Trends in

Parasitology
Review

Leishmaniasis: the act of transmission

Tiago D. Serafim , 1 Iliano V. Coutinho-Abreu , 1 Ranadhir Dey , 2 Ryan Kissinger ,3
Jesus G. Valenzuela , 1,* Fabiano Oliveira , 1,* and Shaden Kamhawi 1,*

The contribution of vector transmission to pathogen establishment is largely Highlights

underrated. For Leishmania, transmission by sand flies is critical to early survival The feeding behavior of sand flies is inti-
involving an irreproducible myriad of parasite, vector, and host molecules acting mately connected to vector competence
and success of Leishmania transmis-
in concert to promote infection at the bite site. Here, we review recent break-
sion. In the sand fly, a postinfected
throughs that provide consequential insights into how vector transmission of blood meal prevents loss of parasites
Leishmania unfolds. We focus on recent work pertaining to the effect of gut mi- and promotes transmissible infections.
crobiota, sand fly immunity, and changes in metacyclogenesis upon multiple More blood meals increase sand fly in-

blood meals, on Leishmania development and transmission. We also explore fectiousness through retroleptomonad
parasites. In addition, prolonged host
how sand fly saliva, egested parasite molecules and vector gut microbiota, and bleeding induces heme oxygenase-1
bleeding have been implicated in modulating the early innate host response to (HO-1) in macrophages, which dampens
Leishmania, affecting the phenotype of neutrophils and monocytes arriving at inflammation and promotes disease
tolerance.
the bite site.
Sand fly gut microbiota is critical to
Leishmania development and pro-
The Leishmania–sand fly–host triad motes transmission through host
Leishmaniasis is a vector-borne parasitic disease with a global footprint. It is caused by inflammasome-mediated interleukin
protozoa of the genus Leishmania and manifests in a variety of clinically distinct diseases. (IL)-1β that amplifies neutrophil
recruitment.
Despite the polymorphic nature of leishmaniasis, most naturally acquired infections are initi-
ated by inoculation of parasites into host skin via the bite of a blood-feeding phlebotomine Sand fly yellow proteins in saliva are
sand fly. bona fide neutrophil chemoattractants
that promote infection.
Leishmania transmission occurs at the parasite–vector–host interface and its success is de-
Neutrophils are central to acute inflam-
pendent on a dynamic multifactorial process influenced by all three. In the sand fly, Leishmania mation caused by bite-specific media-
parasites develop from amastigotes to infectious metacyclic promastigotes within the digestive tors, but dermal resident macrophages
tract [1]. At this point, the sand fly is infectious and can transmit the parasites as it attempts to are gaining attention as participants that
harbor Leishmania parasites early in
take another blood meal. These parasites are not transmitted alone. Over the past few de-
infection.
cades, several groups have demonstrated that vector-derived factors of both sand fly and
Leishmania origin, such as sand fly saliva [2,3] and the promastigote secretory gel (PSG)
(see Glossary) [4], are an integral part of vector transmission and facilitate parasite survival
and establishment in the host. The host immune response to a bite from an infectious sand
fly also contributes to transmission success, as highlighted by the ‘Trojan horse’ role of re- 1
Vector Molecular Biology Section,
cruited neutrophils in parasite survival [5]. Here, we update the literature on what is new in Laboratory of Malaria and Vector
Research, National Institute of Allergy
Leishmania development within the sand fly, expand the repertoire of the infectious inoculum, and Infectious Diseases, National
and reveal the importance of multiple blood meals in infection and transmission. We also dis- Institutes of Health, Rockville, MD
cuss recent advances in our understanding of the early interaction of Leishmania parasites 20852, USA
2
Laboratory of Emerging Pathogens,
with their host cells in the skin at the bite site. Division of Emerging and Transfusion
Transmitted Diseases, Center for
Newly identified determinants for Leishmania parasite growth and development Biologics Evaluation and Research,
Food and Drug Administration, Silver
inside the insect vector Spring, MD 20993, USA
Sand fly gut microbiota 3
Visual and Medical Arts Unit, Research
It is well established that, for sand flies to become 'infectious' with Leishmania, the parasite has to Technologies Branch, Rocky Mountain
Laboratories, Institute of Allergy and
develop inside the sand fly gut into infective metacyclic promastigotes. Once a sand fly takes a Infectious Diseases, National Institutes
blood meal from a Leishmania-infected reservoir host the parasite must survive the physical of Health, Hamilton, MT 59840, USA

976 Trends in Parasitology, November 2021, Vol. 37, No. 11 https://doi.org/10.1016/j.pt.2021.07.003

Published by Elsevier Ltd.
Trends in Parasitology

and biochemical barriers imposed by the vector and by-products of blood digestion [1]. *Correspondence:
jvalenzuela@niaid.nih.gov
Recently, sand fly gut microbiota was established as another critical determinant for parasite (J.G. Valenzuela),
growth and development [6,7]. The bacterial communities present in the gut of different sand loliveira@niaid.nih.gov (F. Oliveira), and
fly species, either from laboratory or field specimens, have been recently reviewed [8,9]. As a skamhawi@niaid.nih.gov (S. Kamhawi).

