolated from Plasmodium vivax
patients’ plasma was shown that induce NF-κB-associated upregulation of
ICAM-1 in human spleen fibroblasts, probably favoring the adhesion of
parasites to the human spleen microvasculature (Toda et al., 2020). In another
study, EVs from P. falciparum gametocytes was found that delay the erythroid
differentiation allowing to reach the maturation before their release from the
host bone marrow and probably, contributing to the development of anemia in
patients with malaria (Neveu et al., 2020).
In addition to transfer parasite components to other RBCs, EVs from
RBCs infected with Plasmodium spp. can promote activation of innate
immune cells upon internalization and engage of cell receptors. Thus, EVs
from in vitro-infected RBCs and from infected mice was shown to induce the
TLR4-dependent expression of the co-stimulatory molecule CD40 and TNF-
α in bone-marrow derived macrophages as well as the expression of
proinflammatory cytokines IL-1β, IL-2 and IL-6 and the regulatory IL-10 in
monocytes-derived macrophages and plasma blood mononuclear cells
(PBMCs). This effect was independent of an inflammatory state in mice as
EVs from Plasmodium berghei-infected RBCs of INF-γ-/-, TNF-α-/-, IL-2-/-
and RAG-/- mice were also able to induce the expression of CD40 and TNF-
α in bone-marrow derived macrophages (Couper et al., 2010; Mantel et al.,
2013). Parasite proteins in reticulocyte-derived EVs from Plasmodium yoelii-
infected mice include the merozoite surface proteins 1 and 9, the serine-repeat
antigens, enolase, GADPH, cysteine proteases, aldolase, HSPs and others
(Martin-Jaular et al., 2011). Genomic DNA has been identified as one of the
P. falciparum EVs cargo that activates human monocytes. The parasite DNA
is sensed by stimulator of interferon genes (STING), which in turn, activates
the TANK-binding kinase 1 (TBK1) / interferon regulatory factor 3 (IRF3)
pathway resulting in STING-dependent gene expression (Sisquella et al.,
2017). Interestingly, EVs from uninfected RBCs were not uptake by
monocytes, suggesting upregulation of specific recognition molecules in the
infected cells (Sisquella et al., 2017). Activation of NK cells by P. falciparum

RNA contained in EVs from infected RBCs has also been reported, resulting
in the melanoma differentiation-associated protein 5 (MDA5)-dependent
upregulation of INF-γ and granzyme expression which have been associated
with the lysis of infected RBCs (Ye et al., 2018).
Studies comparing the immunostimulatory capacity of EVs of different
stages of Plasmodium have shown that EVs of late stages in RBC
(trophozoites) are more likely to activate inflammatory states such as
transcriptional changes in human monocytes, than earlier stages (ring state),
implying the former in the interaction with the host's immune system
(Sampaio et al., 2018). Recently, internalization of RBCs-derived EVs by
human microglia that resulted in the upregulation of pro-inflammatory TNF-
α concomitant with down-regulation of regulatory IL-10 has been reported,
suggesting a contribution of EVs in the inflammation of cerebral malaria
(Mbagwu et al., 2019).
EVs from Trichomonas, Giardia and Entamoeba parasites

The studies on EVs from extracellular parasites include the effects of the
environmental conditions under which they are formed, and how changing
them, affect their number, size, and cargo. A first association of Trichomonas
vaginalis tetraspanin 6 with adherence and relocation of this protein into
vesicles was described in 2012 by the Johnson group (de Miguel et al., 2012).
They observed that the expression of this protein, one of the well-known
markers of exosomes, increased significantly after contact of the parasite with
vaginal ectocervical epithelial cells and that more adherent strains express
higher levels of tetraspanin than poor adherent ones (de Miguel et al., 2012).
The same group demonstrated the release of EVs from the parasites,
containing RNA and around 215 proteins (identified by proteomic analysis)
that were delivered to ectocervical cells by fusion, having an impact on the
immune response as pro-inflammatory cytokines IL-6 and IL-8 were elicited
in these cells (Twu et al., 2013). A preferential cargo of small RNAs, mainly
5' tRNA halves, has recently been reported, suggesting their possible
involvement in the regulation of gene expression in host target cells and other
parasites (Artuyants et al., 2020). Noteworthy, viable T. vaginalis was also
shown to release EVs larger than 1 µm and that, like small EVs, they interact
with other parasites and host cells (Nievas et al., 2018). Those studies showed
the role of EVs in parasite-parasite communication conferring properties as
adherence and parasite-host cells communication modulating innate
immunity. Studies by other groups support the above, showing that the
addition of EVs of T. vaginalis to cultures of RAW264.7 macrophage cell line

results in an increase of up to 15 times in the expression of IL-10 and of 2
times in IL-6 and TNF-α (Olmos-Ortiz et al., 2017). They also showed that
the treatment of BALB/c mice with 50 µg/ml of EVs by intravaginal route
before infection with T. vaginalis altered the levels and fluctuation of IL-6,
IL-10, IL-13, and IL-17 cytokines during the infection, and reduce the vulvar
inflammation, favoring the survival of the parasite (Olmos-Ortiz et al., 2017).
More recently, internalization of the T. vaginalis EVs by host cells was shown
to be mediated by the binding of EVs surface 4-α-glucanotransferase to
heparan sulfate in the target cell, in a clathrin-dependent endocytosis that
requires host´s cell cholesterol and caveolin-1 (Rai and Johnson, 2019).
Finally, in a very recent study it was demonstrated that EVs derived from T.
vaginalis natively bearing endosymbiont viruses show immunosuppressive
properties compared to EVs from an isogenic strain lacking the virus.
Specifically, the EVs of parasites with viruses inhibited the NF-κB-dependent
expression of IL-18 and RANTES by cervical uterine cells, the release of IL-
8, IL-6 and TNF-α by monocytes, and the inflammatory signaling of a
mycoplasma-derived TLR ligand (Govender et al., 2020). These results
suggest that the symbiosis of parasites with viruses can bring advantages to
the parasite by modifying the charge of the EVs derived from the parasite,
making them more likely to suppress the immune response.
In the case of Giardia parasites, EVs were reported to be produced by
trophozoites of Giardia intestinalis, which induce the maturation of DCs by
upregulation of CD25 (Evans-Osses et al., 2017). As reported for
Trichomonas, EVs derived from G. intestinalis also favor the adherence of the
parasite to epithelial cells. The same study also showed that EVs were
optimally induced at pH 7.0 while decreased at pH 8.0, the latter a condition
that trigger encystation, suggesting that are released by trophozoites but
inhibited in cysts. Their uptake by target cells was shown to depend on host
cholesterol, probably mediated by lipid rafts (Evans-Osses et al., 2017).
Exosome-like vesicles from G. lamblia was shown to have the same size,
shape, and protein and lipid composition as exosomes described for other
eukaryotic cells (Moyano et al., 2019). More recently, a population of large
vesicles (in addition to the small one) was reported to be produced by
trophozoites of G. intestinalis, which seem to be responsible of promoting
adherence of the parasite to host cells, suggesting that the two population of
vesicles may have different role in host/parasite interplay (Gavinho et al.,
2020). Finally, G. duodenalis-derived EVs was found to induce the release of
pro-inflammatory cytokines IL-1β, IL-6, IL-12, IL-17, IL-18, IFN-γ, TNF-α,
and the chemokines CCL20 and CXCL2 in mouse peritoneal macrophages,

involving the signaling by TLR2 and activation of the NLRP3 inflammasome
(Zhao et al., 2021).
Regarding the E. histolytica amoeba, there is only one report on EVs
derived from the trophozoite stage of this parasite. In this work, in addition to
demonstrating the production of EVs by the amoeba, it was demonstrated that
these EVs package small antisense RNAs, which can impact communication
between parasites and with target cells. Interestingly, EVs derived from
amoebae in the process of encysting were able to promote encystation, while
EVs derived from trophozoites inhibited it, suggesting that EVs can transfer
information that promote differentiation of the parasite (Sharma et al., 2020).
Effects of Helminth EVs on Host’s Innate Immunity

Helminths are worm-like invertebrate organisms that exhibit very complex life
cycles, usually involving definitive and intermediary hosts that harbor the egg,
adult, and larval stages, in which the parasites reproduce sexually. There are
both hermaphroditic and bisexual species. Many infections by helminths are
eliminated by host defenses whilst others become established and may persist
for years successfully modulating host defenses in a variety of ways.
EVs from Nematode Parasites

The first report of EVs derived from a nematode had to do with studies about
secretion products from the mouse nematode Heligmosomoides polygyrus, a
model of hookworm helminths (Buck et al., 2014). They reported that the
nematode release EVs that transfer small RNAs to mammalian cells
suppressing the type 2 innate response. Thus, the administration of EVs by
intranasal route in mice suppressed the allergic response to the fungus
Alternaria evidenced by the decrease of eosinophilia and downregulation of
IL-5 and IL-13 cytokines, concomitant with suppression of the IL-33 receptor
expression in the innate lymphoid cells ILC2 (Buck et al., 2014). In another
study, inhibition of the IL-33 receptor expression by EVs of H. polygyrus or
their proteins was also reported in alternatively activated macrophages (AAM;
Coakley et al., 2017). Inhibition of classically activated M1 macrophages was
also observed by downregulation in the production of inducible nitric oxide
synthase (iNOS), IL-6, IL-12 and TNF-α. These results suggest that H.
polygyrus EVs modulate the host's immune response for evasion, inhibiting
the activation and differentiation of macrophages, thereby favoring the
establishment of the parasite in the mouse intestine by suppressing the type 2
innate response that is the responsible for expelling the worms. However,
when those EVs were used as vaccine, a decrease in egg expulsion and worm
burden was observed, a protection associated to the induction of specific
antibodies (Coakley et al., 2017). Differences in lipid composition between H.
polygyrus EVs and EVs from mammalian cells was also found, mainly
showing that EVs release by the nematode is 9 to 62 folds enriched in
plasmalogens respect to EVs from murine cells, but deficient in cholesterol
and sphingomyelin, which could have to do with their immunosuppressive
properties (Simbari et al., 2016).
Regarding whipworms, the release of small RNA-containing EVs from
Trichuris suis larvae and the release of EVs from Trichuris muris containing
parasite/host proteins have been reported (Hansen et al., 2015; Shears et al.,
2018). In the latter, vaccination of mice by subcutaneous route with purified
T. muris EVs conferred protection against the infection associated with the
induction of specific IgG2a/c antibodies, response that was suppressed when
an EV-depleted fraction was used. In contrast to the EVs from Nipostrongylus
brasiliensis that protected against trinitrobenzene sulfonic acid-induced colitis
in mice by decreasing IL-1α, IL-6, IL-17a and INF-γ expression (Eichenberger
et al., 2018), EVs from T. muris showed no effect, which was interestingly
related to the number of miRNAs identified in the EVs (29 in N. brasiliensis
vs 17 in T. muris). These results suggest that the EVs from different nematodes
have different immunoregulatory potential.
EVs from Trematode Parasites

Studies on EVs in trematodes have been carried out mainly in Schistosoma
and Fasciola species. EVs have been identified from the adult fluke and the
schistosomula of Schistosoma mansoni, a migrant form of the parasite within
the host that forms once the cercaria loses its tail (Sotillo et al., 2015; Nowacki
et al., 2015). Proteomic studies of those EVs have shown the presence of small
non-coding RNAs, miRNAs and tRNAs, in addition to more than 100 proteins,
both from the host and the parasite, including vaccine candidates (Enolase,
HSP-70, Glutathione-S-tranferase, tetraspanin TSP-2, among others) and
virulent factors (Nowacki et al., 2015; Sotillo et al., 2015). S. mansoni-derived
miRNAs has also been identified in exosomes isolated from sera of infected
mice (Samoil et al., 2018). EVs from Schistosoma japonicum eggs containing
miRNAs has also been reported, suggesting that are produced by all the life
cycle stages and all of them having regulatory effects on the host immune
response (Zhu et al., 2016). For example, after treatment of DCs with S.
japonicum soluble eggs antigens (which usually carry EVs), these immune
cells release EVs that, injected by intraperitoneal route in mice, ameliorate the
pathology of experimental colitis induced by dextran sulfate sodium (Wang et

al., 2017), suggesting a modulation of the EVs cargo release by the innate cells
once exposed to the parasite. In a recent study, it was shown that S. japonicum
EVs are enriched in the host miRNA miR-148a, which once taken up by
macrophages regulate their cytokine production (Giri and Cheng, 2019). In
addition, EVs from S. mansoni was shown to be mainly internalized by
monocytes, but also by NK cells and T and B lymphocytes. Moreover, the in
vitro incorporation of EVs miRNAs into macrophages, stimulated their
proliferation and production of TNF-α. This result was reproduced in infected
mice where innate activation was associated to pathology, suggesting a
participation of the EVs in parasite survival (Liu et al., 2019). Uptake of S.
mansoni EVs into human monocyte-derived DCs seems to be mediated by
DC-SIGN, and not by the mannose or DCIR receptors, which drive the
expression of the costimulatory molecules CD80 and CD86, the regulatory
marker programmed cell death 1 ligand 1 (PD-L1), and the cytokines IL-12
and IL-10 (Kuipers et al., 2020). In human umbilical vein endothelial cells
(HUVECs), S. mansoni EVs induced the expression of genes involved in the
intravascular establishing of the parasite affecting coagulation, contraction of
the vascular endothelium, arachidonic acid production and innate immune
response (Kifle et al., 2020). In contrast, EVs from HUVECs decreased the
liver injury caused by S. japonicum in mice improving their survival,
supporting their role in host resistance against this parasite (Dong et al., 2020).
Regarding Fasciola, several studies have been carried out on EVs from F.
hepatica, with more or less similar results to the above-mentioned studies on
Schistosoma EVs. It should be added that two types of EVs have been
identified in this trematode; some small EVs that seem to participate in
immunomodulation processes, and other larger ones that have to do with
feeding processes of the parasite (reviewed in Corral-Ruiz and Sánchez-
Torres, 2020).
EVs from Cestode Parasites

EVs have also been reported in cestodes, including the human tapeworm
Taenia asiatica, the mouse parasite Taenia crassiceps and the zoonotic
parasite Echinoccocus multilocularis, the last two EVs described to contain
parasite miRNAs and proteins as well as host antibodies and complement
components (Galán-Puchades et al., 2016; Ancarola et al., 2017). EVs from E.
granulosus was shown later that downregulate the nitric oxide synthase in
RAW macrophages, inhibiting the production of NO as well as the
proinflammatory cytokines IL-1α and IL-1β (Zheng et al., 2017). Moreover,
E. granulosus EVs were also taken up by DCs, in which they increased CD86
while downregulated MHC-II expression, probably damping the antigen
presentation function (Nicolao et al., 2019). Recently, three subpopulations of
EVs were identified by ultracentrifugation in hydatid fluid of E. granulosus.
The subpopulation called 110 K (which contained more proteins) was further
shown that contained miRNAs that stimulated peripheral blood mononuclear
cells of sheep to release TNF-α and IL-10 while downregulated IL-1β, IL-17
and CD14 (Yang et al., 2021), suggesting a role of those EVs in the parasite:
host communication, leading to activation and/or evasion of the host innate
immune response.
Exploitation of EVs from Innate Cells by Viruses
The case of the study of EVs in viral infections has run into difficulties that
have to do with the fact that viral particles have similar size, density and, in
the case of enveloped viruses, membrane as EVs. Some viruses even use the
same biogenesis pathways from which EVs originate (Cantin et al., 2008;
Purvinsh et al., 2021). Therefore, the difficulty of separating them during the
purification process could have affected the interpretation of results and
conclusions, especially in the first reports in which efficient separation
strategies had not yet been developed. However, from these analyzes very
interesting aspects of intercellular communication during virosis have
emerged, such as the ability of viruses to use EVs as Trojan horses that carry
infective particles from an infected to an uninfected cell, or that EVs can carry
the genetic material of the virus that could allow the assembly of particles in
an uninfected target cell (Purvinsh et al., 2021).
Like infectious processes caused by bacteria and parasites (such as those
mentioned above), EVs derived from cells infected by viruses exert
immunomodulatory effects on innate immunity cells via both receptor-
dependent and receptor-independent pathways, favoring and/or suppressing
the host immune response which influence the outcome of the viral infection.
As such, the associated immunomodulation mechanisms are, with few
exceptions, like those described above, so in this section we will focus on
describing cases of exploitation of innate immunity cells by viruses.
EVs during HIV Infection
The Trojan horse hypothesis was first postulated as a mechanism for retrovirus
dissemination, mainly HIV, by hijacking the exosome biogenesis and
secretion pathways to assemble new particles and spread them to uninfected
cells independently of Env viral recognition (Gould et al., 2003). Besides,
viruses such as herpesvirus, hepatitis, filoviruses, arenaviruses, rhabdoviruses,
among others, use ESCRT and Rab GTPases for their secretion (Chen and
Lamb, 2008). Using proteomic, lipidomic and cell biology approaches, HIV
particles were shown to be associated with exosomes aggregates, allowing
rapid and efficient infection of human-monocyte-derived macrophage where
the virus replicates robustly (Kadiu et al., 2012). Noteworthy, EVs was also
shown to carry cytokines such as IL-3, IL-4, IL-8, IL-17, TNF-α and leptin
that were protected from proteolytic degradation allowing them to be stable
for several days, contributing to the recruitment of innate immune cells that
favor dissemination of HIV infection and pathogenesis (Kadiu et al., 2012).
Exosomes released by HIV-infected cells transport RNA and viral proteins to
neighboring uninfected cells and even, the exploitation of this system is so
sophisticated that exosomes from uninfected cells stimulate the replication of
latent HIV-1 transcripts in infected cells through phosphorylation of RNA
polymerase II (Barclay et al., 2017). On the other hand, activation of the innate
response by EVs from HIV-infected cells has also been reported. In addition
to carry cytokines, chemokines and viral components that are recognized by
receptors in the target cells, the EVs from virus-infected cells can also carry
products of cytosolic sensors, such as the second messenger 2´3´-cyclic GMP-
AMP (derived from the cyclic GMP-AMP synthase), which can activate
innate immunity and antiviral defenses in uninfected target cells (Gentili et al.,
2015).
EVs during Hepatitis Virus Infection
Another example of well-known exploitation of the EVs secretory system by

viruses is the case of Hepatitis A (HAV, a piconavirus) and Hepatitis C (HCV,
a flavivirus) viruses. Unlike HCV, HAV is a non-enveloped RNA virus that
has been demonstrated to be incorporated into exosomes once assembled in
the infected cells; in contrast, in the case of the enveloped HCV virus, are both
the full-infectious HCV RNA or the whole virions that have been detected in
exosomes, in any case allowing evasion of the viruses from antibody-mediated
immune response (Ramakrishnaiah et al., 2013; reviewed in Longatti et al.,

2013). Moreover, packaging of Hepatitis virions inside EVs promote the
likelihood of infecting cells other than hepatocytes (reviewed in Longatti et
al., 2013). Paradoxically, both exosomes carrying the assembled viral particle
or the full RNA sequence are also necessary for the TLR7-dependent
activation of plasmacytoid DCs (viral particles alone fail to activate them),
which drive an innate immune response that includes type-I IFN production
(Dreux et al., 2012; Feng et al., 2015). Accordingly, exosomes from TLR3-
activated macrophages inhibited HCV replication in the hepatocyte cell line
Huh7 through the delivery of anti-HCV miRNAs (Zhou et al., 2016).
EVs during Herpes Virus Infection
Herpes Simplex Virus (HSV-1)-infected cells release exosome-like EVs

known as L-particles which do not contain infectious particles but contain host
and viral transcripts (coding and non-coding RNAs) and proteins (including
immune factors as STING) that prepare target cells for subsequent infection
by inducing the transcription of HSV-1 promoting proteins in the target cells
while decrease the expression of MHC-II and CD83 in DCs and other antigen
presenting cells (APCs) leading to concomitant immunosuppression (Kelly et
al., 2009; Temme et al., 2010; Heilingloh et al., 2015). In contrast to L-
vesicles, MVs from infected cells seems to shuttle whole virions favoring the
spreading of the HSV-1 by increasing the range of target cells (Bello-Morales
and López-Guerrero, 2018).
EVs during Epstein-Barr Virus Infection
In the case of Epstein-Barr Virus (EBV), the exosome cargo selectively

contains the viral membrane protein and oncogene LMP1 which is transferred
to target cells, including lymphocytes B where it causes transformation, or
lymphocytes T CD4+ and natural killer cells where it induces extensive
apoptosis (Kaye et al., 1993; Dukers et al., 2000; Hurwitz et al., 2017). As
much as 25% of the total miRNAs of a cell has been detected in EBV-derived
exosomes which are thought to perform control of post-transcriptional
functions including pathogenesis and evasion of the innate immune system
(Pegtel et al., 2010; Grundhoff and Sullivan, 2011). Thus, the downregulation
of the NLRP3 inflammasome activation and IL-1β/CXCL11 release by
miRNAs contained in EVs from EBV-infected cells has also been

demonstrated (Xia et al., 2008; Haneklaus et al., 2012), supporting their role
in the evasion of the host immune response and the pathogenesis of BEV
tumor-associated diseases.
EVs during SARS-CoV-2 Virus Infection
Very recently, it was found that SARS-CoV-2 infection induces the release of
EVs carrying tissular factor in blood that, in turn, activates endothelial cells,
platelets, macrophages and neutrophils, contributing to the common
occurrence of thrombosis in COVID-19 patients (Mackman et al., 2020).
Proteomic analyses showed that EVs from COVID-19 patients had more
abundant markers than EVs from healthy donors, being coat complex subunit
beta 2 (COPB2) the marker with the best prediction value between mild and
severe cases (Fujita et al., 2021). Moreover, the presence of SARS-CoV-2
RNA in the circulating exosomal cargo has also been identified, suggesting
that this virus may use the endocytosis route to spread as other viruses.
Therefore, circulating exosomes may play an essential role in inflammation,
coagulation, and immunomodulation during SARS-CoV-2 infection (Barberis
et al., 2021).
Overall, viruses have evolved to exploit the secretion and exosome
biogenesis pathways in order to favor their spreading to uninfected cells by: i)
evading the host innate and adaptive immune system by hiding inside the EVs;
ii) allowing the transferring of whole infectious viral particles or their
complete infectious nucleic acid; iii) delivering miRNAs, mRNAs and viral
proteins that hinder the cell antiviral defenses and; iv) delivering of viral
miRNAs and proteins involved in pathogenesis, contributing to the
establishment of the viral disease.
Effects of Tumor-Derived Extracellular Vesicles

on Innate Immunity
Tumor cells are known to continuously produce and release EVs as a

mechanism to promote its growth and modulate its microenvironment (Qian
et al., 2015). By their size and biogenesis route, tumor-derived EVs (TD-EVs)
are usually termed as exosomes (Whiteside, 2016), although larger tumoral
EVs (OcMs) have also been found to participate in tumor growth (Minciacchi
et al., 2015). In contrast to EVs derived from nonmalignant cells, TD-EVs

carry oncogenic membrane proteins or genomic material, which favor tumor
progression (Qiao et al., 2019).
While tumor cells shed EVs in a continuous manner, the production rate
of TD-EVs is known to increase significantly after cancer treatment, and it can
be a major contributor to chemoresistance in prostatic and pancreatic cancer
(Aubertin et al., 2016; Qiu et al., 2018). Additionally, the oncogenic cargo of
TD-EVs (including growth factor receptors, proteins, and miRNAs) has been
proved to favor tumor progression (Kalluri, 2016) by promoting cell
proliferation, epithelial‑mesenchymal transition, angiogenesis, and the
development of premetastatic niches. There is ample evidence that, along with
modulating the local microenvironment and distributing oncogenic cargo to
distant locations, TD-EVs can repress the antitumor immune response,
favoring the survival of malignant cells (Greening et al., 2015; Lowry et al.,
2015).
Exosomes can modify the characteristics of normal cells, by entering new
information. A clear example has been reported in gastric cancer. The
characteristics of exosomes depend on the donor cells and the circumstances
of their formation. High levels of miR-27a have been reported in exosomes
secreted by cells of gastric cancer patients (Chen et al., 2021). These exosomes
favor the conversion of fibroblasts into cancer-associated fibroblasts, by
targeting cysteine- and glycine-rich protein 2 (CSRP2). Moreover, fibroblasts
transfected with miR-27a overexpress constructs and significantly increase
fibroblast motility. Their up-regulation also increases fibroblast proliferation
and the invasion capacity of gastric cancer cells. This indicates that tumor-
derived exosomes can induce critical modifications in normal fibroblasts
(Wang et al., 2018).
EVs can also act through by modifying the microenvironment, as reported
in nasopharyngeal carcinoma. Non-malignant immune cells like T and B
lymphocytes, macrophages, and DCs can be rapidly replaced by malignant
cells, influenced by the secretion of cytokines and different substances by
EVs, promoting a suppressor microenvironment, enriched with regulatory T
cells (Tregs) that favor tumor progression (Luo et al., 2021). EVs can
exacerbate tumor growth using other targets, as observed in melanosomes,
where extracellular particles released from melanoma cells that express the
FasL resulted in the induction of apoptosis in lymphoid cells, inhibiting anti-
tumor immunity (ying-yang). It has also been reported that melanoma can
modulate the anti-tumor immunity by producing EVs, through a mechanism
that seems to be mediated by the PD-1/PD-L1 immune check point (ying-
yang). Exosomal PD-L1 released from metastatic melanoma cells inhibits

cytotoxicity, favoring tumor growth (CD8þ T cells) and melanoma
progression (Chen et al., 2018).
The mechanisms underlying the regulation of antitumor immune response
by TD-EVs constitute an active field for research, and many details are still
unknown. However, there is consensus that TD-EVs establish a complex
interaction with both the innate and the acquired immune responses
(Whiteside, 2013; Whiteside, 2016). The net effect of this interaction will
determine the fate of the tumor (Yamauchi and Moroishi, 2020).
On one hand, TD-EVs have been proved to engage with cellular
components of the innate and acquired immunity through surface proteins with
ligand activity, binding receptors on immune cells. For instance, the epidermal
growth factor receptor (EGFR) and human epidermal growth factor receptor
2 (HER-2) on TD-EVs surface can promote the survival of tumor-derived
monocytes and the formation of tumor-associated macrophages (TAMs)
(Song et al., 2016). PD-L1, also detected on the membrane of TD-EVs, has
shown ALIX-mediated immunosuppressive effects along with EGFR in breast
cancer cells (Monypenny et al., 2014; Chen et al., 2018). Melanoma cell TD-
EVs express FasL, which exert immunoinhibitory effects by inducing
apoptosis in lymphoid cells (Andreola et al., 2002). Heat-shock proteins
(HSPs) on the TD-EV membrane are known to bind TLR2, TLR4, lectin-like
oxLDL receptor 1 (LOX-1), and other receptors on immune cells; thus, HSP72
binds TLR2 on myeloid-derived suppressor cells, activating the STAT3
signaling and promoting the production of IL-6 (Chalmin et al., 2010; Youn
et al., 2010), while HSP70 was reported to activate the NF-κB signaling by
binding TLR2 on mesenchymal stem cells (MSCs) (Li et al., 2016).
On the other hand, TD-EVs interact with the immune cells through their
cargo, either releasing it to the circulation or being internalized by their target
cells. MICA*008, a circulating variant of the MHC-I polypeptide-related
sequence A released by TD-EVs, binds the NKG2D receptor on NK cells,
inducing its downregulation and inhibiting NK cytotoxicity (Ashiru et al.,
2010). EVs derived from gastric cancer cells and containing the high mobility
group box-1 (HMGB1) activate the TLR4/NF-κB signaling, inducing a tumor-
promoting neutrophil activation (Zhang et al., 2018).
Various miRNAs contained in TD-EVs suppress the host immune system,
favoring metastasis. In a particular case, miR-21 and miR-29a in EVs derived
from the lung cancer cells in BALB/c mice were reported to promote the
production of the pro-metastatic inflammatory cytokine TNF-α by binding
TLR7/8 in the endosomes of immune cells (Fabbri et al., 2012). Pancreatic
cancer-derived EVs containing miR-212-3p decrease the expression of MHC-

II, suppressing antigen presentation in DCs. Similarly, MiR-212-3p inhibits
the mRNA expression of the regulatory factor X-associated protein, required
for the transcription of MHC-II (Ding et al., 2015). EVs derived from
epithelial ovarian cancer cells contain miR-222-3p that RIGcan polarize
macrophages into TAMs (Ying et al., 2016). The miR-222-3p inhibits mRNA
expression of suppressor of cytokine signaling 3 (SOCS3), promoting STAT3
activation and triggering inflammatory cascades, favoring TAM polarization.
While TD-EVs are noted by their immunosuppressive effects, they have
also been demonstrated to exert immunostimulatory actions. The HLA-B-
associated transcript 3 (BAT3) in TD-EVs was reported to activate NK cells
by binding the natural cytotoxicity receptor 3 (NKp30), enhancing their
cytotoxic activity (Pogge von Strandmann, 2007). The release of BAT3-
contanining EVs from melanoma cells upon activation of the innate immune
receptor retinoic acid-inducible gene I is activated (RIG-I) resulted in a potent
anti-tumor activity in an animal model (Daßler-Plenker et al., 2016). Rather
paradoxically, TD-EVs containing HSP70 can stimulate NK cells (Gastpar et
al., 2005) and macrophages (Vega et al., 2008).
TD-EVs carrying dsDNA can induce anti-tumor immunity. DNA-
containing TD-EVs can activate DCs through the cGAS/STING pathway
(Kitai et al., 2017). Additionally, EVs derived from irradiated breast cancer
cells present dsDNA to DCs, stimulating the production of type-I Interferon
via cGAS/STING (Diamond et al., 2018). TD-EVs containing nucleic acids
released by various cancer cell lines after deleting the Hippo pathway kinases
LATS1/2 triggered the TLR signaling pathway, inducing a strong type-I IFN
anti-tumor immune response (Moroishi et al., 2016).
Tumor-Derived Extracellular Vesicles in Cancer Diagnosis

and Treatment
TD-EV specific molecules have been proposed as biomarkers for various

cancer types, in an approach termed as “liquid biopsy.” Some of the most
important are shown in Table 1.
Additionally, various therapeutic approaches based on TD-EVs have been
proposed. First, treatments targeting TD-EVs to mitigate or offset their tumor-
promoting effects. For instance, an approach to block the uptake of melanoma-
derived EVs in vivo by administering reserpine, reducing cell education and
the formation of premetastatic niches (Ortiz et al., 2019). Second, the use of
engineered TD-EVs to deliver anti-cancer drugs to specific tumor targets is a

promising approach. For instance, the use of nanometric vehicles based on
EVs derived from cancer stem cells has been explored (Wang et al., 2017).
Third, the use of TD-EVs as vectors for anti-cancer vaccines or as antigenic
candidates has shown encouraging results (Zitvogel et al., 1998).
Table 1. EVs associated biomarkers in various tumors
TD-EV proteins Tumor Body fluid Year Ref

NY-ESO-1 Lung Plasma 2016 Sandfeld-Paulsen
et al., 2016
PKG1, RALGAPA2, Breast Plasma 2017 Chen et al., 2017
NFX1, TJP2
Her2 Breast Plasma 2017 Fang et al., 2017
Glypican-1 Breast Serum 2016 Melo et al., 2015
Glypican-1 Pancreatic Serum 2016 Melo et al., 2015
Glypican-1 Colorectal Plasma 2017 Li et al., 2017a
CEA Colorectal Serum 2017 Yokoyama et al.,
2017
AMPN, VNN1, PIGR Cholangiocarcinoma Serum 2016 Arbelaiz et al.,
2017
PSA Prostate Plasma 2017 Logozzi et al.,
2017
GGT1 Prostate Serum 2017 Kawakami et al.,
2017
CD24, EpCAM, CA- Ovarian Plasma 2016 Zhao et al., 2016
125
Modified from Li et al., 2017.
Effects of Extracellular Vesicles in Autoimmune Diseases
Autoimmune diseases (ADs), like rheumatoid arthritis (RA), systemic lupus

erythematosus (SLE), systemic vasculitis, dermatomyositis, systemic
sclerosis, mixed connective tissue disease, autoimmune hemolytic anemia,
autoimmune thyroiditis, and ulcerative colitis, constitute a heterogeneous
group of conditions, characterized by an aberrant immune response to
functioning structure of the cell (Wang et al., 2015). Under normal
circumstances, factors such as central and periphery tolerance, peripheral
anergy, T regulatory cells (Tregs), and the immune homeostasis orchestrated
by the action of cytokines, chemokines, and their receptors ensure the
distinction of self from non-self (Crimeen-Irwin et al., 2005). When this
balance is altered, however, autoantibodies and autoreactive cells may induce
chronic inflammation, causing damage to tissues and organs. Thus, chronic

inflammation is the hallmark of ADs (Duan et al., 2019).
The precise etiology of most ADs is still unknown, but genetic and
environmental factors, including infectious agents, xenobiotics, and the
composition of microbiota, are recognized as participating in the onset and
progression of various ADs (Wang et al., 2015). However, two clear
components have been described as underlying the physiopathology of ADs:
autoimmunity and cellular stress (Harris, 2016). The cellular stress response
involves various signaling pathways, including the generation, and sensing of
ROS, activation of the unfolded protein response (UPR), and autophagy
initiation (Higa and Chevet, 2012; Hetz, 2012; Lapaquette et al., 2015). While
cellular stress pathways evolved to restore homeostasis after a noxious event,
their overactivation could result in inappropriate immune responses to the
affected tissue (Lapaquette et al., 2015; Martinon and Glimcher, 2011;
Hasnain, 2012).
As mentioned above, a major communication channel between tissue cells
and immune cells are EVs; immune cells are known to release EVs and to
receive cellular-derived EVs, which modulate the immune response
(Lindenbergh and Stoorvogel, 2018). A significant increase in EV trafficking
is observed when healthy cells are subjected to stress. For instance, when
subjected to oxidative stress, bovine granulosa cells released EVs containing
anti-oxidative molecules (Saeed-Zidane et al., 2017), while serum-deprived
MSCs show a 22-fold increase in EV exchange with neurons than healthy
MSCs (Haraszti et al., 2019). Thus, EV-mediated intercellular communication
is thought to have a key role in the onset and progression of autoimmunity.
Conversely, understanding the role of EVs in antigen presentation could open
new perspectives to treat ADs, not merely alleviate their symptoms. In other
words, a growing body of evidence indicates that EVs play a key role in the
modulation of the immune response and may be involved in the pathogenesis
of autoimmune diseases. On the other hand, EVs offer a novel target to
develop new alternatives for the diagnosis and therapy of autoimmune
diseases.
EVs are known to play a role in the pathogeny of various ADs. For
instance, SLE is characterized by the accumulation of immune complexes
(ICs), which cause tissue damage. ICs are formed from the interaction of EVs
containing surface antigens with IGs or extracellular matrix proteins (Fendl et
al., 2018; Fortin et al., 2019) and induce a complement-mediated immune
response (Buzas et al., 2018). Additionally, ApoBDs could promote the
production of type-1 IFN, leading to further damage (Rasmussen and
Jacobsen, 2018). The pathogeny of lupus nephritis, a common complication in

SLE patients, has been linked to EVs containing autoantibodies and
complement proteins, promoting the formation of immune complexes (ICs),
triggering inflammatory responses, and leading to renal damage (Zhao, 2020).
In type-1 diabetes (T1D), it has been hypothesized that cellular stress
results in the release of EVs containing autoantibodies; these autoantibodies
trigger the activation of autoreactive CD8+ T cells, which in turn destroy
insulin-producing β-cells in the pancreas (Dai et al., 2017). In addition, EVs
derived from pancreatic islet cells contain immunostimulatory cytokines like
IFN-γ, which stimulate monocytes and autoreactive B and T cells, further
destroying pancreatic tissues (Bashratyan et al., 2013; Rahman et al., 2014;
Rutman et al., 2018).
The synovial fluid and joint spaces of RA patients have been found to
contain increased EV concentrations with respect to healthy controls (Fu et
al., 2018; Viñuela-Berni et al., 2015; Gyorgy et al., 2012). Leukocytes,
fibroblast-like synoviocytes (FLS), and especially platelets seem to be the
main EV source in RA (Fu et al., 2018; Cloutier et al., 2013; Distler et al.,
2005; Boilard et al., 2010). Platelet-derived EVs (PDEVs), whose levels are
correlated with RA severity, present IL-1 on their surface, which stimulate
FLS to release IL-6 and IL-8, as well as extracellular matrix (ECM)-degrading
enzymes that contribute to joint damage (Fu et al., 2018; Boilard et al., 2010).
Citrullinated proteins in RA-related EVs can act as autoantigens and form ICs,
which stimulate leukotriene secretion by neutrophils, worsening joint
inflammation (Cloutier et al., 2013; Skriner et al., 2006).
In vitiligo, ROS-mediated oxidative stress damages melanocytes, which
release EVs containing tyrosinase and Melan-A, a melanosome antigen
capable of activating DCs and recognized by CD8+ T cells and lead to the
activation of an autoimmune response (van den Boorn et al., 2011; van den
Boorn et al., 2009; Harris, 2016).
The inflammatory bowel disease (IBD) is believed to be due to a
dysregulation of intestinal immune homeostasis. Under normal conditions,
intestinal epithelial cells release EVs, which then interacts with DCs,
promoting a tolerogenic environment. There is a complex crosstalk between
epithelial cells, immune cells, and microbioma cells mediated by EVs. Under
stress conditions, higher amounts of ATP and other nucleotides are released
into the small intestine lumen; these nucleotides are dephosphorylated by
CD39, leading to increased levels of nucleosides, which disrupt the purigenic
signaling and the delicate balance, shifting to an inflammatory environment.
Activated T cells destroy the epithelial cells, which only increases the pro-
oxidant damage (Longhi et al., 2017; Ayyar and Moss, 2021). Circulating EVs
are also involved in the pathogenesis of IBD by altering the activity of
macrophages, a cell type critically involved in maintaining the homoeostasis
of the intestinal environment. EV-related proteins, most of which are acute-
phase proteins and immunoglobulins, related to complement activation and
coagulation cascade, were found in serum samples from mice treated with
dextran sulfate sodium, a model of IBD (Miao et al., 2021).
It has been reported that EVs in the cerebrospinal fluid from multiple
sclerosis (MS) patients has toxic effects on astrocytes in vitro. These results
strongly suggest that EVs have a role in the pathogeny of MS (Ragonese et al.
2020).
Extracellular Vesicles in the Diagnosis and Treatment of

Autoimmune Diseases
Currently, about 100 different conditions have been described as ADs (Wang
et al., 2015). Some of them are organ-specific, like autoimmune thyroiditis or
primary biliary cirrhosis (PBC), whilst other diseases, like SLE, involve
multiple organs (Yu et al., 2014), and are regarded as non-organ-specific.
Some of the most common ADs and their markers in EVs with potential for
diagnosis are shown in Table 2.
On the other hand, various approaches for the therapeutic applications of
EVs in ADs have been proposed recently. Their role in the pathogeny of
various ADs, as well as their capacity to deliver bioactive cargos to specific
cells makes them either a therapeutic target or an efficient drug vehicle. For
instance, EVs derived from M2 macrophages have been assayed to attenuate
colitis in a murine model (Yang et al., 2019). Umbilical cord-derived EVs and
human adipose cell‐derived EVs were used against experimental autoimmune
encephalomyelitis (Kim et al., 2020; Jafarinia et al., 2020). The use of IL-35-
producing EVs derived from i35-Breg cells has shown interesting results
against neuroinflammation and autoimmune uveitis (Kang et al., 2020).
Similarly, oligodendrocyte-derived EVs were used against autoimmune
neuroinflammation in mice (Casella et al., 2020). Stem cell derived EVs were
assayed against various autoimmune and neurodegenerative disorders.
Finally, mesenchymal cell-derived miRNA-150-5p-expressing EVs were
assayed in a RA murine model (Chen et al., 2018).
Table 2. EVs-associated biomarkers in some autoimmune diseases
Autoimmune EV Biomarkers References

disease
Primary Sjögren’s Calmodulin Aqrawi et al., 2017;
syndrome Copine 1 Sellam et al., 2009;
Platelet-derived MPs Bartoloni et al., 2015
Tyrosine-protein phosphatase nonreceptor
type substrate 1
Guanine nucleotide-binding protein
subunit alpha-1
Endothelial MPs
Leukocyte-derived MPs
Systemic lupus Total MPs, PMPs, CD25 + MPs, EMPs, López et al., 2017; Fortin
erythematosus monocyte-derived MPs, and T cell-derived et al., 2019; Viñuela-
MPs Berni et al., 2015; Duval
CD41+ MPs harboring IgG and CD41– et al., 2010
MP harboring IgG
CD14+ monocyte-derived MPs
Total EMPs, CD54+ EMPs, CD54− EMPs,
and CD54+ EMPs/total EMPs
Type-1 diabetes Insulin-containing exosomes, exosomal Garcia-Contreras et al.,
mellitus islet autoantigen and GAD65 2017; Korutla et al.,
MiR-16-5p, miR-574-5p and miR-302d- 2019
3p
Behçet’s syndrome Total MPs, CD14 + MPs, Granulocytes- Khan et al., 2016
derived MPs, and tissue factor + MPs
Oral lichen planus MiR-34a-5p, miR-130b-3p and miR-301b- Peng et al., 2018
3p
MPs: microparticles; EMPs: endothelial microparticles; T1DM: type-1 diabetes mellitus.
Modified from Xu et al., 2020.
Immunotherapy Based on EVs
As we have reviewed so far, EVs are released by practically any cell and their
study has made possible to establish their role during health and disease.
Thanks to the characterization of surface molecules and cargos present in EVs,
they have been defined as elements with a very important therapeutic potential
since the molecules that they contain help the body to recover homeostasis
(EL Andaloussi et al., 2013; Rani et al., 2015; Wiklander et al., 2019). EVs,
mainly MVs and exosomes, have been used for different therapeutic purposes;
one of them is their use to induce proliferation and regeneration after damage,
which have been explored for the repair of various cells and tissues including
bone, cartilage, neurons, or liver (Doeppner et al., 2015; Qin et al., 2016; Vonk
et al., 2018; Cao et al., 2019; Xu at al., 2019; Madison and Robinson, 2019;
Huang et al., 2020). In this section we review some of the therapeutic
applications of EVs derived from innate immune leukocytes, as well as the
therapeutic use of vesicles from other sources on these cells.
Mesenchymal Stromal/Stem Cell-Derived EVs for Immunotherapy
Stem cells have been widely studied for their ability to self-renew and
differentiate into multiple cell types, which makes them ideal candidates for
use in tissue repair (Wang et al., 2013; Maranda et al., 2017; Hassanshahi et
al., 2019). Embryonic stem cells are obtained from blastocysts; however, their
use is limited by the ethical implications related to the handling of embryos
(King and Perrin, 2014; de Miguel-Beriain, 2015). However, stem cells can
also be isolated from adult tissue and are referred as mesenchymal
stem/stromal cells (MSCs). MSCs can be isolated from different sources such
as adipose tissue, endometrium, bone marrow or umbilical cord. The relative
ease of obtaining MSCs has positioned them as a very important study model,
not only in the field of medicine, but also in other disciplines (Ding et al.,
2011; Fu et al., 2019). It has been described that MSCs interact with cells of
the immune system by cell-cell contact or through the secretion of multiple
chemical mediators, many of them contained in EVs. This is the reason why
the immunomodulatory properties of MSCs-derived EVs (MSC-EVs) have
been studied (Wang et al., 2014; Lai et al., 2015; Li and Hua, 2017). Here we
mention some of the immunotherapeutic applications of these structures on
the innate immune response.
Applications of MSC-EVs in Inflammation Control