permanent resident of the sand fly midgut, every time a sand fly takes an infectious blood
meal, the ingested Leishmania parasites will encounter these microbes [8]. In laboratory condi-
tions, a significant loss in diversity of sand fly gut microbiota occurred as a result of infection
with Leishmania [6]. Sand flies given a Leishmania infantum-infected blood meal showed a
gradual loss of gut microbial diversity over time coincidental with an increasing parasite burden
[6]. Strikingly, elimination of the sand fly gut microbiota by antibiotic treatment resulted in rapid
impairment of Leishmania multiplication, preventing the development of infectious metacyclic
promastigotes [6,7]. It has been proposed that removing the microbiota changes the concen-
tration of sugar present in the sand fly gut to an osmolarity that is lethal for the Leishmania par-
asite [7]. Furthermore, Campolina et al. [9] showed that in vivo coinfection experiments with
wild-caught Lutzomyia longipalpis that combined Leishmania major, L. infantum, or Leishmania
amazonensis parasites with Serratia, Lysinibacillus, or Pseudocitrobacter bacteria resulted in a
significant reduction in the number of surviving parasites compared with controls. This
indicates that using specific bacteria as paratransgenic agents to impair parasite growth
has potential as a tool for biological control [8–10]. Overall, these new findings demonstrate
the critical role of the insect gut microbiota in parasite growth, development, and vector
competence.

Sand fly immune genes and Leishmania

Induction of immune genes in mosquitoes, including the immune deficiency (IMD)
pathway, the Toll pathway, and the JAK/STAT pathway, were critical for Plasmodium
clearance [11–13]. However, ookinetes traverse the mosquito midgut epithelium, damaging
it in the process, while Leishmania parasites remain extracellular in the lumen of the sand fly
midgut [1]. Despite the presence of a large number of Leishmania parasites, the sand fly
midgut is not immunologically responsive to Leishmania infection when compared with
blood-fed sand flies [14–16]. The differential expression of sand fly midgut genes in
Leishmania-infected sand flies was assessed by RNA-Seq [14–16]. Surprisingly, only 113
differentially expressed midgut genes were observed upon Leishmania infection when com-
pared with blood-fed sand flies [15]. Importantly, immune genes from the insect gut were
not significantly upregulated in the presence of Leishmania [14–16]. On day 6 after infection
and onwards, multiple genes related to the metabolism of lipids and detoxification of xenobi-
otics were upregulated [15]. Overall, Leishmania seems to manipulate midgut genes from day
2 to day 4 after infection in order to survive midgut barriers related to structure and metabo-
lism, whereas it behaves as a commensal at day 12 to 14 after infection when the midgut im-
poses little difficulty for parasite development [14–16]. The presence of blood upregulates
transcripts from the Toll (Spätzle and GNBP3) and IMD pathways in sand flies [16].
However, these immune genes are not significantly modulated by the presence of
Leishmania [14–16] and are most likely reacting to the observed increase in sand fly gut bacteria
in the presence of blood [6]. Interestingly, knockdown of the negative regulator of the IMD pathway,
caspar, reduced Leishmania mexicana survival in Lu. longipalpis [17]. Similarly, knocking down the
transforming growth factor-beta (TGF-β) pathway, another negative regulator of the insect innate
immune response, resulted in L. infantum suppression in Lu. longipalpis [18]. One can postulate
that activation of the innate immune system by removing caspar and TGF-β may result in a
significant decrease in gut bacteria and consequently affect parasite growth and development.
The connection between the sand fly immune system, gut microbiota, and Leishmania survival
needs further investigation.