MSCs have been characterized by their ability to regulate inflammatory
responses, activity that has recently been seen to be mediated in part by the
release of EVs. MSC-EVs injected intravenously in a murine model of acute
kidney injury decreased the inflammation associated with the damage, an
effect that was associated with the transfer of mitochondrial transcription
factor A and mitDNA trough EVs to the damaged cells (Zhao et al., 2021).
Similar results have also been observed in a porcine model of kidney damage
(renal artery stenosis caused by metabolic syndrome), where the MSC-EVs
injected co-localized with macrophages and tubular cells in the kidneys,
reducing local inflammation. In this case, IL-10 present in the EVs was
associated with the immunomodulatory effect as IL-10-depleted EVs showed
less effect (Eirin et al., 2017). MSC-EVs have also shown good results in
reducing inflammation during temporomandibular joint osteoarthritis by
suppressing the synthesis of MMP13 and NO mediated by IL-1β (Zhang et al.,
2019). Additionally, MSC-EVs has shown efficacy to reduce inflammation
associated with spinal cord injury by reducing expression of the pro-
inflammatory cytokines IL-6 and IL-1β, an effect that was even greater than
the inoculation of intact MSCs (Romanelli et al., 2019). The protective effect
of MSC-EVs has also been reported in a mouse model of lung damage due to
radiation exposure by reducing the infiltrate of neutrophils and macrophages
into the lungs thanks to the increase in IL-10 synthesis and the decrease of
proinflammatory cytokines (IL-1β, IL-6 and TNF-α) (Lei et al., 2020). Similar
results have been obtained in murine and rat models of liver damage where
MSC-EVs reduced the inflammation by decreasing the number of Kupffer
cells and synthesis of pro-inflammatory cytokines (Ohara et al., 2018) or
preventing neutrophil recruitment as well as ROS production (Yao et al.,
2018). Consistent with the above, MSC-EVs decreased allergic inflammation
by reducing macrophage and neutrophil infiltration after allergen exposure
(Cruz et al., 2015) as well as by inhibiting the function of innate lymphoid
cells 2 (Fang et al., 2020). Finally, there is in vitro and in vivo evidence that
shows that MSC-EVs reduce neuroinflammation thanks to the decrease in the
synthesis of pro-inflammatory mediators (Drommelschmidt et al., 2017;
Thomi et al., 2019).
Application of MSC-EVs in Macrophage Polarization

Despite the diversity of cargos that MSC-EVs possess, in most cases their
immunomodulatory capacity has been linked to direct action on macrophages
diminishing their activation. Thus, in vitro studies suggest that MSC-EVs
reduce inflammation and promote tissue repair due to the M1 macrophage
polarization (pro-inflammatory) to M2 phenotype (anti-inflammatory) (Lo
Sicco et al., 2017). In a mouse model of abdominal aortic aneurysm, MSC-
EVs were found to decrease inflammatory cell infiltration and pro-
inflammatory cytokine production by macrophages (Spinosa et al., 2018). In
vivo experiments in an osteochondral defect model have shown that MSC-
derived exosomes increase tissue repair due to M2 macrophage infiltration and
a reduction in the M1 macrophage population with the consequent less
production of proinflammatory cytokines IL-1β and TNF-α (Zhang et al.,
2018). Similarly, intranasal administration of MSC-EVs in a model of
Alzheimer's disease reduced inflammation and showed a neuroprotective
effect related to the polarization of microglia towards an anti-inflammatory
phenotype characterized by the inhibition in the synthesis of IL-1β and IL-6,

as well as restoration of IL-10 production (Losurdo et al., 2020).
Protective effect of MSC-EVs by macrophage polarization has been also
observed in many pathologic models such as myocardial ischemia-perfusion,
bronchopulmonary dysplasia, acute respiratory distress syndrome, colonic
inflammation, chronic inflammation, and peripheral neuropathy (Zhao et al.,
2019; Willis et al., 2018; Morrison et al., 2017; Liu et al., 2019; Ti et al., 2015;
Fan et al., 2019). Interestingly, a similar effect has also been observed in
monocyte polarization towards an anti-inflammatory phenotype in a
pulmonary fibrosis model (Mansouri et al., 2019). Finally, it is important to
mention that MSC-derived exosomes increase the effector functions and
lifespan of other innate cells such as neutrophils. Thus, they promoted
proliferation, functionality, and lifespan in neutrophils of patients with severe
congenital neutropenia, which may be a strategy to reduce the incidence of
infections in these people (Mahmoudi et al., 2019).
Application of Non-MSC EVs and EVs-Educated Innate Cells

in Tissue Repair
In addition to the ability of EVs to regulate the inflammatory response, they

also promote tissue regeneration through various mechanisms that involve the
innate immune cells. An alternative strategy to the use of EVs in tissue repair
was proposed by Chamberlain et al., (2019). The authors treated macrophages
with MSC-EVs obtaining cells with an M2 phenotype, denominated EVs-
educated macrophages, which were then injected into a murine model of
Achilles tendon rupture. Interestingly, the EVs-educated macrophages
promoted tendon healing showing a more effective effect compared to the
inoculation of MSC-EVs (Chamberlain et al., 2019). Unfortunately, to our
knowledge, no further studies on innate immune cells educated by EVs have
been reported.
On the other hand, the use of EVs derived from other non-MSC cells have
also yielded positive results, opening the possibility of their use as
regenerative therapy. An example is the use of EVs in bone repair. In vitro
experiments have shown that EVs released by DCs recruit bone marrow
derived-MSC to initiate the regeneration process (Silva et al., 2017).
Moreover, the inoculation of EVs from M0 and M2 macrophages exacerbated
bone regeneration in a rat model due to miRNA transfer (Kang et al., 2020).
Amnion epithelial cell-derived exosomes also have regenerative capacity in a
lung fibrosis model showing a reduction in fibrosis associated with a reduction

in myeloperoxidase activity by neutrophils, as well as a reduction in cellular
infiltrate (Tan et al., 2018). Repair after an acute infarction is also stimulated
by the inoculation of EVs from stem cell-derived cardiovascular progenitors
due to the effect they have on innate immune cells, reducing the presence of
inflammatory macrophages, monocytes and neutrophils, as well as causing a
decrease in the synthesis of IL-1α, IL-2, and IL-6 and an increase in the
production of IL-10 (Lima Correa et al., 2021).
Application of Pathogen-Derived EVs for Immunotherapy
Although most studies on pathogen-derived EVs have focused on establishing

their relationship with pathogenicity or their role in immune evasion that
favors the establishment of the infectious agent (as described above in point
4), their immunomodulatory properties have also open the possibility of its use
for therapeutic purposes. For example, EVs derived from the nematode
parasite Trichinella spiralis (T. spiralis-EVs) have been used to reduce the
pathology associated with 2,4,6-trinitrobenzene sulfonic acid-induced colitis
in a murine model. Inoculation of T. spiralis-EVs reduced inflammation by
decreasing cellular infiltrate, the production of Th1 cytokines (IL-1β, TNF-α,
IFN-γ, IL-17A) and increasing the synthesis of Th2 cytokines (IL-10, TGF-β,
IL-4) (Yang et al., 2020). Similar results have been reported by other authors
indicating that, in addition to changes in cytokine synthesis, T. spiralis-EVs
prevent the development of colitis due to the inhibition of macrophage
polarization towards an M1 phenotype and favoring recruitment of M2
macrophages (Gao et al., 2021). In another study, EVs derived from N.
brasiliensis (Nb-EVs) have also shown effectiveness in reducing the
inflammation observed in murine chemical colitis, because the intraperitoneal
inoculation of Nb-EVs prevented production of pro-inflammatory cytokines
and increased the synthesis of IL-10 (Eichenberger et al., 2018). In another
case, exosomes secreted by H. polygyrus have been successfully used to
prevent the inflammatory response associated with allergy to Alternaria
fungus in a murine model by suppressing the eosinophilia associated with this
pathology, confirming the importance of helminth EVs as therapeutic agents
(Buck et al., 2014). But it is not only the EVs of helminthic parasites that have
been shown to have anti-inflammatory properties. The EVs derived from M.
tuberculosis have shown efficiency as immunomodulatory agents since the
subcutaneous inoculation of these in a murine model increases the synthesis
of IL-10 without affecting the synthesis of IFN-γ, positioning them as a

powerful anti-inflammatory therapy (Tavassol et al., 2020).
Another therapeutic effect of EVs derived from microorganisms is their
ability to prevent infections, that is, they serve as prophylactic agents.
Lactobacilli-derived EVs prevented HIV virus infection in ex vivo tissue and
susceptible cells by reducing the attachment of virions to cells, which makes
these EVs a possible alternative to reduce the transmission of this virus (Ñahui
Palomino et al., 2019). Finally, the EVs secreted by pathogens have served as
vaccines, since they limit the establishment of pathogens through the
production of protective antibodies. Some examples are the EVs from
Opisthorchis viverrini, T. muris and S. aureus (Shears et al., 2018; Wang et
al., 2018; Chaiyadet et al., 2019).
Conclusion
The field of EVs has become one of the most impressive in cell biology of our
time. The fact that chemical and immune mediators are specifically loaded
into membranous structures and through them travel between cells and tissues,
implies that the cells of the organism can communicate with each other, or
with the cells of other organisms, sharing information necessary for the
maintenance of homeostasis in steady state and disease. Immunology is one
of the areas where EVs have had the greatest impact. These messengers induce
many effects on immune cells including activation, differentiation, and
proliferation. EVs produced during the steady state regulate different
checkpoints in the immune system, either by avoiding unnecessary leukocytes
activation, or eliminating potentially harmful cells (such as cancer).
Inasmuch as EVs are released by all tissues and are found in practically
all body fluids, it is to be expected that their presence at infection sites play a
very important role in the outcome of infections. In the first instance, pathogen
EVs are important players in promoting virus and bacterial spreading, parasite
differentiation, and in general, contributing to pathology in the infectious
diseases by delivering virulent factors, evading the immune system by hiding
antigens and PAMPs from the humoral and cellular PRRS, and transferring
molecules that have a detrimental effect, downregulating the host´s innate
immunity. On the other hand, host EVs contribute to activate the host immune
response by triggering immune pathways in innate cells. However, EVs from
the host can be manipulated by pathogens in their favor, and EVs from
pathogens can activate the immune system. The outcome will depend on the
combination of a multitude of factors, where the EVs are of the main actors.
The use of EVs for therapeutic purposes implies a series of great
advantages over other therapies. Firstly, EVs are relatively easy to obtain,
secondly, by their nature, chemical mediators are protected and stable within
these structures, and thirdly, they can specifically and directly deliver the
mediators into the target cells of interest. Since EVs has a great
immunomodulatory capacity, a wide window of opportunities has been
opened for the treatment of tissue damage that accompanies inflammatory
processes. Therapeutic applications have focused on the utility of MSC-EVs
as direct promoters of anti-inflammatory states or selectively polarizing
macrophages towards phenotypes of anti-inflammatory nature. Thus, MSC-
EVs are close to becoming an alternative to the immunomodulatory drugs
currently available. On the other hand, the possibility of using pathogen EVs
as a prophylactic tool to prevent infectious diseases by using EVs as vaccines
has also drawn great attention.
Although not comprehensive, the evidence highlights the complex
interaction of tumor-derived EVs and the components of the innate immune
system. While in general the effects of these EVs tend to favor tumor
progression and dissemination, in some instances it can elicit or boost an anti-
tumor response. Additionally, the potential diagnostic and therapeutic
applications of tumor-derived EVs in various types of cancer are very
promising. Since tumor EVs are found in various biological fluids (including
blood, saliva, and urine), these liquid samples can be used for cancer detection,
staging, and monitoring. “Liquid biopsy” has emerged recently as a
noninvasive, convenient approach for cancer diagnosis (Li et al., 2017).
However, their use as cancer biomarkers is still in its infancy, and its wide
application depends on the development of suitable isolation and
characterization methods and their clinical validation. Beyond their diagnostic
applications, tumor EVs can be used in cancer therapeutics. For instance, the
use of modified EVs as drug delivery carriers loaded with anti-tumor drugs or
tumor-targeting RNAi as well as immunogenic agents against cancer are under
research. In a way, it is inspiring that TD-EVs, often used by tumor cells as a
strategy to survive, could offer the key for cancer cure.
Acknowledgments
This research was funded by Programa de Apoyo a Proyectos de Investigación

e Innovación Tecnológica (PAPIIT-UNAM), grant number IN208020 (to
J.C.C.). César Díaz-Godínez is a student of the Programa de Ciencias
Bioquímicas, UNAM, and is a recipient of a scholarship from Consejo
Nacional de Ciencia y Tecnología (CONACyT), Mexico (742064).
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Chapter 6
Enhanced Innate Immune Response:

A Novel Strategy to Interrupt
the Transmission of Diseases by Mosquitoes
Jorge Cime-Castillo, Fabiola Claudio-Piedras,

Valeria Vargas-Ponce de León
and Humberto Lanz-Mendoza
Centro de Investigaciones Sobre Enfermedades Infecciosas,
Instituto Nacional de Salud Pública, Cuernavaca, Mexico
Abstract
The immune system is an interweaving of molecules, signaling

pathways, transcription, and modulation of effectors whose purpose is to
control, mitigate or eradicate what is not proper. In vector-borne diseases
mosquitoes, the innate immune system has been characterized as highly
efficient in combating foreign organisms but sufficiently benevolent to
tolerate vector-borne diseases. These implications lead to replication,
dissemination, and ultimately the transmission of the pathogenic
organisms when feeding on a host. In recent years, it has been discovered
that the innate immune response of insects, including mosquitoes, can
trigger an enhanced immune response to the stimulus of a pathogen that
it previously had an encounter. This phenomenon has been called
immune priming. This defense mechanism allows effector molecules of
the immune response to be alert and act efficiently as an immune boost,

Corresponding Author’s E-mail: humberto@insp.mx.

ISBN: 978-1-68507-510-1
236 Jorge Cime-Castillo, Fabiola Claudio-Piedras et al.
which has earned it the name of "innate immune memory." In this

chapter, we will describe immune priming and its implications as an
enhancer of the immune response to control diseases transmitted by
mosquitoes, vectors of diseases of medical importance, and reduce the
tolerance of mosquitoes to the main organisms that they transmit.
Likewise, we emphasized its use as a possible tool for protecting
mosquitoes against a second encounter with a pathogen. The differences
between immune priming, classical immune memory, and the finding of
trained immunity in vertebrates will be put into context.
Keywords: cancer, inflammation, metastasis, tumor-associated neutrophil,

elastase, neutrophil extracellular traps, immunotherapy, Priming, vector-
borne-disease, mosquito, innate immune, immune memory, trained
immunity
Introduction
Vector-Borne Diseases
Among the diseases transmitted by vectors, those transmitted by mosquitoes

are the most important worldwide. Due to their mortality rate and associated
morbidity, malaria and dengue stand out for putting a high percentage of the
population at risk worldwide and by the number of deaths caused, without
neglecting the outbreaks of Zika, Chikungunya, Yellow fever, Mayaro, West
Nile, among other diseases. Data from the World Health Organization show
that in 2019 there were an estimated 229 million cases of malaria worldwide,
with approximately 409 thousand deaths, with children under five years of age
being the most affected population sector with 67% of deaths from malaria
worldwide (WHO). Dengue cases have increased considerably in recent years.
It is estimated that 390 million infections occur worldwide with an estimated
prevalence of 3.9 billion people (WHO), reported dengue cases exceeded
505,430 cases in the year 2000 to more than 2.4 million in 2010 and 4.2 million
in 2019 with death cases from 960 in 2000 to 4032 in 2015 (WHO). Currently,
the WHO has classified dengue as a neglected tropical disease since it has
received little attention and is delayed in public health priorities, infections are
associated with poor housing and sanitation conditions, and malaria, children,
are the most affected vulnerable group. Given the current panorama of cases,
coupled with the gradual development of an efficient and effective vaccine for
the prevention of vector-borne diseases, for years, the strategy to mitigate the
Enhanced Innate Immune Response 237
growing number of cases have been vector control through insecticides,

larvicides, and more recently with the use of biological control (Wolbachia)
or with the use of genetically modified, sterile organisms or a combination of
methods, with promising results.
Recently, the economic costs of the biological invasion of certain
organisms have been evaluated, being invertebrates the ones that generate the
most significant expenses in financial resources with a cumulative cost of
US$416 billion and a mean annual cost of US$8.7 billion from 1970 to 2017,
which is estimated to increase up to US$23.8 billion per year in 2017
With the figures mentioned above, it is not so surprising to find that the
taxa that generate the most significant cumulative damage at the cost level of
the health impact are Aedes aegypti, the primary vector of dengue, zika, and
chikungunya, with an incremental cost of almost US$150 billion (Diagne et
al. 2021).
Blood Feeding
Of the approximately 3,500 species of mosquitoes integrated into 39 genera,

only three stand out for their importance and medical and veterinary relevance,
Aedes, Anopheles, and Culex, for being responsible for the worst vector-borne
disease. Due to his imperative need for hematophagy to continue his
gonotrophic cycle, these mosquitoes acquire the pathogens of the blood that
they feed, transmitting it in a new feeding to another vertebrate. Among
mosquitoes, there are specific domestic and peri-domestic species that prefer
to feed on human blood; such is the case of Ae. aegypti, An. gambiae, An
albimanus, Culex quinquefasciatus, and to a lesser degree in preference to
humans Ae. albopictus, Ae. japonicus, An. darlingi between others. The
contact rate between mosquitoes and their hosts results from a complex
sequence of mosquito behaviors, including flight activation, attraction,
landing, and probing (Lehane 2005). It has been documented that the
preference for human blood is tightly linked to an increase in the expression
and ligand sensitivity of the odorant receptor AaegOr4. This molecule may
help mosquitoes distinguish humans from non-human animals by increasing
behavioral sensitivity to the signature human odorant sulconate and
geranylacetone (McBride et al. 2014). It has also been implicated like
attractive compounds to carbon dioxide with differential effects on Ae. aegypti
activation rate (Dekker and Cardé 2011); lactic acid which is present until five
times higher in samples from humans than in other mammals (Dekker, Geier,
and Cardé 2005), being attractive in isolation to Ae. aegypti or blend with CO2
in the case of An. gambiae or with octanoic acid for Ae. albopictus. Other
attractant compounds identified are ammonia; carboxylic acids, and
particularly fatty acids, the relative proportions of aldehydes such as nonanal,
decanal, or octanal, which are regularly found in volatile skin extracts, have
also been proposed to play a key role in the attraction of anthropophilic
mosquitoes (review in Dormont et al. 2021). Behavioral assays and
electrophysiological experiments have shown that mosquitoes, predominantly
Anopheles and Aedes, can also distinguish between odors emanating from
different people. Interestingly, bacteria living on the human skin also
contributes in the mosquito-human attraction, as well as, has been documented
that mosquitoes Anopheles infected with Plasmodium or Aedes with dengue
virus are more persistent at biting and to feed more frequently than non-
infected mosquitoes (review in detail in Martinez et al. 2020).
On the other hand, it has been seen that mosquitoes infected with
Plasmodium or dengue virus can modify feeding behavior in response to
olfactory attractants, an effect in which the immunity of the insect mounted by
the infection of the pathogen may be involved (Cator et al. 2012; 2013; Platt
et al. 1997). As a whole, the combination between the components that
emanate from the food source and the mosquito's response to these attractants
converge in the feeding preference, being marked in some particular species
whose preference is channeled to obtain human blood. Once the mosquito
detects its target, a silent, cautious, and camouflage behavior helps feed
copiously.
After recognition and preference for human blood, the mosquito lands on
the skin (preferably above the ankles) and tries to feed on several occasions,
spilling saliva and with it a variety of pharmacologically active molecules that
will prevent the blood from clotting (inhibiting some coagulation factor,
platelet aggregation or some vasoactive amino component or a multiple
inhibition of these pathways. These compounds modulate vertebrate
hemostasis, immunity, and inflammation during the insect's feeding process.
In this way, they are managing to ingest food that will help with the
vitellogenesis and development of the eggs. If this person is infected with
some pathogen (for example, Plasmodium or some flavivirus of medical
importance) this is acquired with blood ingestion. Therefore, the rate at which
mosquitoes encounter and bite humans is a key determinant of their capacity
for diseases transmission.
Interaction with Pathogens and Immune Response Pathways
Among the main mosquito-borne viruses, flaviviruses (Dengue, Zika, and

West Nile viruses) and alphaviruses (Chikungunya viruses) are most
important. It is known that each of them has a different extrinsic incubation
period (period of time in which the virus is in the host mosquito) reaching the
central mosquito tissues at different times. The path of entry and transmission
is common between them. First, the virus is taken from an infected vertebrate
and the food bolus. Later, it reaches the intestine, facing the first immunity
barrier mounted by the mosquito. Once it leaves the intestine, it spreads to the
hemocele, infecting tracheolae, fatty tissue, Malpighian tubules, and finally,
the salivary glands. In this tissue, it has been documented that there is a second
period of viral replication. Vira particles accumulate to be inoculated together
with the saliva in the second period of the mosquito's feeding.
During their passage through the mosquito, pathogens face the insect's
immune response. Which is made up of various response pathways, the Janus
kinase/signal transducer (JAK-STAT), the Toll pathway, the immune
deficiency (IMD), and RNA interference pathways. The last is the main
antiviral route in mosquitoes and is separated into the small interfering RNA
(siRNA); the micro RNA (miRNA) and the P-element-induced wimpy testis
(PIWI)-interacting RNA (piRNA) pathway. PIWI pathway has been
implicated in the protection of immunity in Drosophila and mosquitoes
against the encounter with the same pathogen (detailed later) in a phenomenon
known as immune priming. Although the mechanisms of this immune priming
are not yet well characterized, there are recent findings that glimpse the way
in which a mosquito can respond in a better way to the encounter with a
previously recognized pathogen, in a manner of innate immune memory.
Immune Memory
Immune memory has been defined as the enhanced protection resulting from
past experiences with pathogens (Kurtz 2005). This definition includes
experimental procedures in homologous and heterologous infections in both
vertebrates and invertebrates. We refer to the phenomenon of immune priming
in mosquitoes analogous to immune memory in vertebrates. A previous
stimulus capable of inducing better protection of the host in terms of an
immune response, removing or reducing pathogens, in turn, increases the
survival of the host after a second exposure of the pathogen, which can occur
within and between generations (Little and Kraaijeveld 2004; Little,

Hultmark, and Read 2005). It has been suggested that at least three main
components must be considered, as immune memory: (i) specific immune
resistance, (ii) long-lasting and prolonged protection, and (iii) a biphasic
immune response (Figure 1) (Kurtz 2005; Schmid-Hempel 2011). However,
recent studies have shown that these characteristics are not always met.
In the first component, the specificity of host immune memory can
distinguish between genetically related pathogens and pathogens that are not
related (Kurtz and Franz 2003). These scenarios reveal that the immune
memory may be specific if the host is confronted twice with immune
challenges homologs. However, one issue that has not been tested is the effect
of a heterologous challenge since, under natural conditions, species face
different immune challenges with pathogens (Schmid-Hempel 2011). On the
other hand, specificity can be an expensive attribute for mosquitoes.
Therefore, some researchers have proposed a nonspecific or generalized
immune memory; however, this idea is under discussion. Concerning the
second component, long-lasting improved resistance, it has been shown that
an improved immune response can persist throughout life in animals
previously exposed to a pathogen, thus covering different stages of
development. Concerning immune memory through the generations, its effect
should protect offspring in terms of the immune response, either in eggs,
larvae, or adults. However, a critical question that has not yet resolved is how
and where information from a prolonged protection is stored over a previous
immune challenge. Finally, the third component is the biphasic response,
which required as a functional mechanism in order to be consider immune
memory, analogous to immune memory in vertebrates. Although the biphasic
immune response can occur both in homologs such as heterologous
(nonspecific), challengers only in the homologous are stronger and/o faster
(Figure 1). Currently, few studies demonstrate a biphasic immune response
during immune memory in mosquitoes (Contreras-Garduño et al. 2015;
Vargas et al. 2016). This biphasic effect suggests the existence of a memory
mechanism associated with subsequent exposures to a given pathogen.
However, more studies are necessary to determine whether the biphasic
response is maintained through generations.
If the specificity, long-lasting protection, and biphasic response are
confirmed in the mosquito, its immune memory could be considered
analogous to the immune memory of vertebrates. If all scenarios do not coexist
in a single species, we must ask, for example, how biotic or abiotic factors
determine the outcome of immune memory. It is very important to note that it
is necessary to show an optimal response against the pathogen in question

(Viney, Riley, and Buchanan 2005). Depending on their ecological
circumstances and physiology, immune memory can become a cost for some
species, favoring another type of immune response, such as a sustained
response, tolerance, or non-immunological barriers.
Figure 1. Biphasic and persistent model of immune memory in invertebrates.
Evidence of Immune Memory in Mosquitos
The cellular immune response, particularly in phagocytosis, appears to play

an important role in immune memory (Pham et al. 2007). The parasite
Plasmodium berghei induces an immune response in the mosquito An.
gambiae. The invasion of the mosquito's midgut due to Plasmodium ookinetes
alters the barriers that normally prevent the gut microbiota from making direct
contact with epithelial cells. Microbiota triggers a long-lasting response
characterized by increasing abundance of granulocytes and increased
immunity to bacteria, reducing the survival of reinfected Plasmodium
parasites. It has been suggested that differentiation of hemocytes was
sufficient to generate an exacerbated immune response against P. berghei
(Rodrigues et al. 2010). However, without evidence of specific protection, nor
of long duration and lacking a biphasic response, it cannot be excluded the
possibility that the bacteria themselves and not the immune memory are
responsible for the elimination of pathogens. On the other hand, it has not been
identified which are the molecules and genes of hemocytes that are modified
during immune memory.
In the humoral response, several effectors in immune memory include
antimicrobial peptides, reactive oxygen and nitrogen species, lysozymes, etc.
In the case of the mosquito Ae. aegypti, there are few works that have
demonstrated immune memory. Vargas et al. 2016, showed increased
expression of antimicrobial peptides (Attacin, Cecropine, Defensin) and an
increase in survival in mosquitoes previously infected with E. coli compared
to the control group. In addition, a limited specificity was shown since the
immune memory was specific for immune challenges homologous with E. coli
and nonspecific in heterologous infections with Staphyloccocus aureus.
Likewise, it has been demonstrated immune memory against E. coli in the
adult state of these mosquitoes, reporting a decrease in the mortality rate and
an increase in the activity of phenoloxidase and nitric oxide in individuals who
had the first infection with E. coli compared to the control group (Moreno-
Garcia, Lanz-Mendoza, and Córdoba-Aguilar 2010).
Possible Mechanism of Immune Memory
Endoreplication
Some researchers suggest cell proliferation as a mechanism of immune

memory; however, its involvement has not been proven so far. On the other
hand, cell proliferation can be expensive for insects because it requires a large
amount of energy and resources for their process. Endoreplication is a process
that requires little energy for the insect because multiple copies of the genome
or amplicons can be made in hemocytes or cells of different tissues without
the need to enter into mitosis. In primary cell cultures of several tissues of An.
albimanus, intensive DNA synthesis has been reported in response to
inoculation with Saccharomyces cerevisiae (Hernández-Martínez, Salvador;
Román-Martínez, U; Martínez-Barnetche, J; Garrido, E; Rodríguez, M.H.;
Lanz-Mendoza 2006). DNA synthesis and the forming of polytene
chromosomes could be a mechanism for increasing genes' activity to
synthesize large amounts of proteins involved in the insect's immune response,
leading to an increase in the number of sequences available for transcription
(Contreras-Garduño et al. 2015). This DNA synthesis has been observed in
epithelial cells that may be in contact with pathogens such as the intestinal
epithelium and pleural membrane. During the immune memory of An.
albimanus, increased expression of the hnt gene has been demonstrated. This
gene has been involved in changes of cell cycle mitosis in the Drosophila
melanogaster endocycle (Sun and Deng 2007). The presence of the hnt gene
has been determined in Ae. aegypti challenged with inactive dengue virus
(DENV) showing a lower viral load upon a second oral DENV challenge.
Interestingly, priming with inactive DENV can induce DNA synthesis levels
comparable to the control group in this study. Likewise, hnt and delta-notch
relative expression levels are higher in priming mosquitoes than in control
(Serrato-Salas et al. 2018). In An. albimanus cell line LSB -AA695BB, a
decrease in cell proliferation and activation of the hnt gene after immune
memory against Plasmodium or zymosan was observed; as well as an increase
in DNA synthesis and an increase in the number of copies of phenoloxidase
and TEP genes, two crucial immune response molecules of the mosquito in
the presence of Plasmodium. This information suggests that DNA synthesis
by endoreplication can generate copies of genes that can be quickly
transcribed to respond to the presence of the antigen in a second challenge
(Cime-Castillo et al. 2018).
iRNA and Endogenous Viral Elements (EVEs)
As far as is known, somatic diversification processes produce a repertoire of

receptors without direct induction against the antigen. However, interfering
RNA (RNAi) participates in resistance against viruses, providing sequence-
specific immunity directed by the nucleotide sequence of a pathogen. The
three main pathways of small RNA (RNAs) driven RNA silencing in
mosquitoes are siRNA, microRNA (miRNA), and PIWI-interacting RNA
(piRNA) pathways (Mukherjee et al. 2019). siRNAs are a potent antiviral
mechanism against RNA and DNA viruses, from single-stranded RNA
(ssRNA), double-stranded (dsRNA), single-stranded and double-stranded
DNA (Mukherjee et al. 2019). This pathway begins with the recognition of
dsRNA of viral origin in the cytoplasm by the enzyme RNase III Dicer-2
(Dcr2) (Palmer, Varghese, and Van Rij 2018); the dsRNA is then cleaved by
Dcr2 into siRNA duplexes of approximately 21 nucleotides (Samuel,
Adelman, and Myles 2018). One strand of the siRNA duplex is incorporated
into the RNA-induced silencing complex (RISC), while the other strand is
degraded (Rand et al. 2005). Subsequently, the guide strand is methylated at
the 3 ' end by the methyltransferase DmHen1 to form a mature RISC
(Förstemann et al. 2007). Argonaute-2 (Ago2) is the catalytic core of RISC,
so that Ago2 binds to the guide strand within the protein complex through
interactions between the 3' and 5' ends of the guide strand (Chen et al. 2011).
siRNAs bound to Ago2 guide RISC to complementary RNAs in the cytoplasm
in a sequence-specific manner through nitrogen base pairing. The antiviral
nature of the siRNA pathway is clearly demonstrated by the finding that
inactivation of Dcr2 or Ago2 results in improved virus replication and
increased host mortality in a variety of systems (Van Rij et al. 2006; Poirier et
al. 2018).
In the context of enhanced immune, during RNA virus infections, DNA
molecules are generated, which serve as the origin of siRNA and remain even
after the viral infection has been eliminated, in a type of immune memory
(Goic et al. 2016; Tassetto et al. 2019). Virus-specific siRNA responses are
amplified via the reverse transcription of viral RNA to viral DNA (vDNA)
produced during RNA virus infection. vDNA is present in both linear and
circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that
confer partially protective immunity when challenged with a cognate virus in
a kind of immune priming. cvDNAs bear homology to defective viral genomes
(DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis.
Furthermore, vDNA synthesis is regulated by the DExD/H helicase
domain of Dicer-2 in a mechanism distinct from its role in siRNA generation.
It has been suggested that, analogous to mammalian RIG-I-like receptors,
Dicer-2 functions like a pattern recognition receptor for DVGs to modulate
antiviral immunity in insects (Poirier et al. 2018). Prevention of vDNA
formation during virus infection in Ae. albopictus, decreases the abundance of
virus-specific siRNA and increases insect mortality.
Interestingly, cvDNAs can be integrated into the host genome in the form
of EVEs during virus replication using transposons not yet described. In this
way, favor the immune response through the piRNA pathway. It is
hypothesized that EVEs could provide the determinants of the specificity of a
long-lived similar, and inherited nucleic acid silencing system. In support of
this, EVEs in Ae. aegypti and Ae. albopictus were found to enrich within
piRNA pools and give rise to piRNA (Palatini et al. 2017; Whitfield et al.
2017). It has even been seen that this mechanism in Ae. aegypti transmit
antiviral immunological memory to their progeny throughout generations
(Mondotte et al. 2020).
Finally, since RNAi is an evolutionarily ancient and conserved
mechanism, it is possible that it is involved in the process of immune memory
in invertebrates.
Signaling Pathways
The immune systems of invertebrates are very diverse, consisting of

evolutionary components that preserve specific parts of each taxon. Therefore,
immune memory can be explained by evolutionary elements held in the
invertebrates' immune system. For example, the Toll and IMD and JAK/STAT
signaling pathways allow immunological reactions to be induced and, to some
extent, are also specific to some insects (Lemaitre, Reichhart, and Hoffmann
1997). The Toll pathway is activated by Gram(+) bacteria and fungi. In
contrast, the IMD pathway is activated by Gram(-) bacteria (Lemaitre and
Hoffmann 2007), resulting in the expression of different antimicrobial
peptides (Gambicin, Cecropine, Dipterisin, Defensin, and Drosomisin). The
activation of the Toll pathway, through the expression of genes encoding
antimicrobial peptides, could be limiting the viral replication of DENV in the
midgut of mosquito Ae. aegypti (Jose L. Ramirez and Dimopoulos 2010).
Therefore, it is suggested that activation of the Toll pathway could be involved
as an immune response mechanism against DENV.
The specific function of the JAK/STAT pathway is believed to be
involved in antiviral activity. In the mosquito Ae. aegypti signaling pathways
intersect each other to give rise to the synthesis of antimicrobial peptides (Xi,
Ramirez, and Dimopoulos 2008).
This ability to distinguish between different pathogens is based on a set
of pathogen recognition receptors (PRRs), such as different variants of
peptidoglycan recognition proteins (PGRPs), which are specific to pathogen-
associated molecular patterns (PAMPs) that are shared between different types
of pathogens. It should be noted that other insect species may show different
associations of PGRPs with Toll and IMD signaling pathways and, therefore,
different activation patterns.
Once activated, the Toll and IMD signaling pathways can produce
different molecules affecting the immune response, such as reactive oxygen
species (ROS), melanization made through the enzymatic cascade of
profenoloxidase and antimicrobial peptides. Overexpression of Attacin,
Gambicine, and Cecropine has been reported in the An. albimanus mosquito
after a second immune challenge with P. bergei and under a biphasic effect
similar to that seen in immune memory in vertebrates (Contreras-Garduño et
al. 2015). The biphasic effect on Atacina, Defensin and Cecropine was also
observed in the Ae. aegypti mosquito against E. coli; however, an immune
memory with limited specificity was shown (Vargas et al. 2016). Notably, it
isn't easy to imagine that antimicrobial peptides are effectors of a specific
response since they act against many pathogens. The localized activation may
contribute to the specific response of these effect molecules (Pham and
Schneider 2008). However, it is suggested that a synergistic response between
combining recognition molecules with immune effectors in a specific immune
challenge could improve diversity to combat pathogens during immunization
(Schulenburg, Boehnisch, and Michiels 2007; Kurtz and Armitage 2006).
Dscam
Vertebrate immune memory depends on the somatic recombination

mechanisms of the immune receptors of T and B lymphocytes. Lymphocytes
undergo clonal proliferation and selection after antigen-specific recognition.
The invertebrates lack mechanisms of somatic recombination; however, there
are some receptor systems that also have the potential to develop somatic
diversification processes (Schulenburg, Boehnisch, and Michiels 2007). The
most prominent examples are the Down syndrome cell adhesion molecule
(Dscam) in arthropods, which is probably the most debated candidate for
specificity in the immune memory of mosquitoes, because this receptor is
somatically diversified by alternatively splicing. Such diversification can
produce a set of combinations of up to 31,920 extracellular isoforms in An.
gambiae, conferring an additional mechanism of diversity that allows specific
recognition and probably protection against pathogens (Dong et al. 2012;
Dong, Taylor, and Dimopoulos 2006).
Moreover, the IMD and Toll signaling pathways appear to regulate
variants of Dscam alternative splicing. However, this would not improve the
degree of specificity beyond the arbitrary capacity of these two signaling
pathways. At the same time, the considerable number of variants due to
Dscam's alternative splicing could theoretically be more specific. These
findings may support the theory of a mechanism for generating diversity in
invertebrates (Cooper and Alder 2006). In An. gambiae Dscam diversity
increase with P. falciparum exposure. However, higher Dscam expression
diversity was not observed (P. H. Smith et al. 2011); therefore, so far, there is
not enough experimental evidence to support Dscam's involvement in immune
memory.
The unchanged patterns in splicing in pathogen infections do not exclude
that Dscam splicing variants do not serve as pathogen-specific receptors. The
different individual splicing variants could lead to a great diversity of
receptors expressed in the population. This observation resembles receptors
that diversify in the germline, but the selection pressure would be different for
polymorphic receptors compared to receptors by alternative splicing
(Milutinović and Kurtz 2016). It is believed that such receptors can be released
or secreted either in the hemolymph or exposed on the surface of the
hemocytes for subsequent recognition of the antigen, similar to an anticipatory
response. However, it is unknown if there is an increase in the number of
copies of the messenger RNA or the gene to generate more Dscam molecules,
and much less is known to be over-expressed after a second challenge.
Lipoxin/Lipocaline Complex
Although the immune response appears to be sustained and does not adapt to
a biphasic memory response, the complex lipoxin/lipocalin favors an increase
in the immune response. It has been established that when Plasmodium bergei
invades the midgut of An. gambiae mosquito, it allows contact of the intestinal
microbiota with epithelial cells (Rodrigues et al. 2010). This event induces the
systemic release of a hemocyte differentiation factor (HDF) consisting of
Lipoxin A4 bound to Evokin (a lipid carrier of the lipocalin family), increasing
the proportion of circulating hemocytes in the hemolymph (Ramirez et al.
2015). As well as, the mosquito midgut epithelial cells produce and release
prostaglandin E2 (PGE2), which attracts hemocytes to the midgut surface and
enhances antiplasmodial immunity by triggering HDF production (Ana
Beatriz F. Barletta et al. 2019). In this context, the role of prostaglandins has
also been associated with a decrease in components of the Toll pathway and
IMD, this when the synthesis of PGs is decreased, increasing the replication
of the dengue virus in Ae. aegypti intestines (Ana Beatriz Ferreira Barletta et
al. 2020).
Fibrinogen-Related Proteins (FREPs)
Alternative splicing can generate somatic diversity in invertebrates at immune

receptors, such as FREPs (S. M. Zhang et al. 2004). FREPs have been
proposed as immune receptors that participate in the immune memory of
invertebrates since it has been shown that these molecules containing the
domain of immunoglobulin superfamilies and that can be induced in snails
after infection by trematodes, in addition to being able to diversify somatically
(Hanington et al. 2010). In the mosquito An. gambiae were found 59 putative
members of FREPs genes (Dong and Dimopoulos 2009). Like Dscam, FREPs
are unknown if there is an increase in the number of copies of genes to generate
more FREPS molecules after a second immune challenge.
Classical Memory Immune Response and Its Differences

with Immune Priming in Mosquitoes
As described above, it has been consistently stated that the immunological

memory must possess the following features: (i) specific immune resistance,
(ii) long-lasting and prolonged protection, and (iii) a biphasic immune
response. The changes observed in immune reactivity to specific stimuli, and
the persistence of these adjustments that protect against disease throughout the
individual's life in the absence of antigen, have been the guidelines for
studying the immune memory. Despite the above, we have witnessed an
increasing number of reports in which these features are not always fulfilled
in recent years. While the cellular basis of classical immune memory resides
in T and B cells, recent evidence on the extension of memory to cell types of
the innate immunity such as monocytes, macrophages, dendritic cells and NK
cells (Beaulieu, Madera, and Sun 2015; Dominguez-Andres and Netea 2019;
Domínguez-Andrés and Netea 2020; Farber et al. 2016; Hamada et al. 2019;
Peng and Tian 2017; Xing et al. 2020) opens the possibility to explore the
cellular basis of the memory phenomenon reported in invertebrates. In
conciliation, we consider it necessary to take a step back and revisit the
conceptual basis of cellular memory to understand its fundamental nature. We
have collected the distinctive and convergent features of memory cells in
vertebrates to contrast them with the features observed in circulating mosquito
cells as representative of the invertebrate models.
As a phenomenon, immunological memory has been recognized since the
time of the ancient Greeks, and its conceptualization was first proposed nearly
2500 years ago to explain protection against reinfections (Farber et al. 2016;
Jacob and Baltimore 1999; Zinkernagel 2002). Historically, immunologists
have been fascinated by the immune system's ability to store and recall
information about previous infections, immunizations, or altered self-
components and develop an enhanced immune response against a second
similar challenge. With the progress of technology, it became possible to study
the cellular basis of immunological memory that had remained inaccessible
for more than half a century, when the humoral basis of immune memory
began to be formally investigated.
Immune memory is functionally defined as the event that occurs when an

individual re-encounters an antigen to which somebody was exposed during
an infection or immunization and develops a response that is stronger and
faster than the first encounter, preventing reinfection or significantly reducing
the severity of the clinical disease (Jacob and Baltimore 1999; Zanetti,
Castiglioni, and Ingulli 2010)⁠. This implies the indelible imprinting of the
encounter information at the cellular level and its long-term maintenance,
extending even throughout the individual's life.
In mosquitoes, cell-mediated immunity is mediated by hemocytes,
epithelial and epidermal cells. However, the effector capacities of hemocytes
at the systemic and regional levels are outstanding. Hemocytes are circulating
cells (~75%) and tissue-adherent cells (~25%), adhering primarily around the
heart valves or ostia (periostial hemocytes), intestine, and Malphigian tubules,
followed by the thorax, maxillary head palps, and legs (Hillyer and Strand
2014; King and Hillyer 2013; Sigle and Hillyer 2018). Individual adult
mosquitoes are estimated to contain around 500 to 4000 hemocytes (Hillyer
and Strand 2014)⁠. Adult females of An. gambiae and Ae. aegypti have three
types of hemocytes, distinguishable by morphological and functional markers:
granulocytes, oenocytoids, and prohemocytes (Castillo, Robertson, and Strand
2006; Hillyer and Strand 2014), however, previous and recent evidence
indicates a greater hemocyte population complexity (Araújo et al. 2008;
Hillyer and Christensen 2002; Kwon et al. 2021; Raddi et al. 2020).
Granulocytes are the most abundant cell type (80-95%) (Castillo, Robertson,
and Strand 2006; Hillyer and Strand 2014)⁠, they are polymorphic, with a
developed endoplasmic reticulum, abundant mitochondria and vesicles
(Araújo et al. 2008)⁠, their phagocytosis and melanization response is as fast as
5 min after bacterial inoculation (Hillyer, Schmidt, and Christensen 2003)⁠.
Oenocytoids and prohemocytes represent less than 10% of the hemocyte
population. Oenocytoids have homogeneous cytoplasm, abundant rough
endoplasmic reticulum, small smooth endoplasmic reticulum, and
characteristically express phenoloxidase (Hillyer and Christensen 2002;
Hillyer and Strand 2014). Prohemocytes are the smallest circulating cells (4-6
um in diameter), with large nuclei involved in phagocytosis and with
proliferative capacity (Araújo et al. 2008; Hillyer and Strand 2014; Kwon et
al. 2021).
Activation
Hemocytes mediate the rapid clearance of bacteria (Hillyer, Schmidt, and