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The critical role of sand fly behavior and blood acquisition in Leishmania Glossary
development Concordant sand flies: sand flies that
The midgut life cycle after a single blood meal complete a gonotrophic cycle before
Decades of research had established the occurrence of a complex developmental process that taking another blood meal, that is, they
take one blood meal between egg
involves several well-defined morphological and functional Leishmania stages in the sand fly [1]. layings. This is in contrast to discordant
Based on experimental studies conducted after a single blood meal, the life cycle of Leishmania sand flies that do not need to complete a
parasites in the sand fly midgut was established as follows: amastigotes differentiate into gonotrophic cycle before taking another
procyclic promastigotes; this parasite stage then transforms to nectomonad promastigotes, blood meal, that is, they take more than
one blood meal between egg layings.
large forms that escape the deteriorating peritrophic matrix (PM) and anchor to the insect midgut CXCL1: a member of the CXCL class of
[1,19,20]. Anchoring to the midgut wall is a crucial event in the parasite life cycle and determines chemokines. This protein is involved in
vector competence for restrictive sand fly vectors through parasite lipophosphoglycan (LPG)- the inflammatory process and induces
chemotaxis of neutrophils and other
midgut receptor-specific interactions [1,19]. This is less clear for permissive vectors, in which
immune cells.
attachment has been proposed to occur through other interactions with O-glycans bearing Dermal resident macrophages
N-acetyl-D-galactosamine residues that are common on the midgut surface of permissive (DRMs): the most abundant resident
sand fly vectors [1,19]. However, blocking LuloG, an O-linked glycoprotein in the midgut of immune cell type in the skin. They are
involved in resistance against
Lu. longipalpis, transiently reduced but did not prevent the development of parasites in the mid-
pathogens, attraction of immune cells,
gut [21]. In another study, using LPG-deficient (Δlpg1 mutant) parasites in in vitro experiments, and tissue repair.
Coutinho-Abreu et al. demonstrated that L. infantum attachment to the midgut epithelium of Exosome: a type of extracellular vesicle
Lu. longipalpis is LPG-mediated, but that both wild-type and Δlpg1 mutant parasites devel- that contain cell constituents, including
protein, DNA, and RNA of the cells that
oped comparable mature infections in Lu. longipalpis [22]. These studies indicate that attach-
produced them.
ment to the midgut epithelium may not be crucial for retainment of parasites in the gut of G-protein-coupled receptor
permissive vectors [21]. As most of the parasites are lost when the digested blood is defecated, (GPCR): a cell-surface, seven-
only a few nectomonad promastigotes remain to continue the life cycle. Nectomonad transmembrane receptor that binds
extracellular ligands and transmits
promastigotes then differentiate to leptomonad promastigotes, a stage that multiplies and col- signals inside the cell to regulate
onizes the anterior part of the sand fly gut prior to differentiating to metacyclics, the infective processes including cell proliferation,
stage of Leishmania. A transcriptomic analysis of gene expression by the different Leishmania survival, and motility.
stages inside the sand fly gut was recently reported for L. major in Phlebotomus papatasi [23] IL-1β: interleukin-1 beta is a cytokine
(protein) produced by macrophages,
and for L. infantum in Lu. longipalpis sand flies [15] and reviewed in [24]. These studies identi- natural killer cells, monocytes, and
fied stage-specific differentially expressed transcripts that may be useful in assessing the neutrophils, that promotes inflammation.
maturity of infections in field-collected specimens. Recently, Giraud et al. [25] developed a This protein is processed to its active
form by the enzyme caspase 1, a
real-time qPCR based on small hydrophilic endoplasmic-reticulum-associated protein (sherp)
component of the inflammasome.
amplification from RNA to quantify infectious metacyclic stages in sand flies or skin. After Immune deficiency (IMD) pathway:
validation in field settings, such tools can help to elucidate transmission cycles in disease foci. a signaling pathway in insects that is
responsible for the recognition of Gram-
negative bacteria and which regulates
The impact of multiple blood meals on parasite development, growth, and sand fly infectiousness
the antibacterial defense response.
This account of the life cycle has been accepted for decades. However, it did not take into ac- JAK/STAT pathway: Janus kinase
count the recurrent blood-feeding behavior of sand flies and its potential impact on parasite de- and signal transducer and activator of
velopment. The bloodmeal is a nutritious source of proteins to produce eggs. In nature, sand flies, transcription (JAK/STAT) is a key
pathway responsible for induction of the
and other vectors of disease, take multiple blood meals throughout their life span [26,27]. It has
insect innate immune system and
long been appreciated that multiple blood feeding is of epidemiological relevance because it in- processes such as melanization and
creases the frequency of pathogen transmission and amplifies the pool of infected vectors by in- phagocytosis.
creasing the likelihood of feeding on an infected host. However, the effect of a second blood meal Neutrophil: a type of granulocyte which
is a phagocytic white blood cell that
on pathogen development in vectors has been underappreciated until recently. It has been pre- forms part of the innate immune system
viously observed that a second blood meal increases the presence of metacyclics in the thoracic and is the first type of cell to arrive at the
midgut of Leishmania-infected sand flies, potentially enhancing sand fly infectiousness [28]. More site of infection.
recently, multiple blood meals were shown to have a great impact on various stages of NLRP3 inflammasome: a protein
complex inside the cells involved in the
Leishmania development in the sand fly midgut, leading to the discovery of the process of reverse inflammatory process and responsible
metacyclogenesis and the characterization of a new developmental form, the retroleptomonad for cleavage of caspase 1 needed to
[29]. This resulted in a revision of the Leishmania life cycle inside the vector [29,30]. Figure 1 illus- produce active IL-1β.
trates the revised life cycle focusing on the impact of multiple blood meals in parasite survival and

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Permissive sand fly vectors: sand

flies that support the development of
multiple Leishmania species.
Promastigote secretory gel (PSG): a
proteophosphoglycan-rich gel-like
solution secreted by Leishmania
parasites inside the sand fly gut.
Restrictive sand fly vectors: sand
flies that support the development of
one specific Leishmania species.
Toll pathway: a signaling pathway in
insects that is responsible for the
recognition of fungi and Gram-positive
bacteria and the induction of
antimicrobial peptides.
Vector competence: the ability of a
disease vector to support pathogen
development to maturity, culminating in
its successful transmission to a host.
Vectorial capacity: a measurement of
the potential of a vector of disease to
transmit a pathogen.

Trends in Parasitology

Figure 1. Leishmania development throughout the life span of a sand fly vector.

For a Figure360 author presentation of Figure 1, see the figure legend at https://doi.org/10.1016/j.pt.2021.07.003
Each panel depicts parasite development over 6 days, approximating the period between a blood meal and egg laying.
(A) After a sand fly takes its first infected blood meal (BM1), amastigotes are released into a blood bolus confined by a
peritrophic matrix (PM). Amastigotes differentiate into procyclic promastigotes, a sluggishly dividing form that gives rise to
nectomonad promastigotes (NPs). NPs exit a disintegrating PM and anchor to the midgut epithelium. NPs differentiate to
leptomonad promastigotes (LPs). LPs divide efficiently, establishing the midgut infection. A few haptomonad
promastigotes (HPs), whose origin remains uncertain, attach to the chitinous stomodeal valve (SV). (B) The infected sand
fly takes another blood meal (BM2), promoting LP multiplication. LPs differentiate into infectious nondividing metacyclic
promastigotes (MPs) that accumulate in the thoracic midgut; more HPs attach to the SV. (C) The sand fly takes another
blood meal (BM3), egesting MPs into the host (transmission). Fresh blood causes MPs to differentiate into rapidly dividing
retroleptomonad promastigotes (RPs). RPs represent the majority of midgut parasites over the next 3 days. RPs and
residual LPs differentiate to metacyclic promastigotes, amplifying sand fly infectiousness. Numerous HPs are now
anchored to the SV, forming the haptomonad parasite sphere (HPS), blocking the SV, and further enhancing transmission.
(D) If a sand fly takes more blood meals (BM4+), intake of fresh blood reinitiates the cycle of MP amplification through RP
division and enlarges the HPS, further blocking the SV. Sequential events are largely based on [22].