Christensen 2003; Sigle and Hillyer 2016; 2018)⁠, viruses (Lamiable et al.
2016; Leite et al. 2021; Nakamoto et al. 2012; Yano et al. 2008)⁠, and parasites
(Hillyer, Schmidt, and Christensen 2003; Raddi et al. 2020; R. C. Smith,
Barillas-Mury, and Jacobs-Lorena 2015) ⁠ due to their high abundance, their
easy localization in infection foci, and their ability to mount effector functions
(Dushay 2009; Eleftherianos et al. 2021; Kim et al. 2020; Yan and Hillyer
2020)⁠. In response to viral and bacterial infections, hemocytes are activated
by Toll-7 or PGRP-LE through the interaction with PAMPs, and can enter
autophagy, restricting viral replication and pathogenesis (Lamiable et al. 2016;
Nakamoto et al. 2012; Yano et al. 2008). Additionally, Ae. aegypti hemocytes
respond to DENV and ZIKV infection by aggregating in the midgut of
mosquitoes and restricting systemic viral shedding (Leite et al. 2021)⁠.
Proliferative capacity. Considerable evidence indicates that hemocytes
can expand and differentiate their populations in Anopheles and Aedes
mosquitoes (Bryant and Michel 2014; King and Hillyer 2013; Kwon et al.
2021; Raddi et al. 2020). These cells are activated within minutes during acute
infections, aggregate. They remain associated with sites of pathogen entry,
switching to a tissue-resident cell phenotype, the location at which their
effector capacity against bacterial infections is manifested (Sigle and Hillyer
2016; 2018; Yan and Hillyer 2020).
Molecular markers. Of the 27 immune-related gene families present in
mosquitoes, hemocytes show high expression of immune transcripts encoding
the Toll pathway, IMD (Immune deficiency pathway), SRRP (Small RNA
regulatory pathway), APHAG (Autophagy), CASPs (caspases), IAP (Inhibitor
of apoptosis), PRDX (Peroxiredoxin), SCR (Scavenger receptor), GALE
(galactoside binding lectins), BGBPs (1,3 beta D glucan binding proteins),
CLIP (Clip domain serine protease), FREPs (Fibrinogen related proteins),
PPO (Prophenol oxidase), SRPNs (Serpins), ML (Myeloid differentiation 2-
related lipid recognition protein), TEP (Thioester protein) and LRR (Leucine
rich repeat), AMPs (antimicrobial peptides), and lysozymes (Kwon et al.
2021; Lombardo et al. 2013; Raddi et al. 2020; Rani et al. 2021).
Until recently, little was known about the epigenetic control of gene
expression in mosquitoes. The understanding of central epigenetic processes
such as nucleic acid methylation, histone modifications, and reorganization of
chromatin structure in mosquitoes remains an area of intense research
(Compton, Sharakhov, and Tu 2020; Gulati et al. 2020; Lezcano et al. 2020;
Lukyanchikova et al. 2020; Sharakhov and Sharakhova 2015)⁠. Although to

date there are no reports of epigenetic regulation in hemocytes, in general,
Anopheles and Aedes mosquitoes have the elements required for chromatin
remodeling, histone acetylation/deacetylation, histone methylation/
demethylation, DNA/RNA methylation, among others; characteristic of the
regulation and maintenance of gene expression profiles, as well as phenotypic
reprogramming (Claudio-Piedras et al. 2020; Gómez-Díaz et al. 2014; Jenkins
and Muskavitch 2015; Lezcano et al. 2020; Lukyanchikova et al. 2020; Ruiz
et al. 2019). In response to viral and parasitic infections, transcription of
epigenetic regulatory elements is increased, indicating a possible involvement
during the mosquito immune response (Claudio-Piedras et al. 2020;
McFarlane et al. 2020; Ruiz et al. 2019; Ye et al. 2013; G. Zhang et al. 2013).
In cells of the innate immunity, the sustained expression profile in memory
monocyte-macrophages is mediated by epigenetic modifications and
chromatin remodeling (Arts et al. 2016; Bekkering et al. 2018; Keating et al.
2020; Netea et al. 2020; Saeed et al. 2014)⁠. Possibly these mechanisms are
conserved in mosquitoes.
Effector Functions
As the principal cells of immunity, hemocytes mediate phagocytosis, lysis,

encapsulation, and melanization, in addition to hemolymph clotting and
wound healing, avoiding dissemination, septicemia and high mortality
(Dushay 2009; Eleftherianos et al. 2021; Kim et al. 2020; Yan and Hillyer
2020). In Anopheles and Aedes mosquitoes, hemocytes aggregate around heart
valves in response to infection, acquiring a function analogous to vertebrate
lymphoid organs, as specialized sites where immune responses are mounted
(Yan and Hillyer 2020)⁠. The hemocytes are an important source of many
humoral molecules. Hemocytes and other cells secrete cytokines or small (~5-
20 kDa) cytokine-like proteins that mediate intercellular communication
between different cell types, promoting hemocyte differentiation and
regulating hematopoiesis, immunity, and an inflammation-like response. The
main organs intercommunicated through the cytokine-receptor system are
hemocyte-to-fat body, fat body-to-hemocyte, muscle-to-hemocyte and gut-to-
fat body (Reviewed in (Eleftherianos et al. 2021). Cytokine/cytokine-like
orthologs and their receptors (cytokine/receptor) are present in Aedes and
Anopheles mosquitoes, and have been extensively studied in Drosophila: (i)
Eiger (eda-like cell death tiger) and its receptor Wengwn: (Ae. aegypti;
AAEL010524/AAEL025438), (An. gambiae; AGAP006771/AGAP000728);

(ii) Pvfs (PDGF- and VEGF-related factor, 1-3) and its receptor Pvr: (Ae.
aegypti; AAEL013840, AAEL007902/AAEL003928), (An. darlingi;
ADAC003613/ADAC000753), (An. gambiae; AGAP000574,
AGAP009549/AGAP008813); (iii) Spätzle and its Toll receptor: (Ae. aegypti;
AAEL013434/AAEL015018), (An. darlingi; ADAC003608/ADAC001785),
(An. gambiae; AGAP000346/AGAP007060); and (iv) Unpaired and its
receptor Domeless (Dome): (Ae. aegypti; AAEL028247/AAEL012471), (An.
darlingi; ADAC100533/ADAC000118), (An. gambiae; AGAP013506/GAP0
29053).
Thoughtfulness for Inducing Immune Memory
Cells destined to become memory cells are the survivors of activation-induced

cell death and undergo lineage differentiation to give rise to two equivalent
subpopulations: effector and central memory cells.
The first consideration is for antigen exposure, which is only necessary
for a short period (20-24 hours) to initiate the cellular effector response and
probably to initiate a cellular developmental "program" (Kalia, Sarkar, and
Ahmed 2010)⁠. Suboptimal stimulation may lead to limited expansion of
memory T cells. In contrast, too much antigen activates a significant fraction
of available precursor cells and may cause their elimination by exhaustion
(Zanetti, Castiglioni, and Ingulli 2010)⁠. In addition, high antigenic stimulation
increases susceptibility to apoptosis. Finally, cells that receive the highest
stimulus magnitude possess the lowest potential to survive and differentiate
into memory cells (Kalia, Sarkar, and Ahmed 2010). This is the case under
conditions of high inflammation (Zanetti, Castiglioni, and Ingulli 2010)⁠. Thus,
the potential of effector cells to differentiate into memory cells progressively
decreases with increasing antigenic stimulation.
The second consideration relates to the criterion of memory maintenance
independently of antigen stimulation or persistence. Reviews of the subject
suggest that the persistence of antigen in the host over a long time increases
the memory cell population and enhances protection (Zanetti 2007). Small
amounts of antigen present by mild infections that are not cleared but
controlled (tolerance) or persisting in follicular dendritic cells in the order of
picograms are sufficient to maintain long-lasting and effective memory
responses (Zanetti 2007). However, prolonged antigenic stimulation leads to
gradual loss of memory cells y telomeric depletion, and erosion (Henson and
Akbar 2010)⁠, impairs long-lasting memory generation potential and drives

cells towards a differentiated effector phenotype (Kalia, Sarkar, and Ahmed
2010).
The third consideration revolves around antigen-independent cell
longevity and is related to the previous consideration. The postulate concerns
the maintenance and turnover of the memory cell pool. The rate of generation
of new memory cells should be equal to the rate of memory cell death in the
absence of antigen. However, antigen-induced sporadic cell division at low
doses can enhance the persistence of cell populations for months and, in some
cases, years (Zanetti 2007; Zanetti, Castiglioni, and Ingulli 2010)⁠.
Zanetti (Zanetti, Castiglioni, and Ingulli 2010)⁠ proposes three basic
principles for the induction of protective memory cell responses about antigen,
co-stimulation, and inflammation.
1. Priming with low antigen dose; prolonged antigen presentation: (i)

increased expansion of resting memory cells after antigen
withdrawal, (ii) increased commitment to central memory cell lineage
generation vs. memory effector cells, and (iii) possible protection
against activation depletion and accelerated senescence.
2. Cell cooperation during priming: (i) cooperation increases the degree
of protection and probably favors differentiation to central memory
cells.
3. Priming avoiding excessive inflammation (TLR agonists, viral
vectors): (i) reduces presenter cell activation by limiting conditions
that favor effector generation and differentiation of the memory
effector cell lineage; (ii) prevents restriction of memory cell
expansion when antigen is removed; (iii) slows replicative
senescence; (iv) regulation of transcription factor which in turn
regulates memory cell potential.
Regarding the influence of the environment on memory generation,

micro-environments that provide a unique repertoire of cytokines, co-
stimulation, accessory immune cells, and antigen persistence have been shown
to influence the programming of cells to acquire different trafficking
properties, implying that particular environmental cues possibly dictate the
memory outcome (Kalia, Sarkar, and Ahmed 2010)⁠.
From the above considerations, we would like to highlight the
appreciation of the need for a balance between the generation of effective
cellular responses that immediately fight infections and the generation of
memory responses that enhance protective immunity against reinfections

(Butler and Harty 2010)⁠. While short-duration antigenic stimulation favors the
generation of central memory cells, longer stimulation promotes the
generation of effector and effector memory cells (Kalia, Sarkar, and Ahmed
2010)⁠.
In addressing the above considerations, we find coincidence with the
resistance/tolerance hypothesis developed in insect immunology, where life-
threatening infections induce potent effector responses that eliminate the
infection. In contrast, other mild infections tend to be tolerated. This strategy
makes sense taking into consideration the reproductive strategy of insects. In
mosquitoes, being short-lived, with high reproduction rates, it might be more
convenient to tolerate a non-threatening infection and allocate the resources to
produce a high amount of offspring than to invest the limited amount of
resources at the cost of spawning less offspring. If the infection is, on the
contrary, life-threatening, then a robust immune response is mounted.
Otherwise, no offspring could be spawned. This may come with the
disadvantage of not generating any memory, which might not be needed. If an
insect successfully dealt with and survived a life-threatening infection, why
wouldn't it survive it again? On the other hand, if the infection is not life-
threatening, it may be tolerated, and a certain memory acquired to generate
enhanced protection against reinfection, stronger and potentially life-
threatening.
In summary, central phenotype cell responses correlate with protective
and long-lasting effects. These cells can develop as a stable lineage under the
following conditions: (i) a range of low precursor frequencies, (ii) under
conditions of low antigen presentation and (iii) with apparently low antigen
dose (Zanetti, Castiglioni, and Ingulli 2010)⁠⁠. The expansion and contraction
phases appear to be programmed by activation and are proportional to the
antigen dose, the strength of antigen presentation, and the antigenic context.
On the other hand, contraction is independent of the magnitude of expansion
(Kalia, Sarkar, and Ahmed 2010)⁠.
Priming and Tolerance to Arboviral Infection
Despite the various immune response strategies that a mosquito uses, viral
infections remain long-term, in a replicative and infectious state, so
mosquitoes tolerate viral infections without any apparent setback. It has been
described that this tolerance to dengue and chikungunya viruses by Aedes
mosquitoes could be due to the viral replicative cycle that generates vDNA
particles, a mechanism not fully described but that would help mosquito
survival and viral tolerance (Goic et al. 2016).
Although the mechanism that governs the priming phenomenon has not
been fully elucidated, in recent years, considerable progress has been made in
explaining it, notwithstanding the mechanism, the final result of increasing the
immune response until a second stimulus has generated interest in the field. It
has been possible to decrease the threshold tolerance phenomenon and obtain
mosquitoes unable to transmit viral particles. The induction of priming in Ae.
aegypti larva with inactive dengue virus results in adult mosquitoes with an
enhanced antiviral-immune response, a reduction in the load and replication
of RNA of dengue virus, and a decline in viral infective particles production
(Vargas, Cime-Castillo, and Lanz-Mendoza 2020). Adult mosquitoes
previously primed during larval stages over-expressed Ago2 and Dcr2. The
mosquitoes primed during larval stages appear to control the infection,
reducing the viral load. The over-expression of interferon-like factors
(VAGO) and Ago2 in the pupa stage suggests a fast activation of antiviral
mechanisms after immune priming in larvae, creating a condition in which
adult mosquitoes are less tolerant to the pathogen in the posterior exposure
(Vargas, Cime-Castillo, and Lanz-Mendoza 2020). Recently has been
hypothesized that reduce mosquito tolerance is more sustainable than increase
resistance to arbovirus infection (Lambrechts and Saleh 2019). This is why
immunological priming could be an excellent strategy for the control of
vector-borne diseases.
Perspective
The phenomenon of immune memory and its possible mechanisms have been
studied in taxa, where there is currently no clear evidence of the phenomenon,
and there are no yet precise mechanisms for many taxa where there is evidence
of immune memory. It is essential to consider that there may be different
mechanisms to produce and conserve immune memory. It is necessary to use
cellular, molecular, and population strategies to establish each group's general
and particular mechanisms or taxon. Insects and mosquitoes can be an
excellent group to address this problem as many are easy to maintain in the
laboratory and can be challenged with natural pathogens. In addition to being
the main vectors of some arboviruses such as DENV, CHIKV, ZIKA, and
parasites such as malaria, it is of great medical importance to know the
mechanisms of the immune response to develop alternative strategies to

control these diseases. However, there is evidence (some clearer than others)
of the possible mechanisms and molecules that could be involved. The
pathways Toll, IMD, Dscam, FREPs receptors of lectin type C give us
palpable ideas of a wide repertoire of receptors of great diversity of the
pathogen recognition, but how it is that they are involved in immune memory,
how they are selected and how this information is stored is unknown. In
addition to these possible mechanisms involved in immune memory,
endoreplication and epigenetic modifications are in the process of
development. They could shortly give us a broader explanation of the
phenomenon of immune memory in invertebrates.
Conclusion
The immunological memory represents a powerful aspect of the immune

response and a key attribute of the immune system for protecting the
individual (acquired memory) and the species (transmissible memory) against
pathogens and other molecules capable of causing damage.
Multiple authors have highlighted immune memory as an evolutionary
adaptation in light of its importance for the survival of individuals and species,
providing individuals with faster and more effective responses against life-
threatening antigens. While immunological memory is acquired throughout an
individual's lifetime, it is notable for its importance in being transmitted to the
offspring during the perinatal and neonatal periods. In general, immunological
memory is transmissible in mammals through the passage of immune factors
(antibodies) through the transplascentary route and the intestinal absorption of
the factors present in colostrum (Zanetti 2007)⁠. In birds, immunological
memory is transmissible through antibodies and in invertebrates, possibly
through cytoplasmic inheritance of various immune effectors (Houri-Zeevi et
al. 2020; Mondotte et al. 2020). With the understanding that the functional
correlation of the anatomical and cellular structures of the mosquito immune
system is still limited, the importance of immunological memory in this group
of animals is best understood in light of its role in the survival of the individual
species.
The view of immunological memory as a phenomenon has broadened and
is now considered to extend from innate to adaptive immune memory, both
being emergent properties of the immune system. Currently, the definition of
immune memory is changing. Although our understanding of the molecular
mechanisms of its generation and maintenance is still limited, the traditional

view of immune memory as represented by individual elements of the immune
system (exclusively by B and T lymphocytes and antibodies) needs to be
reconsidered in light of recent evidence in cells of the innate immunity.
The phenomenon of immunological memory can be studied in divergent
immune systems as an emergent property of the particular system, i.e., as the
product of the interaction of the various immune elements, whose individual
and simple functions together result in complex property. In this chapter, we
urge the reader to appreciate immune memory as a property of the immune
system as a whole, of a gradual nature, complex and multidimensional, which
cannot be explained by the individual study of the elements of the immune
system.
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Appendix: Glossary
 Immune memory: An individual's ability to improve their immune

response against a pathogen, after having witnessed a previous
contact with the immune response activator.
 Transgenerational immune memory: An individual's ability to inherit
or transmit to his offspring a better immune response against a
pathogen, in which the parent has had prior contact with the pathogen
throughout his or her life.
 Immune memory in ontogeny: Ability of an individual to improve
their immune response against a pathogen, having witnessed a
previous contact with the immune response activator in early stages
of host development.
 Homologous immune challenges: Infection of an individual with a
subletal dose of a pathogen and repeat the immune challenge but with
a lethal dose of the same pathogen in the same generation.
 Heterologous immune challenges: Infection of an individual with a
subletal dose of a pathogen and repeat the immune challenge but with
a lethal dose of another pathogen in the same generation.
 Horizontal transmission: Transmission of a pathogen between
members of the same species that does not have a parental
relationship (parent-child).
 Vertical transmission: Transmission of a pathogen between members
of the same species that has a parental relationship (father-child).
 Costs: Commitment suffered by an individual in a given scenario, due

to the efficiency of the use of optimal resources or information
processing.
 Epigenetic: Regulatory mechanism that controls the expression of
genes that do not involve changes in DNA sequences and that are
heritable.
 Endoreplication: Process in which the cell cycle enters the phase of
DNA synthesis, without going through the phase of mitosis.
 Lipoxin/Lipocaline Complex: Hemocytes differentiation factor
composed of an inhibitor derived from arachidonic acid and a lipid
carrier(lipoxin and evokin).
 Signaling pathways: Immune pathways capable of being activated to
regulate genes involved in the effectors of the immune response.
 Antimicrobial peptides: Small natural proteins that have antibiotic,
antifungal and antiviral properties.
 Dscam: Cell adhesion molecules belonging to the superfamily of
immunoglobulins.
 FREPs: Immune receptors composed of proteins related to
fibrinogen.
 RNAi: interfering RNA, a process of genetic silencing mediated by
RNA molecules.
Chapter 7
Modulation of the Innate Immune System

by the Endocrine Disrupting Compounds
Bisphenol A (BPA) and Phtalathes
Karen Elizabeth Nava-Castro1,

Margarita Isabel Palacios-Arreola1,
Victor Hugo Del Río-Araiza2,
Mariana Segovia-Mendoza3
and Jorge Morales-Montor4,
1Laboratorio de Biología y Química Atmosféricas,
Departamento de Ciencias Ambientales,
Instituto de Ciencias de la Atmósfera y Cambio Climático,
Universidad Nacional Autónoma de México, Ciudad de México, México
2Departamento de Parasitologia, Facultad de Medicina
Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico,

Ciudad de Mexico, Mexico
3Departamento de Farmacología, Facultad de Medicina,
Universidad Nacional Autónoma de México México,

Ciudad de México, Mexico
4Departamento de Inmunología, Instituto de Investigaciones Biomédicas,
Universidad Nacional Autónoma de México,

Ciudad de Mexico, Mexico

Corresponding Author’s E-mail: jmontor66@hotmail.com.