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development inside the gut. Apart from a few species [26], most sand flies are concordant in
their feeding behavior, and from observations of colony reared sand flies, females lay their eggs
5–8 days after blood feeding [31,32]. Taking this into consideration, we made two assumptions
in illustrating the revised cycle in Figure 1: the sand fly is concordant and it lays its eggs 6 days
after each blood meal. Under this scenario, when a sand fly takes a Leishmania-infected blood
meal, the Leishmania parasite develops from the amastigote to the leptomonad stage
(Figure 1A). A small number of leptomonad promastigotes and a small number of attached
haptomonad promastigotes are observed at this time point. The influx of fresh blood from a sub-
sequent meal induces parasite multiplication [29], and this ensures that the few leptomonads re-
maining after egestion of the initial infected blood meal successfully colonize the insect midgut
(Figure 1B). We would like to emphasize that, if a sand fly species takes longer to lay its eggs,
there is a small possibility that it may become infectious prior to taking its second blood meal.
Similarly, if a sand fly takes more than one blood meal before laying its eggs, it may become infec-
tious earlier. These unknown variations will impact sand fly infectiousness, highlighting the need
for field-based studies to establish the blood feeding behavior of vector species. Another dra-
matic change is observed when an infectious sand fly takes a blood meal (Figure 1C). The
metacyclic promastigote, once considered to be terminally differentiated, transforms into a
retroleptomonad promastigote in a process named reverse-metacyclogenesis [29]. One key as-
pect of retroleptomonad promastigotes is their replicative capacity that results in a significant in-
crease in the number of parasites in the sand fly gut, and consequently a greater population of
infective metacyclic promastigotes [29]. As both retroleptomonad and leptomonad
promastigotes are replicative, the contribution of each stage to the enrichment of metacyclics
will depend on the percentage of metacyclics in the infected sand fly prior to taking a blood
meal. Enhancement of metacyclic parasites was shown to increase the infectivity of sand flies;
a single fly that has taken a subsequent blood meal produced a higher frequency of cutaneous
leishmaniasis (CL) lesions in rodents as compared with one that has only taken the initial infected
blood meal [29], further reinforcing the relevance of multiple bloodmeals for vectorial capacity of
the sand fly vector. Multiple blood meals also impacted the formation of the haptomonad parasite
sphere (HPS) [29]. The size and location of the HPS indicates that it is a major contributor to par-
asite blockage of the sand fly foregut (Figure 1C). Blockage of the anterior part of the thoracic mid-
gut caused by PSG and the HPS, combined with the destruction of the stomodeal valve by
chitinases [33], is critical for Leishmania transmission. Though experimental evidence was not ob-
tained due to the difficulty of keeping infected sand flies alive after blood feeding in laboratory con-
ditions, the replicative nature of the retroleptomonad stage implies that the infectiousness of a
sand fly is likely being perpetually enhanced by more blood meals, transforming our understand-
ing of its transmission potential (Figure 1D). This is of particular relevance in light of the variable
efficiency by which sand flies transmit under experimental conditions [25,34–36]. Additionally,
the number of parasites that initiate a sand fly infection may also be variable, influenced by a
patchy parasite distribution [37,38]. The effect of multiple blood meals on sand fly infections
under these conditions needs to be evaluated since experimentally infected sand flies that were
blood-fed twice produced homogeneous infections that were less variable compared with
those that were fed only once [29]. In fact, a probabilistic model for disease transmission incorpo-
rating parasite patchiness and multiple blood meals showed that the former is less important for
successful transmission by longer-lived sand flies that take multiple blood meals [39]. Then, for a
successful transmission event to occur, or to increase the frequency of successful transmission
events [29,39], a subsequent blood meal becomes a critical part of the parasite's life cycle in na-
ture. This becomes even more relevant in natural transmission scenarios considering the low
Leishmania infection rates observed in field vector populations [40–42]. Interestingly, limited
data provided in Ashwin et al. [34] did not find a significant difference in the number of metacyclics
between single- and double-fed sand flies. This further highlights the need for more experimental

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and field-based studies of different vector–parasite pairs by independent groups to fully under-
stand the scope and significance of this phenomenon on Leishmania development in sand
flies. Nevertheless, the impact of a subsequent blood meal on enhancing vector infectiousness
is not limited to sand flies and Leishmania [28,29,43] and was recently demonstrated for other
pathogens including mosquitoes infected with viruses [44] and Plasmodium parasites [45].

Early immune events at the bite site govern Leishmania survival and disease
outcome
The skin serves not only as a physical barrier against external insults but also harbors a wide va-
riety of immune cells that patrol it and mount an immune response upon challenge [46]. Skin-res-
ident innate immune cells are considered as the first line of defense against invading pathogens.
Innate cells armored with an array of pattern-recognition receptors (PRRs) detect signals from
tissue injury and pathogens alike and are among the most studied with respect to infectious
diseases [47–49].

During a bite from an uninfected or Leishmania-infected sand fly, several sequential events occur
at the skin interface. Irrespective of infection status, sand fly bites result in multiple proboscis-
induced microinjuries to the skin caused by laceration of capillaries and vascular leakage aug-
mented by vasodilators and anticoagulants in saliva [2]. Additionally, infected sand flies deliver
parasites together with gut bacteria and Leishmania-derived exosomes bathed in PSG [50].
As a consequence, an acute inflammatory response develops after a Leishmania-infected sand
fly bite (Figure 2). Observed within minutes, this bite-specific response results in an intense and
sustained recruitment of neutrophils and monocytes to the wound and has been implicated in
the early survival and establishment of Leishmania parasites [51–53]. Potent neutrophil attrac-
tants to the bite site have been recently identified, including interleukin (IL)-1β and CXCL1
from the host, and yellow salivary proteins from the sand fly, emphasizing the redundancy of
this system and the central role that neutrophils play in the early inflammatory response to vector
bites [51–54]. Similarly, enhanced neutrophil recruitment promoted virus dissemination only after
needle inoculation of virus in the presence of mosquito bites [55], suggesting that exploitation of
the innate immune response may be common in blood-feeding disease vectors and their
pathogens.

Bite-specific mediators of inflammation

As part of a complex infectious inoculum, the sand fly regurgitates some of its gut bacteria
along with Leishmania parasites, activating the NLRP3 inflammasome in neutrophils and in-
duces IL-1β generation as early as 3–6 h postinfected bites [51] as depicted in Figure 2A,B. Al-
though Leishmania can directly engage NLRP3 activation, bacteria-derived lipopolysaccharide
(LPS) induces the noncanonical activation pathway resulting in active IL-1β production [56,57].
Thereafter, IL-1β provides an autocrine signal that further recruits neutrophils following
infected-vector bites [51,53], identifying it as a major driver of acute inflammation after sand
fly bites (Figure 2B). Other than IL-1β, Leishmania-infected sand fly bites induce chemokines
such as CXCL1, CCL2, and CCL3, among others [51,53]. CXCL1 induces the recruitment of
neutrophils, while CCL2 and CCL3 target recruitment of monocytes. In leishmaniasis, another
bite-mediator of inflammation is sand fly saliva. The primary function of saliva is to disrupt he-
mostasis and prolong bleeding at the bite site. However, it has been well established that cer-
tain salivary proteins are potent modulators of the host immune response [2,3,50,58].
Recently, it was demonstrated that the spreading factors in Lu. longipalpis saliva, the endonu-
clease Lundep and the hyaluronidase LuloHya, each exacerbated Leishmania infection by pro-
moting inflammatory cell recruitment, particularly neutrophils, to the inoculation site [59].
Blocking LuloHya activity by passive immunization led to a significant reduction in blood

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Figure 2. Vector-derived factors govern parasite establishment in the skin after an infected bite. (A) A sand fly
bites the host skin, lacerating blood capillaries to form a blood pool containing the infectious inoculum (II); the II is
composed of metacyclic promastigotes (MPs) and vector-derived factors, including parasite exosomes, gut microbiota
bathed in a sticky promastigote secretory gel (PSG) that blocks the sand fly foregut; within 3 h, salivary proteins
counteract host hemostasis to prolong bleeding. Neutrophils arriving at the bite site capture microbiota and parasites,
activating their inflammasomes to produce interleukin (IL)-1β. (B) IL-1β attracts more neutrophils to the bite site in a
positive autocrine loop where they shelter MPs from the acute inflammatory environment. (C) Monocytes arrive at the bite
site by 18 h; together with dermal resident skin macrophages, monocytes phagocytose MPs and collaborate in the clean-
up of extravascular red blood cells (RBCs); RBC ingestion leads to the release of carbon monoxide (CO), an anti-
inflammatory end-product of heme degradation by heme-oxygenase 1; CO reduces levels of IL-1β and other inflammatory
mediators, promoting disease tolerance. (D) Leishmania parasites establish infection in macrophages that are either
retained in the skin or migrate via the lymph nodes and lymphatics to the liver, spleen, and bone marrow, causing
cutaneous or visceral leishmaniasis, respectively. The timeline given is approximative and is largely based on [51,53].

feeding success. Moreover, humoral immunity triggered by vaccination with these molecules
reduced L. major lesion size and parasite load in infected mice ears [59]. Furthermore, a
novel neutrophil chemotactic activity was attributed to members of the sand fly salivary yellow
protein (YLWP) family [54]. Of note, these YLWP proteins lack any known classical motifs or
structural similarities to chemokines or other types of chemoattractants. The sand fly YLWP re-
cruited neutrophils through activation of a still unknown G-protein-coupled receptor
(GPCR) on the neutrophil surface. Importantly, when YLWPs in the salivary gland homogenate
of Phlebotomus duboscqi were neutralized with specific antibodies, the direct neutrophil

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migration was abrogated. To date, two other vector-derived factors of parasite origin have
been identified as consequential for Leishmania infection and disease outcome. Leishmania
exosomes [60] and PSG [61] are actively shed by Leishmania parasites and accumulate inside
the sand fly midgut, and both have been shown to be regurgitated jointly with parasites during
transmission (Figure 2A) and to augment leishmaniasis severity. Leishmania exosomes were
shown to contain parasite virulence factors, including the well-studied GP63 [60,62–64],
while also serving as a safe haven for the Leishmania RNA virus1 [65]. PSG produced by var-
ious Leishmania species in natural or experimentally competent sand fly vectors has been
shown to also exacerbate leishmaniasis outcome [25,66–68]. Within 4 h, PSG injected into
the skin recruits neutrophils and macrophages synergistically with salivary proteins [67]. Fur-
ther, PSG induces expression of several chemokines and proinflammatory cytokines, including
IL-1β, IL-6, and tumor necrosis factor (TNF)-α, while promoting wound repair, by insulin-like
growth factor (IGF-1) secretion, driving alternative macrophage activation via arginase-1 induc-
tion that sustains both Leishmania survival and re-epithelization of the sand fly bite wound [69].