ISBN: 978-1-68507-510-1
272 K. E. Nava-Castro, M. I. Palacios-Arreola et al.
Abstract
As a result of human socio-economic activity, industrial wastes have

increased distressingly. Plastic pollution is globally distributed across the
world due to its properties of buoyancy and durability. A big health
hazard is the sorption of toxicants to plastic while traveling through the
environment. Two broad classes of plastic-related chemicals are of
critical concern for human health—bisphenols and phthalates. Bisphenol
A (BPA) is an endocrine-disruptor compound (EDC) with estrogenic
activity. It is used in the production of materials that are used daily. The
endocrine modulating activity of BPA and its effects on reproductive
health has been widely studied. BPA also has effects on the immune
system; however, they are poorly investigated and the available data are
inconclusive. Phthalates are also EDCs used as plasticizers in a wide
array of daily-use products. Since these compounds are not covalently
bound to the plastic matrix, they easily leach out from it, leading to high
human exposure. These compounds exert several cell effects through
modulating different endocrine pathways, such as estrogen, androgen,
PPARγ and AhR pathways. The exposure to both classes of plastic
derivatives during critical periods of development has detrimental effects
on human health. In this chapter, we have compiled the most important
of their effects on the function of the innate immune system.
Keywords: pollution, microplastics, endocrine disruptors, Bisphenol A,

phthalates, innate immunity, macrophages, dendritic cells, mast cells
Introduction
Environmental Pollution and Its Impact on Organisms
As a result of human socio-economic activity, industrial wastes have increased

alarmingly. All pollutants have direct and indirect effects in almost every
ecosystem. No matter what their impact is, pollution affects every species on
this planet. Wildlife is prone to suffer the same symptoms and diseases
suffered by humans (Lovett et al. 2009). Global warming is changing some
ecosystems faster than the capability of animals and plants to adapt, leading to
possible extinction of a huge number of species (Andrady et al. 2016).
Toxicity of the water, reduced oxygen concentration in deeper layers of bodies
of water and difficulty in adapting to the new substances may damage
indigenous fauna and flora in several ways. Among the worst and most
widespread contaminants in the environment are plastics (Bellard et al. 2012).
The Problem
Plastics have great practical value, and this has allowed them to be readily
accepted by the consumer society.to the point that they are now present in
many of the everyday products we use. Part of the success of plastic materials
is that they are inexpensive, light and resistant. However, their resistance to
corrosion and degradation makes them slow to decompose, leading to a great
environmental problem (Hammer, Kraak, and Parsons 2012). It was believed
that plastics did not react with anything, that is, that they were stable, inert and
therefore did not contaminate; that is not true. Unfortunately, plastics make up
between 60% and 80% of the litter present in the marine environment. The
wind acts on the surface of seas and oceans, acting as the engine to spread
litter over these waters. The currents or turns manage to collect all kinds of
garbage in their path and finally form what are known as garbage islands or
plastic islands (Sharma and Chatterjee 2017).
Endocrine Disrupting Compounds (EDCs)
Endocrine disrupting compounds (EDCs) are substances that exist in the

environment as a result of agricultural and industrial activity. Some examples
are dichlorodiphenyltrichloroethane (DDT), Bisphenol A (BPA), Bisphenol S
(BPS), phthalates, among others, but they also can be found in pharmaceutical
products (ethinylestradiol, diethylstilbestrol (DES)) or naturally in different
plants such as soybean (phytoestrogens, genistein, daidzein, coumestrol).
EDCs may exhibit estrogenic, anti-estrogenic or anti-androgenic activity. In
addition, these compounds are highly lipophilic and can be stored for
prolonged periods of time in the adipose tissues. During pregnancy, the fetus
can be exposed to these compounds through the placenta and at birth by the
lactogenic route (Guzmán-Arriaga and Zambrano 2007; Pergialiotis et al.
2018).
Different studies have shown that, perinatal and neonatal exposure to BPA
or phthalates generate irreversible alterations in the reproductive axis, in the
central nervous system and in the immune system of the offspring of different
species. Although, the exposure to BPA or phthalates during the perinatal and
neonatal stages can lead to the development of different diseases in the adult
life, they may affect health at any age (Sweeney 2002). This fact suggests that
early life exposure to EDCs can increase the predisposition to an organism of
disease conditions. In this book chapter, we will delve into BPA and phthalates
effect on the innate immune system. We will do so, by dividng both compound
effects into two sections.
Bisphenol A (BPA) and Phthalates
One of the two main components of microplastics are bisphenol A (BPA) and
phthalates. BPA is widely used as a monomer in the production of
polycarbonate plastics, epoxy resins and dental sealants (Amaral Mendes
2002). This compound can be released easily from these materials due to
incomplete polymerization or hydrolysis of the polymers that contain it, which
can occur when they are exposed to high temperatures, acidic conditions or
enzymatic process. The main BPA exposure source in animals and humans is
through out food and beverages that have been in contact with materials
manufactured with BPA, which is detached from its matrix and it is ingested
orally (Welshons, Nagel, and Vom Saal 2006) (Figure 1). BPA is classified as
an EDC with estrogenic character since it can bind to nuclear estrogen
receptors (ERα and ERβ) and trigger signaling pathways, even when its
affinity is lower (<1,000) than the endogenous ligand, 17β-estradiol (Kuiper
et al. 1998). In addition, BPA also binds to the estrogen like G-coupled protein
receptor (GPER), the estrogen related receptor gamma (ERRγ), the aryl
hydrocarbon receptor (AhR) (Bonefeld-Jørgensen et al. 2007), the thyroid
hormone receptor (ThR) (Pogrmic-Majkic et al. 2019), the peroxisome
proliferator-activated receptor gamma (PPARγ) (Sargis et al. 2010), the
glucocorticoid receptor and the thyroid hormone receptor (TR) (Pogrmic-
Majkic et al. 2019; Sargis et al. 2010; Moriyama et al. 2002; Wetherill et al.
2007; Alonso-Magdalena et al. 2012; Rochester 2013; Lee et al. 2019; M. J.
Kim and Park 2019). Despite the Food and Drug Administration (FDA) and
the European Food Safety Agency (EFSA) calculated that the Tolerable Daily
Intake of BPA is 50 μg/kg/day, it has been estimated that exposure to BPA per
food package is 0.185 μg/kg/day in adults and up to 2.42 μg/kg/day in children
from 1 to 2 months of age (Pogrmic-Majkic et al. 2019). Besides, it has been
demonstrated that BPA exposure at the tolerable concentrations or below is
related to negative effects in the health of both, humans and rodents (Rochester
2013; Rezg et al. 2014; Seachrist et al. 2016).
Other molecules that are highly found in microplastics are phthalates;

which are alkyl diesters of phthalic acid and are used as plasticizers in PVC
products, as solvents and fixatives in personal care products and as additives
in the enteric coating of some drug tablets. Thus, phthalates are present in a
wide array of daily-use products, from flooring and automotive parts to
medical devices and even children´s toys. Since these compounds are not
covalently bound to the products, they easily migrate from them (Duty et al.
2005), leading to high human exposure (Figure 1). Phthalate exposure has
been estimated by biomonitoring studies, and ranges between 1 and 2 μg/kg
bw/d of a single compound for adults (Wittassek et al. 2007; Koch, Drexler,
and Angerer 2003) and up to 4 μg/kg bw/d for children (Koch et al. 2011).
Figure 1. Human exposure tu BPA and phtalathes.
While oral consumption of contaminated food and dermal absorption

from personal care products are considered the main exposure routes (Meeker,
Sathyanarayana, and Swan 2009; Varshavsky et al. 2018; Wittassek et al.
2011), phthalates are ubiquitously found in environmental matrices such as in
the atmosphere, soil and water bodies (Bergé et al. 2013). Of particular interest
is the presence of phthalates in the atmospheric aerosol, forming part of the
organic content of fine particulate matter (PM2.5). Phthalate concentrations can
range from 1 to 100 ng/m3 outdoors (Ji et al. 2014; Quintana-Belmares et al.
2018) and up to 1,000 ng/m3 indoors (Bergh et al. 2011; Fromme et al. 2004),
being Bis-(2-ethylhexyl) phthalate (DEHP) and Di-n-Butyl phthalate (DBP)
the most common. Phthalates exert sexually dimorphic effects, affecting in
different ways to male and female individuals. Based on epidemiological data,

elevated levels of DEHP urinary metabolites were linked to female patients
experiencing premature thelarche (Colón et al. 2000). On the other hand,
phthalates effects on males are grouped into the so called “phthalate
syndrome,” which is a type of testicular dysgenesis featuring cryptorquidism
hypospadias, incomplete testicular descend, reduced ano-genital distance, low
sperm count and/or quality, elevated risk of infertility and testicular cancer
(Duty et al. 2005; Swan et al. 2005; Swan 2008; Joensen et al. 2012), caused
by a reduced testosterone synthesis.
BPA Effects on Innate Immune Cells
Macrophages are one of the main phagocytic cells that play an important role
in the maintenance of organism homeostasis. Furthermore, they express the
two ER isoforms (ERα and ERβ) whereby BPA could exert its effects
(Campesi et al. 2017; Wang et al. 2005). On line with that, different studies
have evaluated the BPA stimulatory effects on macrophages. Hong et al.
(2004) reported that BPA at a concentration of 43 nM potentiated nitric oxide
production (NO) in a murine macrophage cell line (RAW264), after
lipopolysaccharide (LPS) exposure; while interferon-gamma (IFN-γ)
production was not altered (Hong et al. 2004). Other experiments developed
by Yamashita et al. (2005) described that BPA at a concentration of 0.1 μM
stimulated pro-inflammatory cytokines production such as IL-1, IL-6 and IL-
12, also BPA treatment increased the co-stimulatory molecule CD86
expression in murine peritoneal macrophages (Yamashita et al. 2005). In
concordance with this study, mouse peritoneal macrophages under M1 type
conditions that were exposed to low concentrations of BPA (> 1 μM) promotes
polarization toward M1 subtype by the upregulation of IRF5 expression, as
well as TNF-α, IL-6 and MOP-1; while the same BPA exposure to
macrophages under M2 type conditions inhibits the M2 subtype polarization
by the downregulation of IL-10 and TGF-β (Lu et al. 2019). In addition, a BPA
analog [BPA-glycidyl-methacrylate (BisGMA)] stimulated Tumor Necrosis
Factor alpha (TNF-α) production with a concomitant increase of surface
molecules (CD11, CD14, CD40, CD45, CD54 and CD80) expression on a
macrophage cell line (RAW264.7). Like BPA, the BisGMA induced the
production of IL-1β, IL-6, and NO, as well as the inducible nitric oxide
synthase (iNOS) expression and raised the generation of reactive oxygen
species (ROS) intra and extracellular in a dose-dependent manner (Kuan et al.

2012).
The increment of IL-6 and TNF-α secretion stimulated by BPA (0.1 μM)
was also observed in a human macrophage cell line (THP1), on the contrary,
secretion of regulatory cytokines such as IL-10 and TGF-β was decreased by
BPA treatment. Moreover, the treatment of an ER antagonist (ICI 182,780)
reversed this cytokine production pattern, indicating that BPA effects are
carried out by its binding to ERs (Y. Liu et al. 2014). Interestingly, BPA can
induce alternative macrophages activation (M2) by reducing IL-6, IL-10 and
IL-1β production in macrophages derived from human peripheral blood
monocytes (PBMCs), stimulated or not with LPS or IL-4. This work also
reported that BPA treatment increased the IL-10 and decreased the IL-6
production in macrophages classically activated (M1) (Teixeira et al. 2016).
Finally, a work of Yang et al. (2015) reported that BPA increased the
production of NO and ROS in a dose-dependent manner in carp (Cyprinus
carpio) macrophages (Yang et al. 2015).
On the other hand, inhibitory BPA effects on macrophages function have
also been reported. Segura et al. (1999) evaluated the adhesion capacity of the
peritoneal macrophages of rats after BPA treatment, this compound (10 nM)
inhibited the macrophages adherence but it did not have effect on their
viability (Segura et al. 1999). In another study, Kim and Jeong (2003)
evaluated the effect of BPA on the production of NO, TNF-α and iNOS
expression in mice peritoneal macrophages. BPA (50 μM) did not affect the
NO and TNF-α production. On the contrary, when LPS was used as stimuli,
BPA treatment inhibited their production. In addition, BPA decreased the
iNOS expression in a dose-effect dependent manner (J. Y. Kim and Jeong
2003). Supporting this fact, Byun et al. (2005) indicated that mice peritoneal
macrophages, obtained from mice injected with BPA (500 mg/kg/day) for 5
consecutive days during 4 weeks, and cultured ex vivo with LPS, had a marked
reduction in TNF-α and NO production. A similar effect was observed in a
macrophage culture treated with BPA (10 and 100 Μm) (Byun et al. 2005).
The BPA cell inhibitory effect was also observed in RAW 264 macrophage
cell line, where BPA exposure (100μM) inhibited IFN-β promoter activation
after LPS stimulation (Ohnishi et al. 2008). Moreover, this estrogenic
compound also suppressed the NO production and NF-ƙB activation in the
same cell line with the same stimulus. Of note, these effects were blocked by
the estrogen receptor antagonist ICI182,780 (Yoshitake et al. 2008). In line
with that, BPA (200 μM) exposure in RAW 264.7 macrophage cell line also
shown to inhibit the production of NO and induce apoptosis cell death (K. H.
Kim et al. 2014). In summary, BPA alters macrophage functions by decreasing

cytokine secretion, activating of M2 phenotype and stimulate the expression
of adhesion molecules (Figure 2).
Finally, in a recent study Liu et al. (2020), report that BPA (100 μg/L)
generates immunotoxicity in macrophages from common carp (Cyprinus
carpio), by increasing the expression of long non-coding RNAs. This is
reflected in the alteration of some signaling pathways related to the immune
response, such as: NF-κ B, Toll-like receptor, B-cell receptor and the Jak-
STAT signalling pathway (S. Liu et al. 2020).
Dendritic cells (DCs) are the par excellence antigen presenting cells; they
play a fundamental role in the begining of the immunological response and its
polarization and regulation. It has been reported that these cells also express
ERα and Erβ (Kovats 2015). Limited studies have investigated the BPA
effects on DCs. Guo Y and cols. (2010), reported that treatment of BPA in
DCs derived from PBMCs increased the chemokine ligand 1 (CCL1)
expression, the IL-5, IL-10 and IL-13 production as well as the expression of
the transcription factor GATA3 in the presence of TNF-α (Guo et al. 2010),
demonstrating that the exposure to BPA alters the functions of human DCs by
inducing preferentially a Th2 response. Furthermore, BPA affected DCs
differentiation by increasing the class II major histocompatibility complex
(MHC II) and CD69 expression (Pisapia et al. 2012). On the other hand,
Švajger et al. (2016), indicated that BPA at a concentration of 50 μM
decreased the DCs endocytic capacity as well as the CD1a expression (an
important protein mediating antigenic presentation) (Švajger, Dolenc, and
Jeras 2016). BPA at low concentrations (1 nM) also increased the DCs density,
however, the expression of DCs activation markers (HLA-DR and CD86) was
decreased in human DCs derived from PMBCs (Camarca et al. 2016). BPA
had no effect neither in cultivated bone marrow precursors or in the
proliferation of the DCs, regardles of concentration (Chakhtoura et al. 2017).
The works previously described suggest that exposure to BPA affects human
DCs function in a concentration dependent manner (Figure 2).
Granulocytes are the most abundant innate immune cells. They are
divided into neutrophils, which constitutes between 90 and 95% of their
totality, eosinophils from 3 to 5% and basophils less than 1%. In the literature
there are few reports about the BPA effect on these cells. In the case of
neutrophils, Watanabe et al., (2003) evaluated the effect of BPA on the
neutrophilic differentiation induced by dimethylsulfoxide (DMSO) and
granulocyte colony stimulating factor (G-CSF) in a leukemia cell line (HL-
60). They reported that low doses of BPA (10-10 and 10-8 M) increased the
expression of the CD18 integrin protein, the neutrophilic differentiation as

well as superoxide production. Interestingly, the treatment with an estrogen
receptor inhibitor (tamoxifen) in these cells, did not suppress the BPA effect,
suggesting that all the effects above are mediated by an ER-independent
pathway (Watanabe et al. 2003). Another BPA-related compound,
tetrabromobisphenol A (TBBPA), also increased the ROS neutrophils
production in a dose-dependent manner (Reistad, Mariussen, and Fonnum
2005). BPA exposure has also been found to affect eosinophils, which are key
players in allergy and asthma pathogenesis. In vivo experiments, using mouse
models of allergic asthma, described that perinatal BPA exposure enhanced
eosinophilic inflammation and airway responsiveness (Midoro-Horiuti et al.
2010), suggesting that BPA exposure can affect the disease during critical
developmental periods. Similar results were observed when adult mice were
sensitized with ovalbumin combined with BPA treatment, where the result of
the treatment significantly increased eosinophil recruitment in the alveoli and
submucosa of the airways and enhanced eosinophil-produced cytokine and
chemokine levels (He et al. 2016) (Figure 2). These studies suggest that early
or late life exposure to BPA could enhance the severity of immune-mediated
diseases such as allergic diseases and asthma.
Mast cells are resident tissue cells that play a key role in inflammatory
and allergic processes (Ménard et al. 2014). These cells are characterized by
their high content of cytoplasmic granules containing preformed mediators
that include the vasoactive amine histamine, proteases, as well as some
cytokines (O’Brien, Dolinoy, and Mancuso 2014a). Mast cells may be
activated by a variety of stimuli through the numerous receptors on their
surface. Upon activation, mast cells can release preformed as well as a several
distinct newly synthesized mediators (Ménard et al. 2014; O’Brien, Dolinoy,
and Mancuso 2014a). The most studied mechanism of mast cell activation is
the IgE-mediated response, which can be divided into two stages; in the first
stage, IgE produced by plasma cells binds to the FcɣRI on the mast cell
surface, leading to a sensitized state. Later, upon a second encounter with the
antigen, it can bind to the surface-binded IgE, which after crosslink of IgE
triggers mast cell degranulation and release of mediators. Though BPA
exposure has been linked to allergen sensitization, there is limited information
concerning BPA effects on mast cell-response. In a two-generational study,
O’Brien reported that perinatal BPA exposure through maternal diet (ranging
from 50 ng to 50 mg/Kg diet) caused an increase in mast cell-derived
leukotrienes, prostaglandin D2, TNF-α and IL-13, suggesting greater
activation of these cells (O’Brien, Dolinoy, and Mancuso 2014b) (Figure 2).
Further studies will be needed to evaluate the role of the BPA exposure on
mast cell response and its association to allergic diseases.
Figure 2. Effect of BPA on innate immune system cells.
Phtalathes Effects on Innate Immune System
Most studies about the impact of phthalates in the immune system are
developed in murine macrophages, either cell lines or in primary cultures of
peritoneal exudates. In these cells, it was found that some phthalates such as
DEHP, DEP and MEHP promote a pro-inflammatory state characterized by
an increase in the expression and secretion of IL-1, IL-6, TNF-α, and the
chemokine CXCL1 and ROS (Harris et al. 2016). Other studies found that the
secretion of IL-6, IL-10 and the chemokine CXCL8 was enhanced, while
TNF-α was impaired by DEP and DnBP presence during stimulation (Hansen
et al. 2015). They are also capable of increasing the phagocytic index of rabbit
lung macrophages, which is accompanied by an increment in the release of
lysosomal hydrolases (Bally, Opheim, and Shertzer 1980). However, Shertzer
et al., (1985), found that even when the phagocytic index after the infection
with Staphilococcus aureus was increased, there was a reduction in the rate of
pathogen destruction, which may impact as a deficient response in subsequent

exposures (Shertzer, Bally, and Opheim 1985). On the contrary, other
phthalates such as the DBP, BzBP, DEP and DPrP inhibit the production of
IL-1β, IL-6 and IFN-β (Ohnishi et al. 2008; Hong et al. 2004), apparently by
the inactivation of the IFN-β promoter (Ohnishi et al. 2008) (Figure 3).
Regarding other immune cell types, there is also evidence in which the
effect of some phthalates modifies the function of bone marrow-derived
dendritic cells (BM-DC) and basophils. In DC’s, the presence of 10 μM of
DEHP during the differentiation of BM-derived macrophages increased the
expression of maturation markers such as MHC--II, CD80/86, CD11c and
DEC205. It also increased their capacity to induce secretion of IFN-γ, IL-4
and IL-10 by T cells. This effect was also observed when DC’s were
differentiated in normal conditions and DEHP was added during the activation
(Figure 3). Interestingly, at 100 μM of DEHP during activation, the percentage
of maturation/activation markers were reduced and their antigen presenting
activity is not affected as observed by the reduced proliferation of co-cultured
T cells. Authors suggest that the enhancement on these markers and function
might contribute to the aggravating effect of DEHP on allergic disorders,
which also may be useful for the treatment in this kind of pathologies (Koike
et al. 2009).
Finally, since air pollutants have been related to airway diseases, the study
of immune cell populations related to allergies has gained relevance. The
RBL-2H3 cells, a basophil-derived cell line, which has been described also as
a mast cell model, was exposed to different phthalates -DPB, DiPB and
DEHP- at concentrations of 50-500 μM. Authors described that in the absence
of antigen stimulation, none of those compounds induce β-hexosaminidase
release. However, when cells were pre-sensitized and then stimulated with an
antigen, all of the phthalates increased the degranulation of these cells
(Nakamura, Teshima, and Sawada 2002). Another report found an interest
correlation with the number of carbon atoms present in the phthalates and the
histamine-release of human basophils. In this work, PMBC’s were incubated
in the presence of different phthalates and the stimulated with any of the
following stimuli: anti-IgE, the bacterial derived peptide fMLP, a calcium
Ionophore or an allergen (cat hair extract). As described before, there was no
effect on the histamine release in the absence of activation. Interestingly, only
the phthalates and monophthalates with an 8-carbon atoms alkyl chain length
were the stronger histamine-release potentiators, when there were no
differences for the 4-, 9- or 10-carbon chain (Glue et al. 2002) (Figure 3).
Some studies have also assessed the effect of phthalates on human cell
lines and primary cultures. As in the former studies, heterogeneity in exposure
times and concentrations is observed, but some general features can be
distinguished. It is interesting to note that, in general terms, a more reactive or
proinflammatory response is observed in human cells in vitro exposed to
different phthalates, regardless of the heterogeneity of models and exposure
schemes.
Figure 3. Effect of phtalathes on innate immune system cells.
Studies with THP, a macrophage cell line seem to concur in this phthalates
and their metabolites increase the cytokine and chemokine production (Glue
et al. 2002; Bennasroune et al. 2012; Couleau et al. 2015). Tetz et al., (2015),
observed a proinflammatory effect of phthalate exposure, with activation of
COX pathway and increased prostaglandin production (Tetz, Aronoff, and
Loch-Caruso 2015). An interesting study by Teixeira noted that the effect of
phthalate exposure showed differential features, depending on the macrophage
phenotype, either M1 or M2 (Teixeira et al. 2016).
One of the first studies that documented the effect of phthalates on
immune cells was derived from the concern of donated blood being storage in
plastic bags; this study found decreased chemotaxis and bactericidal capacity
of neutrophils (Miyamoto and Sasakawa 1987). However, more recent studies
using human granulocytes have found rather an increase in chemotaxis, IL-8
(Palleschi et al. 2009; Rosado-Berrios, Vélez, and Zayas 2011), and promotion
of inflammatory response (Gourlay et al. 2003) (Figure 3).
Mast cells are of particular interest, given their role in allergic diseases.
Lee and cols. reported an increase in INF-γ and IL-4 production upon DEHP
exposure, which is linked to a more reactive phenotype. However, more
studies regarding mast cells differentiation, migration and function are
desirable (Figure 3).
Conclusion
Endocrine disrupting compounds modulate endogenous steroid responses and

cell functions. Although most studies focus on their reproductive effects, their
potential effects on immune cells and even more, on the immune response
towards pathogens, should draw attention, given the expression of hormonal
receptors by immune cells. Despite the fact that a lot of studies have evaluated
the BPA effect with different variables such as BPA concentration, type of
model, administered BPA dose, life stage or antigenic challenge used, one
common element remains: BPA can differentially modulate the immune
response. Sometimes, BPA treatment helps the host to defend itself against a
pathogen, sometimes BPA treatment it is detrimental and helps the invader.
Thus, BPA not always has to be considered an enemy, but a double edge
sword, that depending on the context of the However, more studies are needed
with the aim to elucidate the possible mechanisms by which this takes place.
The immune system is regulated by a complex crosstalk between its own
soluble mediators and several endocrine pathways. Immune cells not only
express endocrine receptors but synthetize and respond to several hormones
and other endocrine ligands. In this context, perturbations in the endocrine
homeostasis may lead to immune dysregulation. Regarded as EDCs,
phthalates and their metabolites interact with estrogen, androgen, PPARγ and
AhR pathways, and there is vast in vitro evidence of the effects these
compounds exert on immune cells. Furthermore, reports on animal models
confirm these phenomena occur in a whole organism setting. However,
epidemiological evidence is the one that raises the more concerns, since it
points out two relevant aspects of immune dysregulation associated to
phthalates: developmental and long-term effects.
Taken together, the evidence suggests more consciousness should arise

regarding the use of phthalates, especially in daily use personal care items
intended for its use by vulnerable populations. We hope the recognition of the
immunoendocrine modulation network and the role it plays from early
developmental stages will lead a more integrative perspective in toxicological
studies. Regarding phthalates, more studies on immunological imprinting and
potential neurological effects are desirable, since this system is also
profoundly modulated by endocrine signaling.
Acknowledgments
Financial support: Grant IN-209719 from Programa de Apoyo a Proyectos de