Inflammation at the bite site promotes Leishmania establishment

The aftermath of an increased neutrophil recruitment to the bite site is a larger number of viable
transient Leishmania hosts cells [52]. When sand fly chemoattractants YLWPs were coinjected
in mice ears with 500 L. major metacyclics, the resulting cutaneous lesions were aggravated,
with the effect being reversed if YLWPs were neutralized by antibodies or neutrophils were tran-
siently depleted [54]. Similar to CL, abolishing neutrophil recruitment to the bite site either by re-
ducing gut microbiota in L. donovani-infected sand flies or by blocking the effect of IL-1β in mice,
compromised parasite dissemination from the skin to the spleen [51], further highlighting the im-
portance of the neutrophilic response in leishmaniasis. Despite previous work pointing to neutro-
phils as the first cells to be infected during Leishmania transmission by sand fly bite [52], a more
comprehensive picture was reported for CL, in which dermal resident macrophages (DRMs)
appear to be the main infected cells from 1 to 24 h after sand fly transmission of L. major [70]. The
DRMs are present in steady-state mouse skin and carry several M2 activation markers and ex-
press the mannose receptor on their surface [71]. Notably, parasitized neutrophils are taken up
by DRMs, contributing to their high early infection rates. At 5 days and 12 days post-
transmission, the percentage of infected DRMs is reduced in favor of neutrophils, monocyte-
derived dendritic cells, inflammatory monocytes, and eosinophils [70]; eosinophil recruitment to
the skin has been shown to be dependent on DRMs through secretion of CCL24 [72]. Likewise,
in vitro studies have shown that Lu. longipalpis saliva in combination with L. infantum induces IL-
10 and IL-17 secretion from human peripheral blood mononuclear cells (PBMCs), and that in-
fected neutrophils in the presence of bone-marrow-derived macrophages induce the release of
TGF-β and prostaglandin E (PGE2), increasing the number of infected macrophages after cocul-
ture in the presence of Lu. longipalpis saliva [73]. This in vitro study corroborates the role of saliva
in promoting Leishmania infection through interactions between neutrophils and macrophages.
Using intravital microscopy, a discrepancy was observed in bite-site spots, where some had
heavy swarming of neutrophils compared with adjacent areas despite the parasite's presence
in both skin compartments [70]. We could speculate that this could be driven by sand fly-derived
factors, including gut microbiota, PSG, or the salivary neutrophil chemoattractant YLWP
[51,54,67]. Previous reports indicated that L. major remains viable, but not replicative, in parasit-
ized neutrophils prior to transitioning to recruited bone marrow-originated inflammatory mono-
cytes [74]. Chaves et al. revisited these data and suggest that parasites in infected neutrophils
transition to DRMs [70] and that infected cell numbers seem to be stable until day 8 when an ex-
ponential parasite replication starts. At ≥7 days postinfection, in vivo studies show an intricate
picture in which interferon-gamma (IFN-γ) knockout (KO)-mice – infected with L. amazonensis
or L. major via sand fly bites or needle inoculation – are identical to wild-type (WT) animals in

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 Trends in Parasitology

both parasite numbers and cutaneous lesion development despite highly polarized T helper (Th)2
versus Th1 environments, respectively [75]. In this work, the onset of parasite exponential multi-
plication in WT animals occurred in an environment rich in IFN-γ concomitantly with the presence
of alternatively activated macrophages that are MHCII+PDL2+ [75]. The early recruitment of neu-
trophils (<4 days) followed by monocytes to the bite site, and the similarity of the IFN-γ KO mice
and WT responses, indicates that IFN-γ may not be a big player in the early stages of the
L. amazonensis infection. Nevertheless, the monocyte second wave from day 4 onwards
seems to be driven by IFN-γ-inducible chemokines such as MCP-1 and CXCL-9, providing an
additional source of permissive host cells that promote parasite multiplication [75]. Overall,
these studies highlight the complexity of immune events following transmission that transcend
a Th1 versus a Th2 phenotype. Another facet to inflammation and its role in leishmaniasis pathol-
ogy is the observation that infection with L. major induces dysbiosis in skin-residing host micro-
biota that is not limited to lesions but extends to distal sites and other co-housed mice [76].
Importantly, skin dysbiosis exacerbated CL pathology without an effect on parasites due to an
aggravated inflammatory response [76]. This study further emphasizes the complexity of the in-
flammatory milieu in the skin of infected individuals that is distinct from normal skin and may im-
pact vector transmission or pick-up of parasites by sand flies.