Innovación Tecnológica (PAPIIT), Dirección General de Asuntos del Personal
Académico (DGAPA), Universidad Nacional Autónoma de México (UNAM)
and Grant FC2016-2125 from Fronteras en la Ciencia, Consejo Nacional de
Ciencia y Tecnología (CONACYT), both to Jorge Morales-Montor. Grant IA
206220 to Víctor H del Río Araiza, from PAPIIT, DGAPA, UNAM. Carmen
T. Gómez de León was a recipient of a Post-Doctoral fellowship from Grant
FC2016-2125, Fronteras en la Ciencia, Consejo Nacional de Ciencia y
Tecnología (CONACYT).
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About the Editor
Jorge Morales-Montor. His Doctoral Thesis was recognized with the Lola
and Igo Flisser PUIS Award as the best graduate thesis at the national level in
the field of parasitology. He received a fellowship from the Fogarty
Foundation to perform a Postdoctoral Research stay at the University of
Georgia. His scientific production already has 153 articles in international
indexed journals, with more than 3,100 citations, and an H Index of 35. He
has also edited several books and published more than 55 chapters. He is a
member of the Mexican Academy of Sciences, Academy New York Sciences,
American Society of Immunologists, Latin American Academy of Sciences
and The National Academy of Medicine, among others. He has more than 35
awards and 30 distinctions, has graduated more than 35 Bachelor’s, 8 Master’s
and 12 Doctorate students, and has directed more than 7 Post-Doctoral
Researchers. He was President of the Mexican Society of Parasitology and
Mexican Society of Neuroimmunoendocrinology, and he has obtained more
than 20 research grants in his career.
Index
A alphaviruses, 239
acid, 11, 92, 105, 142, 146, 162, 163, 170, alveolar macrophage, 14, 39, 41, 42, 43,
179 73, 119
acquired immunity, 169 angiogenesis, 108, 117, 118, 124, 125, 126,
acquired immunodeficiency syndrome, 15 133, 168, 223, 232
acute respiratory distress syndrome, 178 Anopheles, 237, 250, 251, 258, 259, 260,
adaptive immune response, 34, 69, 74, 223 261, 263, 265, 266
adaptive immune responses, 34, 223 antibody, 20, 24, 34, 78, 79, 81, 108, 139,
adaptive immunity, vii, ix, x, 4, 18, 62, 79, 144, 147, 165
90, 93, 95, 104, 110, 114, 130, 149, 150, antigen, vii, x, 3, 4, 8, 9, 18, 27, 34, 38, 39,
202 44, 76, 79, 93, 97, 108, 125, 129, 139,
adhesion, 93, 107, 122, 133, 140, 142, 146, 154, 164, 166, 170, 172, 173, 187, 217,
147, 148, 157, 184, 189 220, 221
adipose, 11, 12, 15, 19, 26, 132, 174, 176, antigen-presenting cell, 34, 39
184, 202, 207, 233 apoptosis, 6, 12, 14, 34, 37, 77, 109, 134,
adipose tissue, 11, 12, 15, 19, 26, 132, 176, 138, 150, 166, 168, 169, 183, 191, 192,
207 208, 224, 232
Aedes, 237, 250, 251, 254, 257, 258, 259, arthritis, vii, ix, 89, 90, 97, 104, 105, 107,
261, 262, 263, 264, 266, 267, 268 108, 109, 112, 115, 116, 117, 118, 120,
age, ix, 5, 10, 37, 41, 62, 63, 65, 66, 70, 98, 122, 123, 124, 125, 186
117 arthritis rheumatoid, v, vii, 89, 97
air pollutants, 69, 75 articular cartilage, 100
airflow obstruction, 68 aryl hydrocarbon receptor, 7
airway epithelial cells, 231 assessment, 199, 203
airway hyperresponsiveness, 79 asthma, vii, viii, ix, 4, 16, 31, 36, 40, 41,
airway inflammation, ix, 43, 62, 63, 69, 70, 46, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
73, 75, 77, 78, 195 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
airways, 11, 16, 39, 41, 42, 43, 44, 65, 71, 212
75, 80 asthma endotypes, ix, 62, 76, 83, 84
allergen challenge, 39 atopic dermatitis, 15, 16, 27, 78, 152, 201,
allergic asthma, 24, 40, 43, 74, 77, 79, 211 202
allergic inflammation, 24, 177, 183 autoantibodies, 90, 100, 102, 119, 171, 173
allergic reaction, 147 autoantigens, 99, 100, 105, 124, 173, 189
allergy, viii, 4, 15, 24, 179, 202, 212, 224
298 Index
autoimmune disease, x, 34, 97, 99, 107, bronchoconstriction, 14, 68, 70, 74, 79, 80
118, 122, 130, 133, 172, 175, 212, 228,
230 C
autoimmune diseases, x, 118, 130, 133, calcium, 99, 132, 135, 156, 221
171, 172, 174, 175, 212, 228, 230 cancer, viii, x, 4, 15, 16, 17, 19, 23, 25, 27,
autoimmune hemolytic anemia, 171 30, 32, 33, 34, 36, 47, 49, 53, 56, 58,
autoimmunity, 33, 99, 103, 105, 118, 127, 116, 122, 130, 133, 138, 143, 144, 145,
172, 200, 227, 232, 233 168, 169, 170, 171, 180, 181, 184, 188,
189, 190, 192, 195, 197, 199, 203, 204,
B 205, 208, 209, 210, 211, 212, 213, 214,
bacteria, 14, 22, 28, 68, 91, 94, 99, 113, 215, 216, 217,218, 219, 220, 221, 223,
123, 142, 148, 149, 150, 151, 152, 153, 224, 226, 227, 228, 229, 230, 231, 232,
164, 182, 186, 192, 221 233, 236, 276, 288
bacterial infection, 150 cancer cells, 143, 144, 145, 168, 169, 184,
basophils, 74, 77, 94, 224 230
biochemistry, 202, 214, 216, 228, 231 cancer progression, 16, 217
biological fluids, 181 cancer stem cells, 171
biological sciences, 209, 229 candidates, 162, 171, 176, 224
biomarkers, ix, 62, 70, 71, 90, 114, 170, carcinogenesis, 17, 25
171, 175, 181, 183, 220, 227, 230 carcinoma, 16, 145, 150, 205
biphasic immune response, 240, 248 cardiovascular disease, 22
bisphenol A, vi, vii, xi, 69, 271, 272, 273, cardiovascular risk, 15
274, 285, 286, 287, 288, 289, 290, 291, cardiovascular system, 90
292, 293 cartilage, ix, 89, 92, 100, 101, 102, 103,
blood, 16, 17, 29, 35, 37, 41, 43, 74, 76, 106, 113, 123, 175, 232
77, 79, 93, 110, 116, 119, 131, 139, 140, cell biology, 130, 165, 180, 184, 185, 186,
145, 146, 155, 157, 158, 167, 181, 183, 189, 190, 194, 198, 199, 200, 201, 211,
184, 194, 195, 201, 203, 204, 205, 207, 212, 215, 216, 227
210, 214, 215, 216, 225, 226, 229, 233 cell death, 105, 137, 141, 144, 163, 194
blood circulation, 145 cell differentiation, 103, 108, 109, 133, 217
blood monocytes, 41, 119 cell line, 5, 24, 145, 146, 150, 160, 166,
blood plasma, 183, 203, 204, 216 170, 204
blood stream, 139 central nervous system, 208, 233
bloodstream, 140, 141 chemical, 68, 69, 73, 140, 147, 176, 179,
body fluid, 153, 180 180, 181
bone, ix, 34, 37, 38, 39, 89, 100, 101, 102, chemokines, ix, 36, 37, 38, 43, 89, 94, 95,
103, 106, 107, 108, 109, 112, 117, 119, 101, 108, 145, 150, 152, 161, 165, 171
121, 125, 139, 143, 145, 156, 157, 158, chemotaxis, 34, 79, 108, 146
175, 176, 178, 184, 201, 203, 217, 228, cholesterol, 32, 135, 137, 160, 162
230 chronic diseases, 18
bone marrow, 34, 37, 38, 39, 117, 125, chronic obstructive pulmonary disease, 36
139, 143, 145, 158, 176, 178, 184 cigarette smoke, 43, 73, 99, 120, 125
breast cancer, 17, 23, 169, 170, 188, 195, cigarette smokers, 125
221 cigarette smoking, 42
bronchial hyperresponsiveness, 65, 71
Index 299
circulation, 10, 12, 15, 33, 37, 109, 127, destruction, ix, 44, 89, 93, 101, 103, 109,
157, 169 227
clinical application, 217, 218 detection, 76, 90, 106, 132, 141, 181, 233
clinical presentation, 90 developed countries, 63
clinical trials, 42, 78 development, v, vii, viii, ix, x, xi, 3, 6, 7, 9,
coding, 109, 136, 148, 155, 156, 162, 166, 12, 15, 16, 17, 20, 21, 25, 28, 31, 32, 36,
215 39, 45, 46, 51, 56, 71, 74, 75, 80, 85, 89,
colitis, 162, 163, 174, 179, 197 90, 92, 97, 99, 100, 102, 104, 105, 107,
collagen, 100, 108, 116, 186 108, 113, 114, 115, 130, 142, 145, 154,
communication, x, 102, 129, 130, 133, 138, 158, 168, 179, 181, 182, 191, 212, 222,
140, 159, 161, 164, 172, 184, 191, 208, 224, 236, 238, 240, 256, 261, 265, 268,
210, 218, 219, 223 272, 274, 286, 287, 289, 290, 292
complement, 92, 138, 139, 147, 148, 151, developmental change, 154
157, 163, 172, 174, 197 diabetes, 173, 175, 191, 195, 197
complexity, 76, 90, 114, 188 disease activity, 99, 116, 125, 193, 209,
composition, 13, 130, 137, 146, 152, 160, 221
162, 172, 190, 223 disease model, 45, 112
compounds, vii, xi, 68, 69, 71, 73, 92, 125 diseases, vii, viii, x, 18, 31, 33, 43, 46, 75,
corticosteroids, 71, 73, 76, 77, 78, 80, 81 80, 92, 98, 111, 121, 130, 133, 147, 154,
costimulatory molecules, 4, 8, 148, 163 167, 171, 172, 174, 180, 181, 187, 188,
cytokines, viii, ix, 3, 4, 5, 6, 9, 10, 11, 13, 199, 211, 214, 224, 229, 233
15, 16, 18, 26, 36, 37, 38, 40, 62, 71, 73, distribution, 8, 9, 13, 18, 23, 125, 199, 232
74, 75, 77, 78, 80, 81, 89, 91, 93, 94, 95, diversity, ix, 62, 75, 90, 114, 118, 136, 177
96, 98, 101, 102, 104, 106, 107, 108, DNA, x, 4, 34, 95, 98, 105, 109, 118, 120,
110, 112, 126, 139, 140, 143, 144, 145, 122, 125, 127, 129, 136, 138, 141, 150,
146, 148, 150, 152, 156, 157, 158, 159, 151, 152, 157, 158, 170, 186, 193, 204,
160, 161, 163, 165, 168, 171, 173, 177, 206, 223, 224, 225, 228, 233
179 DNA synthesis, 242, 258, 260, 269
cytoskeleton, 134, 135, 142, 156 drug carriers, 199
cytotoxicity, 6, 26, 36, 78, 94, 144, 147, drug delivery, 181, 191
169, 170, 183, 226 drug reactions, 32
drug resistance, 158, 210
D drugs, 32, 70, 76, 157, 171, 181
damages, iv, 17, 173
deficiency, 28, 66, 108, 124 E
degradation, 105, 110, 112, 120, 137, 165, elastase, ix, 32, 44, 50, 73, 89, 101, 142,
204 236
dendritic cells, ix, 4, 23, 34, 38, 39, 40, 47, endocrine disruptors, 82, 272, 285, 290,
48, 50, 51, 52, 54, 56, 57, 62, 71, 74, 75, 291
76, 83, 92, 95, 104, 116, 118, 124, 125, endoreplication, 242, 256, 269
139, 153, 192, 193, 200, 206, 214, 218, endothelial cells, 71, 92, 104, 108, 140,
221, 223, 225, 228, 248, 252, 272, 278, 157, 163, 167, 194, 229
281, 286 environment, xi, 69, 90, 97, 147, 151, 155,
dengue virus, 238, 243, 247, 255, 263, 264, 173, 222, 228
265, 267, 268 environmental conditions, 153, 159, 205
300 Index
environmental factors, 68, 74, 90, 98, 114, F

118, 172 fibroblast, ix, 89, 90, 92, 98, 100, 101, 102,
eosinophil, 11, 14, 16, 27, 71, 77, 78, 147, 104, 107, 112, 113, 115, 118, 122, 123,
187 133, 168, 173
eosinophilia, 71, 74, 161, 179 fibroblast growth factor, 104
eosinophils, ix, 16, 40, 62, 70, 71, 73, 74, fibroblast proliferation, 113, 168
77, 79, 80, 81, 94, 147, 152, 187, 211, fibroblasts, 16, 93, 100, 101, 106, 108, 109,
212, 224 112, 113, 116, 117, 118, 122, 126, 158,
epigenetic alterations, 152 168, 192, 205, 226
epigenetic modification, 97 fibrosis, 16, 66, 178, 179, 210
epithelial cells, 16, 44, 76, 91, 92, 133, flaviviruses, 239
146, 150, 152, 159, 160, 173, 202, 203 fluid, 43, 93, 106, 164, 171
epithelium, 11, 44, 68, 71, 73, 132, 153 formation, 23, 100, 108, 119, 132, 135,
erosion, 101, 102, 107, 108, 123 136, 137, 157, 168, 169, 170, 173, 183,
erythrocytes, 130, 131, 132, 156, 182, 197, 189, 190, 192, 216, 224, 232
219, 229 fungal infection, 22
estrogen, xi, 12, 32, 35, 36, 37, 38, 39, 40, fungi, 13, 91, 94, 149, 186
46 fungus, ix, 62, 161, 179
evidence, ix, 39, 42, 44, 65, 69, 76, 77, 89, fusion, x, 129, 131, 140, 159, 221
96, 100, 101, 105, 107, 110, 112, 113,
168, 172, 177, 181, 206, 216, 219 G
exosomes, x, 109, 129, 130, 134, 135, 136, gastroesophageal reflux, 69
137, 138, 142, 145, 146, 147, 155, 156, gastrointestinal tract, 11, 17, 92
159, 160, 162, 165, 166, 167, 168, 175, gene expression, 34, 36, 38, 109, 146, 158,
177, 178, 179, 182, 184, 185, 186, 187, 159, 204
188, 190, 191, 192, 194, 195, 196, 197, genes, 33, 39, 69, 74, 95, 97, 109, 110,
199, 200, 201, 202, 203, 204, 206, 207, 111, 133, 140, 155, 158, 163, 229
208, 209, 210, 211, 212, 216, 218, 221, genetic background, 45
222, 223, 224, 225, 226, 227, 228, 229, genetic factors, 90, 97, 99, 103, 114
230, 231, 232, 233, 234 glycoproteins, 95, 111, 156
extracellular matrix, 93, 140, 156, 172, granules, 117, 130, 141, 142, 145, 146,
173, 202 190, 229
extracellular vesicles, v, vii, x, 129, 130, growth, 23, 32, 37, 92, 109, 111, 125, 140,
132, 133, 167, 170, 171, 174, 183, 184, 142, 152, 167, 168, 169, 204, 217
185, 186, 187, 188, 189, 190, 191, 192, growth factor, 37, 111, 168, 169, 217
193, 194, 195, 196, 197, 198, 199, 200,
201, 202, 203, 204, 205, 206, 207, 208,
H
209, 210, 211, 212, 213, 214, 215, 216,
hair follicle, 91, 140, 187, 188, 189
217, 218, 219, 220, 221, 222, 223, 224,
health, x, xi, 28, 32, 33, 45, 64, 79, 130,
225, 226, 227, 228, 229, 230, 231, 232,
136, 175, 219
233, 234
hemocytes, 241, 242, 247, 249, 250, 251,
257, 258, 261, 262, 266, 269
hepatocytes, 132, 166, 233
herpes simplex, 204, 225
herpes simplex virus type 1, 204
Index 301
heterogeneity, viii, 32, 62, 70, 76, 90, 93, 256, 257, 258, 261, 263, 264, 266, 267,
114, 190 268, 269, 278, 283, 288, 289
highly active antiretroviral therapy, 15 immune system, vii, ix, x, xi, 4, 10, 33, 35,
HIV, 26, 165, 180, 185, 187, 203, 214 62, 70, 73, 75, 80, 92, 98, 105, 116, 117,
HIV-1, 165, 185, 187, 203, 214 123, 133, 147, 148, 153, 159, 166, 167,
homeostasis, vii, viii, x, 4, 13, 19, 20, 27, 169, 176, 180, 181, 207, 210
36, 37, 41, 91, 92, 130, 132, 141, 145, immunity, ix, x, xi, 19, 20, 24, 26, 27, 28,
153, 171, 172, 173, 175, 180, 184, 189, 29, 62, 74, 80, 90, 91, 92, 94, 115, 118,
190, 232 120, 123, 124, 149, 150, 153, 164, 168,
human neutrophils, 142, 190, 197 170, 189, 193, 202, 206, 218, 220, 221,
human subjects, viii, 31, 33, 45 222, 231
hyperplasia, ix, 16, 89, 100, 106 immunodeficiency, 12, 14, 21, 30
hypothesis, 64, 74, 80, 118, 148, 165, 198 immunomodulation, 151, 163, 164, 167,
hypoxia-inducible factor, 112, 119 229
immunomodulatory, x, 130, 140, 143, 144,
I 149, 150, 154, 164, 176, 177, 179, 181,
identification, ix, 23, 62, 65, 80, 138 205, 209
IFN, viii, 3, 4, 5, 6, 9, 10, 14, 15, 16, 17, immunomodulatory agent, 179
76, 96, 104, 107, 120, 152, 160, 166, immunostimulatory, 159, 170, 173, 186
170, 172, 173, 179, 192 immunosuppression, 166, 188
IL-13, viii, 4, 5, 6, 9, 10, 11, 12, 13, 16, 24, immunosurveillance, 145
27, 36, 40, 71, 72, 73, 74, 78, 79, 81, immunotherapy, x, 32, 76, 81, 130, 133,
146, 160, 161 175, 176, 179, 216, 236
IL-17, viii, 4, 5, 6, 9, 10, 14, 17, 22, 29, 30, in vitro, 24, 37, 39, 141, 152, 156, 158,
41, 79, 103, 107, 108, 109, 124, 160, 163, 174, 177, 194, 202, 209, 210, 216,
162, 164, 165, 179 217, 218
IL-8, 107, 108, 121, 143, 144, 152, 153, in vivo, 39, 122, 131, 141, 151, 156, 170,
154, 159, 165, 173 177, 217
immune activation, 102, 104 inducer, vii, 3, 5, 21, 29, 80
immune defense, 28 induction, 30, 35, 43, 74, 95, 96, 109, 112,
immune function, 142 116, 120, 121, 126, 138, 148, 152, 154,
immune memory, xi, 26, 236, 239, 240, 162, 168, 182, 192
241, 242, 244, 245, 246, 247, 248, 249, infectious agents, 91, 148, 149, 172
252, 255, 256, 257, 263, 268 infectious diseases, 130, 133, 147, 154,
immune modulation, 27, 157 180, 181, 214
immune regulation, 199, 222 inflammation, v, vii, viii, ix, x, 1, 11, 15,
immune response, vii, xi, 3, 9, 13, 14, 18, 16, 17, 22, 24, 27, 28, 30, 31, 32, 33, 36,
27, 33, 34, 37, 38, 39, 40, 42, 45, 47, 50, 38, 39, 40, 41, 43, 45, 46, 47, 48, 49, 50,
53, 69, 70, 74, 75, 76, 81, 92, 94, 95, 51, 52, 56, 57, 58, 59, 62, 63, 65, 67, 68,
115, 116, 138, 139, 147, 148, 149, 155, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80,
156, 157, 159, 161, 162, 164, 166, 167, 81, 83, 90, 92, 94, 97, 101, 103, 107,
168, 169, 170, 171, 172, 180, 187, 198, 108, 109, 113, 115, 119, 120, 121, 123,
205, 207, 219, 223, 235, 239, 240, 241, 125, 130, 132, 138, 140, 141, 143, 144,
242, 244, 245, 247, 248, 251, 254, 255, 147, 148, 157, 159, 160, 167, 172, 173,
176, 177, 178, 179, 183, 184, 186, 190,
302 Index
191, 192, 193, 194, 195, 200, 201, 202, J

206, 208, 209, 213, 215, 219, 220, 226, joint damage, 106, 173
230, 232, 233, 236, 238, 251, 252, 253, joint destruction, 101, 112, 119
258, 279, 286, 288 joint pain, 101
inflammatory bowel disease, viii, 4, 17,
173 K
inflammatory cells, 65, 90, 108 kill, 139, 143, 144, 156
inflammatory disease, vii, viii, ix, 4, 18, killer cells, 23, 36, 230
21, 89, 123, 127, 230
inflammatory mediators, 43, 115, 146, 177
L
inflammatory responses, x, 130, 140, 143,
leucocyte, ix, 89, 101
173, 176
leukocytes, 35, 141, 142, 145, 176, 180
inhibition, 43, 100, 112, 140, 155, 156,
leukotrienes, 8, 94, 102, 146
161, 178, 179
lichen, 175, 187, 192, 216
injury, iv, 44, 133, 163, 176, 192, 193, 225,
ligand, 34, 39, 43, 91, 101, 102, 103, 104,
228, 232
106, 108, 109, 111, 113, 117, 160, 163,
innate immune, v, vi, vii, viii, ix, x, xi, 4,
169, 183, 220, 224
19, 26, 27, 31, 33, 34, 35, 39, 44, 46, 47,
lipids, x, 11, 91, 129, 130, 134, 135, 137,
48, 51, 53, 55, 58, 62, 70, 72, 73, 75, 80,
223
91, 104, 105, 119, 126, 129, 133, 144,
lipoproteins, 94, 113, 149, 150
147, 149, 150, 152, 153, 155, 158, 163,
lipoxin/lipocalin, 247, 265, 269
164, 165, 166, 170, 176, 178, 179, 181,
liver, 7, 11, 24, 35, 36, 41, 132, 133, 163,
186, 198, 206, 235, 236, 239, 258, 259,
175, 177, 190, 192
261, 262, 264, 265, 266, 267, 271, 272,
lung cancer, 19, 36, 169, 220
274, 276, 278, 280, 282, 288
lung disease, viii, 31, 36, 45, 46, 66
innate immune response, v, vi, vii, viii, xi,
lung function, 40, 43, 78, 79
4, 31, 33, 34, 44, 46, 47, 51, 58, 62, 70,
lymphoid, vii, ix, 3, 4, 5, 6, 7, 8, 11, 12, 19,
75, 91, 126, 149, 150, 152, 153, 155,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
163, 164, 166, 176, 206, 235, 264, 266
33, 35, 40, 62, 74, 75, 91, 92, 101, 102,
innate immunity, v, vii, ix, 1, 53, 56, 71,
143, 161, 168, 169, 177, 195
74, 75, 80, 84, 85, 87, 89, 90, 91, 92, 93,
lymphoid tissue, vii, 3, 5, 11, 12, 21, 30
94, 100, 115, 116, 118, 120, 124, 125,
126, 130, 138, 142, 147, 149, 150, 151,
152, 154, 157, 159, 161, 164, 165, 167,
M
180, 186, 187, 188, 193, 200, 211, 233, malaria, 157, 159, 190, 214, 219, 220, 229,
248, 251, 257, 265, 267,272 231
innate lymphoid cells, v, vii, ix, 3, 4, 5, 19, malignant cells, 168
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, mammalian cells, 150, 155, 161, 186, 187,
33, 35, 36, 40, 50, 52, 54, 55, 62, 74, 82, 204
matrix metalloproteinase, 100, 101, 107,
83, 91, 102, 143, 161, 177
113, 115, 192
intercellular adhesion molecule, 71, 93,
125 medicine, ix, 62, 76, 176, 183, 187, 191,
interferon, 34, 93, 96, 104, 119, 124, 127, 192, 208, 209, 212, 213, 225, 226, 228,
145, 151, 158 230, 234
internalization, 38, 141, 158, 159, 160 melanoma, 17, 145, 159, 168, 170
Index 303
mesenchymal stem cells, 169, 184, 202, mucus, viii, 14, 16, 44, 61, 65, 66, 70, 71,
207, 210, 218, 225, 228, 229, 231 73, 74, 79
mice, 6, 7, 9, 10, 11, 12, 13, 14, 15, 20, 22, mucus hypersecretion, 16, 44
34, 35, 36, 39, 40, 41, 43, 79, 107, 108, myeloid cells, 33, 37, 106, 107, 108, 109,
109, 118, 141, 152, 154, 155, 156, 157, 145
158, 160, 161, 162, 169, 174, 185, 187, myocardial ischemia, 178
192, 211, 215, 218, 225, 232
microbiota, 12, 14, 17, 97, 124, 153, 172 N
microorganisms, 8, 9, 12, 68, 73, 80, 91, natural killer cell, 20, 33, 78, 166, 197,
92, 94, 139, 141, 143, 180 217, 230, 231
microparticles, 135, 142, 175, 185, 189, nematode, 14, 161, 179, 186, 223
190, 191, 192, 193, 204, 212, 214, 220, neurodegenerative disorders, 174
221, 228, 229 neuroinflammation, 174, 177, 187, 225
microplastics, 272, 274, 275 neuropeptides, 8, 9, 11, 13, 18
microRNA, 100, 118, 119, 124, 126, 194, neuroscience, 186, 196, 201, 210, 218
197, 202, 207, 220, 224 neutrophil extracellular traps, 32, 142, 202,
microvesicles,innate immunity, 130 236
migration, x, 11, 36, 40, 44, 94, 129, 140, neutrophils, ix, 34, 37, 41, 42, 44, 62, 70,
141, 145, 147, 191, 232 71, 73, 80, 94, 95, 99, 100, 102, 104,
miRNAs, ix, 89, 90, 109, 110, 111, 138, 121, 139, 142, 143, 144, 152, 155, 167,
140, 153, 158, 162, 163, 166, 167, 168, 173, 177, 178, 179, 197, 210, 222, 232
169, 192, 211, 216, 229 nitric oxide, 38, 94, 118, 119, 122, 139,
molecular biology, 227 161, 163
molecular medicine, 184, 189, 203, 204, NK cells, vii, 3, 4, 5, 6, 11, 12, 15, 21, 26,
209 35, 36, 143, 144, 145, 152, 159, 163,
molecular weight, 69 169, 170, 218, 248
molecules, ix, x, 6, 14, 17, 32, 33, 37, 43, nucleic acid, 95, 105, 118, 134, 136, 138,
62, 76, 80, 89, 92, 93, 94, 95, 97, 99, 149, 151, 152, 156, 167, 170, 197
106, 107, 109, 111, 112, 129, 133, 136, nucleus, 110, 141, 146, 148, 150
137, 138, 140, 142, 144, 145, 148, 149,
150, 153, 155, 158, 170, 172, 175, 180, O
190, 224 obesity, viii, ix, 4, 12, 15, 19, 62, 64, 67,
monoclonal antibody, 77, 79 68, 69, 71, 73
monocyte chemoattractant protein, 139 obstruction, viii, 42, 43, 61, 65, 66, 69, 70,
mosquito, 236, 237, 238, 239, 240, 241, 74, 79
242, 243, 245, 247, 248, 251, 254, 255, oenocytoids, 249
256, 257, 258, 259, 260, 261, 262, 263, onset, v, vii, ix, 42, 50, 62, 63, 64, 66, 67,
264, 265, 266, 267 70, 74, 89, 90, 100, 105, 114, 117, 172
mosquitoes, vi, vii, x, 235, 236, 237, 238, overproduction, viii, 61, 65, 70, 74, 106
239, 240, 242, 243, 246, 248, 249, 250, oxidative stress, 112, 123, 172, 173, 220,
251, 254, 255, 258, 259, 261, 262, 263, 231
265, 267
mRNA, 109, 113, 136, 138, 146, 170, 182,
194, 202, 207, 219
304 Index
P 148, 150, 154, 155, 157, 159, 160, 177,

parasite, 14, 153, 154, 155, 156, 157, 158, 179
159, 160, 161, 162, 163, 179, 180, 183, proliferation, 10, 11, 12, 13, 16, 17, 18, 25,
187, 210, 214, 215, 223, 226, 227 35, 36, 77, 100, 103, 104, 111, 112, 113,
pathogen-associated molecular patterns, 4, 122, 126, 138, 141, 142, 145, 163, 168,
33, 34, 75, 94, 150, 245 175, 178, 180, 189, 201, 232
pathophysiology, ix, 62, 69, 71, 224, 233 prostaglandins, 8, 94, 101, 102, 146
pattern recognition, 4, 33, 34, 71, 75, 93, prostate cancer, 189, 192, 204, 208
94 protection, 75, 80, 91, 112, 142, 148, 151,
peptides, x, 12, 91, 95, 99, 100, 127, 129, 152, 156, 162, 222
139, 142 proteomics, 70, 80, 183, 191, 196, 203, 217
peripheral blood, 7, 19, 34, 164
peripheral blood mononuclear cell, 34, 164 R
phagocytic cells, 37, 91, 92, 139 receptor, 8, 9, 11, 12, 16, 20, 35, 39, 42,
phagocytosis, x, 38, 92, 93, 94, 95, 129, 74, 76, 77, 78, 79, 93, 94, 102, 104, 107,
139, 142, 143, 153, 157, 227, 241, 249, 110, 111, 115, 117, 118, 119, 120, 121,
251, 261, 285 123, 124, 125, 131, 132, 140, 141, 161,
phenotype, 13, 18, 26, 32, 38, 41, 43, 71, 164, 169, 170, 200, 216, 217, 220, 233
77, 78, 81, 93, 94, 101, 102, 139, 146, recognition, 33, 34, 44, 65, 68, 71, 92, 94,
147, 151, 177, 178, 179, 183 95, 105, 115, 119, 120, 121, 141, 148,
phosphatidylserine, 135, 144, 195, 196, 150, 158, 165, 195
199, 208, 210, 227, 229 regeneration, 141, 175, 178, 188, 201, 203,
phosphorylation, 95, 96, 111, 127, 135, 217, 228
146, 165 repair, 6, 12, 14, 25, 44, 94, 101, 127, 133,
phthalates, vii, xi, 69, 83, 272, 273, 274, 138, 139, 175, 176, 177, 178, 207, 228,
275, 280, 281, 282, 283, 284, 285, 286, 230, 232
287, 288, 289, 290, 291, 292, 293 resistance, viii, 14, 15, 62, 115, 150, 156,
physiology, 35, 36, 71, 153, 200, 202, 214, 163
219, 224, 227, 228 resolution, x, 16, 28, 130, 139, 199, 226
plasma membrane, 131, 132, 134, 137, response, viii, ix, xi, 4, 9, 10, 11, 13, 14,
149, 182, 214, 228 15, 16, 17, 18, 26, 30, 33, 34, 35, 37, 38,
Plasmodium, 148, 154, 157, 158, 159, 184, 40, 41, 43, 45, 62, 63, 70, 74, 75, 76, 79,
211, 214, 220, 225, 226, 229, 238, 241, 80, 91, 92, 93, 94, 95, 96, 103, 104, 106,
243, 247, 258, 259, 261, 265, 266 109, 110, 113, 126, 133, 139, 143, 149,
platelets, 92, 130, 167, 173, 196, 216, 229 150, 151, 152, 153, 154, 155, 157, 160,
pollution, ix, xi, 62, 64, 65, 68, 74, 114, 161, 162, 165, 172, 173, 178, 179, 181,
272, 285, 289, 291 188, 194, 200, 226, 231, 233
population, viii, 23, 36, 40, 61, 63, 71, 101, reticulum, 124, 200, 211
141, 153, 160, 177, 210 rheumatic diseases, 98, 117
priming, xi, 24, 145, 194, 235, 236, 239, rheumatoid arthritis, ix, 89, 90, 97, 101,
243, 244, 248, 253, 254, 255, 257, 259, 104, 110, 113, 114, 115, 116, 117, 118,
263, 265, 267 119, 120, 121, 122, 123, 124, 125, 126,
pro-inflammatory, 15, 34, 36, 37, 38, 46, 127, 171, 189, 196, 221, 228
73, 78, 93, 98, 106, 140, 141, 143, 144, rheumatoid factor, 97, 100
Index 305
S synthesis, 71, 95, 106, 108, 113, 144, 177,

science, 22, 23, 28, 32, 124, 182, 186, 188, 178, 179
189, 190, 198, 199, 202, 225 systemic lupus erythematosus, 105, 171,
secretion, ix, 11, 20, 62, 71, 93, 101, 107, 209, 218, 221, 232
108, 113, 127, 133, 143, 148, 152, 154,
161, 165, 167, 168, 173, 176, 183, 190, T
201, 211, 212, 214, 215, 220, 222, 223, Th1, vii, 3, 4, 5, 17, 73, 76, 179
225 Th17, ii, vii, 3, 4, 5, 41, 79, 155
sensing, 18, 118, 121, 151, 172 Th2, vii, 3, 4, 5, 16, 40, 71, 74, 75, 76, 79,
sensors, 105, 141, 148, 165, 223 80, 152, 179, 278, 286
serum, 12, 36, 39, 40, 74, 99, 100, 106, therapeutic agents, 179
108, 109, 117, 153, 156, 172, 174, 216, therapeutic approaches, 46, 76, 170, 230
232 therapeutic effect, 180
severe asthma, 26, 40, 41, 42, 74, 76, 78, therapeutic targets, 76, 117, 227
79, 80 therapeutic use, 176
sex differences, 36, 37, 39, 40, 42, 43, 44 therapeutics, 181
sex hormones, vii, viii, 31, 32, 33, 35, 36, therapy, 16, 30, 43, 62, 69, 76, 78, 80, 81,
37, 38, 40, 41, 43, 45, 46, 69 172, 178, 180, 183, 184, 186, 187, 192,
signaling cascade MAPK, 90 204, 210, 217, 218, 221, 225, 230
signaling pathway, ix, x, 13, 89, 91, 94, 95, thrombosis, 148, 167, 183, 204, 208, 213
97, 106, 108, 111, 112, 114, 116, 127, TLRS, v, vii, 89, 90, 96, 105, 110
140, 148, 150, 170, 172, 211, 233 TNF-α, 91, 93, 106, 113, 143, 144, 145,
signaling pathways, ix, x, 89, 91, 94, 95, 146, 152, 154, 155, 157, 158, 159, 160,
97, 108, 111, 112, 114, 140, 150, 172, 161, 163, 164, 165, 169, 177, 179
211, 233, 235, 245, 246, 269, 274, 278, tolerance, xi, 12, 22, 104, 171, 236, 241,
288, 292 252, 254, 255, 260, 262
skin, 8, 16, 17, 91, 120, 126, 152, 201, 202, trafficking, 96, 105, 134, 136, 137, 142,
227 172, 186, 194, 198
stem cells, 140, 176, 187, 191, 192, 200 trained immunity, xi, 236, 257, 258, 260,
stimulation, 14, 34, 35, 39, 74, 108, 126, 261, 264
190 transcription, ix, x, 4, 5, 6, 9, 14, 23, 25,
stress, 124, 172, 173, 191, 200, 228 71, 89, 95, 98, 107, 111, 141, 148, 155,
suppression, x, 109, 112, 130, 140, 151, 166, 170, 176, 182, 185
161, 232 transcription factors, ix, 4, 5, 7, 9, 14, 71,
survival, 9, 14, 37, 77, 93, 100, 101, 111, 89, 107, 111
112, 122, 125, 139, 141, 155, 156, 160, transferrin, 92, 131, 132, 200, 216
163, 168, 169, 189, 210, 222 transformation, 131, 132, 166, 182, 204,
susceptibility, 34, 38, 39, 42, 68, 71, 81, 219
97, 114, 118, 124, 137, 142, 145 treatment, viii, ix, x, 17, 41, 62, 63, 64, 65,
symptoms, 63, 64, 65, 66, 67, 68, 70, 76, 67, 70, 76, 77, 78, 79, 80, 90, 108, 113,
79, 80, 81, 172 114, 130, 151, 156, 157, 160, 162, 168,
synovial fluid, 92, 110, 173 181, 184
synovial membrane, 101, 104 tuberculosis, 14, 19, 122, 151, 179, 193
synovial tissue, ix, 89, 103, 106, 107, 110, tumor cells, x, 4, 6, 130, 133, 168, 181,
113, 119, 124 184, 217
306 Index
tumor growth, 167, 168, 232 vector-borne-disease, 236

tumor necrosis factor, 91, 93, 102, 139 vesicle, 183, 184, 187, 195, 202, 205, 207,
tumor progression, 168, 181 208, 213, 215, 225
tumor-associated neutrophil, 32, 236 viral infection, ix, 35, 37, 62, 65, 164
virology, 200, 201
U virus infection, 180
umbilical cord, 7, 10, 176, 204, 225, 231 viruses, 68, 91, 94, 143, 148, 149, 160,
164, 165, 167, 188
V
vaccine, 155, 162, 221, 224, 228, 234 W
vascular endothelial growth factor, 104, work environment, ix, 62
117 worldwide, 63, 74, 79, 81
vasoactive intestinal peptide, 8, 11

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Immunology and

Immune System Disorders

The Innate Immune System in Health and Disease:

The Innate Immune System in Health and Disease:

Vaccines: Operation Warp Speed, Regulation and Safety

IgG4 Related Disease: Illustrated Pathology of Novel Systemic Disease

Basics and Fundamentals of Immunology

Th17 Cells in Health and Disease

More information about this series can be found at

The Innate Immune System

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

ISBN:  H%RRN

Published by Nova Science Publishers, Inc. † New York

Preface .......................................................................................... vii

Chapter 6 Enhanced Innate Immune Response:

helper ILCs, because their main effector function is the production of

source of the stimulus that triggers the immunopathology; pollution particles,

efficient in combating foreign organisms but sufficiently benevolent to

The Role of Innate Lymphoid Cells

Bibiana Patricia Ruiz-Sánchez1,2,

Instituto Politécnico Nacional, Mexico City, Mexico

Innate lymphoid cells (ILCs) are lymphocytes that lack antigen-specific

In: The Innate Immune System in Health and Disease

Keywords: innate lymphoid cells, pathogen-associated molecular patterns,

Development and Differentiation of ILCs

somatic recombination and T cell development (De Obaldia and Bhandoola

Figure 2. Development and differentiation of ILCs. The development of ILCs in

ILC Receptors and Other Surface Markers

ILCs, through their many receptors, are activated in response to

Table 1. General characteristics of ILCs

Anatomical Distribution of ILCs

Role of ILCs in Tissue Homeostasis

Role of ILCs in the Immune Response to Pathogens

MIP1 (H. Li, Richert-Spuhler, et al. 2014). ILC3 undergo apoptosis in

Role of ILCS in the Immunopathology of Chronic

Obesity is a multifactorial disease with many complications. The chronic

Inflammation is often associated with cancer progression. In a mouse

ILC2 have been associated to the immunopathology of type I hypersensitivity

macrophages and with myeloid-derived suppressor cells (Bie et al. 2014).

Abt, M. C., B. B. Lewis, S. Caballero, H. Xiong, R. A. Carter, B. Susac, L. Ling,

structures." Nat Commun 6: 8280. https://doi.org/10.1038/ncomms9280.

immunity generated by lymphocyte activation." Nature 224 (5214): 38-42.

Immunol 47 (11): 1959-1969. https://doi.org/10.1002/eji.201746961.

antibody-dependent in vitro systems." J Exp Med 143 (4): 772-80.

23 (4): 685-98. https://doi.org/10.1016/j.cmet.2016.03.002. https://www.

R. M. Locksley. 2013. "Type 2 innate lymphoid cells control eosinophil

Satoh-Takayama, N., C. A. Vosshenrich, S. Lesjean-Pottier, S. Sawa, M. Lochner,

allergic lung inflammation." Nature 549 (7672): 351-356. https://doi.org/

The Innate Immune Response

Historically, both basic and clinical biomedical research have mainly

In: The Innate Immune System in Health and Disease

Keywords: cancer, inflammation, metastasis, tumor-associated

Sex and Sex Hormones as Modulators

immunosup-pressive effects of this androgen, there is the dampening of

Sex- and Sex Hormone-Induced Differences

As mentioned above, the innate immune system consists of a diverse network

Natural Killer Cells

In humans, NK cells are commonly defined as CD3−CD56+ cells and

the main function of decidual NK cells is not against infections as their

Group 2 Innate Lymphoid Cells

First identified as non-B, non-T (NBNT), IL-13-producing, and IL-25-

testosterone levels, suggested that these female sex hormones participate in

ISBN: H%RRN