Bleeding controls inflammation at the bite site

Blood-feeding arthropods have evolved complex feeding mechanisms whose primary objective
is to bring more blood from circulation to the bite site. As for many hematophagous disease
vectors, sand fly saliva facilitates blood feeding through redundant inhibition of platelet aggrega-
tion, blood coagulation, and vasoconstriction, and the inoculation of spreading factors such as
endonucleases and hyaluronidases [2,3,50,58]. This ensures continued bleeding as sand flies re-
peatedly probe the skin and lacerate capillaries and vessels to create a pool of blood from which
they feed. This prolonged bleeding, occurring simultaneously with tissue injury, is a mostly
overlooked facet particular to transmission of vector-borne pathogens. Recently, our group dem-
onstrated that leakage of blood into tissue during the biting process results in the production of
heme oxygenase-1 (HO-1), a cytoprotective enzyme that catabolizes heme to mitigate heme-
mediated cell toxicity [53]. Both resident and monocyte-derived macrophages participate in
this process, engulfing extravascular red blood cells (RBCs) or their constituents (Figure 2C). In-
duction of HO-1 by bleeding is distinct from tissue injury and the wound healing response [77],
and is a robust and universal response to bites from hematophagous arthropods [53]. Of note,
saliva of the sand fly Lu. longipalpis was shown to directly induce HO-1 in macrophages partici-
pating in its early induction at the bite site [78]. The induction of HO-1 at the bite site is of conse-
quence to Leishmania parasites. The HO-1 reaction produces carbon monoxide (CO), bilirubin,
and ferrous iron; CO and bilirubin are anti-inflammatory and antioxidant end-products of heme
cleavage, and they participate in control of inflammation and tissue injury [79,80]. The transient
inhibition of HO-1 protracted the acute inflammatory response observed after infected sand fly
bites and aggravated pathology of CL lesions without a direct effect on Leishmania parasites
[53]. Since disease tolerance is defined as a host defense strategy to control disease pathology
without targeting the pathogen itself [81,82], this implicated the early induction of HO-1 after
sand fly bites in disease tolerance to L. major [53]. However, we are uncertain if this is a general-
ized outcome that covers other forms of leishmaniasis. HO-1-mediated disease tolerance has
been reported for several diseases, including visceral leishmaniasis, malaria, Chagas' disease,
and toxoplasmosis [79]. However, these observations were mostly made at later established
stages of disease and as such are distinct from the early induction of HO-1 in the skin observed
after vector bites. Nevertheless, the shared prolongation of bleeding and the clear induction of
HO-1 in skin after arthropod bites places HO-1 at the center of events controlling vector-borne
disease initiation [53].

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Trends in Parasitology

In summary, transmission of Leishmania is facilitated by vector-derived and host factors particular Outstanding questions
to infected sand fly bites. These early events are likely critical to long-term establishment of leish- Are observations of the effect of gut
maniasis (Figure 2D). microbiota of laboratory-reared sand
flies on Leishmania development and
transmission mirrored in wild-caught
Concluding remarks specimens?
A phlebotomine sand fly was once believed to be just 'a messenger' that carries Leishmania par-
asites between hosts. Seminal studies revealed a far more complex picture with a critical role for Can insect microbiota render vectors
refractory to infection, opening an un-
sand fly-derived factors in Leishmania development and transmission success. These include a
tapped source of biological control
new appreciation of the importance of vector gut microbiota to the development of Leishmania strategies to curb leishmaniasis?
parasites, both in the vector sand fly and the mammalian host, potentially providing us with
new strategies for leishmaniasis control (see Outstanding questions). Furthermore, we need to Can we manipulate the sand fly innate
immune system through modulation of
address knowledge gaps in fundamental aspects of sand fly behavior, such as the natural life gut microbiota to indirectly influence
span of different vector species that were shown to be vital to sand fly infectiousness (see Leishmania development in the midgut?
Outstanding questions). Pertinently, we need to use our increasing knowledge of bite-specific
Of consequence for parasite
characteristics of the host immune response following vector-transmission of Leishmania to ex-
multiplication and transmission, how
plore whether it can be modulated to ameliorate disease outcomes (see Outstanding questions). long is the natural life span of sand flies
As most of these observations were made using laboratory-reared sand flies and animal models in their natural biotopes, how often do
of infection, it is also time for us to turn our attention to disease foci to validate whether the signif- they take a blood meal during their life
span, and what happens if a sand fly
icance of vector components, such as sand fly gut microbiota or multiple blood meals, is main-
takes more than one infected blood
tained under natural field conditions. Nevertheless, current research continues to indicate that meal?
vector components may provide unprecedented opportunities for leishmaniasis control.
How do animals that are refractory
to leishmaniasis (such as chickens),
Acknowledgments
but are a common source of sand fly
This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious blood meals, affect the efficacy of trans-
Diseases, and by the Center for Biologics Evaluation and Research, Food and Drug Administration. This publication reflects mission and severity of leishmaniasis?
the views of the authors and should not be considered to represent FDA’s policies or views.
What is the role of the HPS in
transmission?
Declaration of interests
The authors declare no competing interests.
What are the molecular markers
of retroleptomonad compared
References with metacyclic and leptomonad
1. Dostalova, A. and Volf, P. (2012) Leishmania development in the sand phlebotomine fly Lutzomyia longipalpis. J. Biol. promastigotes?
in sand flies: parasite–vector interactions overview. Parasit. Chem. 287, 23995–24003
Vectors 5, 276 11. Meister, S. et al. (2005) Immune signaling pathways regulating Do commensal skin bacteria participate
2. Abdeladhim, M. et al. (2014) What's behind a sand fly bite? The bacterial and malaria parasite infection of the mosquito Anopheles
in the inflammatory response to
profound effect of sand fly saliva on host hemostasis, inflamma- gambiae. Proc. Natl. Acad. Sci. U. S. A. 102, 11420–11425
tion and immunity. Infect. Genet. Evol. 28, 691–703 12. Ramphul, U.N. et al. (2015) Plasmodium falciparum evades mos-
Leishmania at the bite site, and are they
3. Rohousova, I. and Volf, P. (2006) Sand fly saliva: effects on host quito immunity by disrupting JNK-mediated apoptosis of invaded relevant to leishmaniasis outcome?
immune response and Leishmania transmission. Folia Parasitol. midgut cells. Proc. Natl. Acad. Sci. U. S. A. 112, 1273–1280
(Praha) 53, 161–171 13. Garver, L.S. et al. (2012) Anopheles Imd pathway factors and Does skin dysbiosis in Leishmania-
4. Rogers, M.E. et al. (2002) The role of promastigote secretory gel in effectors in infection intensity-dependent anti-Plasmodium action. infected individuals influence the early
the origin and transmission of the infective stage of Leishmania PLoS Pathog. 8, e1002737
events occurring at the bite site, and
mexicana by the sandfly Lutzomyia longipalpis. Parasitology 124, 14. Coutinho-Abreu, I.V. et al. (2020) Leishmania infection induces a
495–507 limited differential gene expression in the sand fly midgut. BMC
would blood feeding on these individ-
5. Peters, N.C. et al. (2008) In vivo imaging reveals an essential Genom. 21, 608 uals interfere with gut microbiota
role for neutrophils in leishmaniasis transmitted by sand flies. 15. Coutinho-Abreu, I.V. et al. (2020) Distinct gene expression pat- composition?
Science 321, 970–974 terns in vector-residing Leishmania infantum identify parasite
6. Kelly, P.H. et al. (2017) The gut microbiome of the vector stage-enriched markers. PLoS Negl. Trop. Dis. 14, e0008014 What is the contribution of damage
Lutzomyia longipalpis is essential for survival of Leishmania 16. Sloan, M.A. et al. (2021) The Phlebotomus papatasi systemic tran-
caused by the proboscis during
infantum. mBio 8, e01121-16 scriptional response to trypanosomatid-contaminated blood does
7. Louradour, I. et al. (2017) The midgut microbiota plays an not differ from the non-infected blood meal. Parasit. Vectors 14, 15 probing and feeding to the sand fly-
essential role in sand fly vector competence for Leishmania 17. Telleria, E.L. et al. (2012) Caspar-like gene depletion reduces bite skin reaction compared with
major. Cell. Microbiol. 19. https://doi.org/10.1111/cmi.12755 Leishmania infection in sand fly host Lutzomyia longipalpis. vector-derived factors?
8. Telleria, E.L. et al. (2018) Leishmania, microbiota and sand fly J. Biol. Chem. 287, 12985–12993
immunity. Parasitology 145, 1336–1353 18. Di-Blasi, T. et al. (2019) Lutzomyia longipalpis TGF-beta has a
Can we change the disease outcome
9. Campolina, T.B. et al. (2020) Tripartite interactions: Leishmania, role in Leishmania infantum chagasi survival in the vector.
by modulating the early immune
microbiota and Lutzomyia longipalpis. PLoS Negl. Trop. Dis. 14, Front. Cell. Infect. Microbiol. 9, 71
e0008666 19. Hall, A.R. et al. (2020) Glycan–glycan interactions determine response to vector components at
10. Diaz-Albiter, H. et al. (2012) Reactive oxygen species-mediated Leishmania attachment to the midgut of permissive sand fly the site at the bite?
immunity against Leishmania mexicana and Serratia marcescens vectors. Chem. Sci. 11, 10973–10983

Trends in Parasitology, November 2021, Vol. 37, No. 11 985


20. Sadlova, J. et al. (2018) Refractoriness of Sergentomyia schwetzi
to Leishmania spp. is mediated by the peritrophic matrix. PLoS
Negl. Trop. Dis. 12, e0006382
21. Myskova, J. et al. (2016) Characterization of a midgut mucin-like
glycoconjugate of Lutzomyia longipalpis with a potential role in
Leishmania attachment. Parasit. Vectors 9, 413
22. Coutinho-Abreu, I.V. et al. (2020) Binding of Leishmania infantum
lipophosphoglycan to the midgut is not sufficient to define vector
competence in Lutzomyia longipalpis sand flies. mSphere 5,
e00594-20
23. Inbar, E. et al. (2017) The transcriptome of Leishmania major de-
velopmental stages in their natural sand fly vector. mBio 8,
e00029-17
24. Alcolea, P.J. et al. (2019) Functional genomics in sand
fly-derived Leishmania promastigotes. PLoS Negl. Trop. Dis.
13, e0007288
25. Giraud, E. et al. (2019) Promastigote secretory gel from natural
and unnatural sand fly vectors exacerbate Leishmania major and
Leishmania tropica cutaneous leishmaniasis in mice. Parasitology
146, 1796–1802
26. Ghosh, K.N. and Bhattacharya, A. (1992) Gonotrophic nature of
Phlebotomus argentipes (Diptera: Psychodidae) in the labora-
tory. Rev. Inst. Med. Trop. Sao Paulo 34, 181–182
27. Mukhopadhyay, J. and Ghosh, K. (1999) Vector potential of
Phlebotomus duboscqi and P. papatasi: a comparison of feed-
ing behaviour, reproductive capacity and experimental infection
with Leishmania major. Ann. Trop. Med. Parasitol. 93, 309–318
28. Elnaiem, D.A. et al. (1994) Development of Leishmania chagasi
(Kinetoplastida: Trypanosomatidae) in the second blood-meal
of its vector Lutzomyia longipalpis (Diptera: Psychodidae).
Parasitol. Res. 80, 414–419
29. Serafim, T.D. et al. (2018) Sequential blood meals promote
Leishmania replication and reverse metacyclogenesis augment-
ing vector infectivity. Nat. Microbiol. 3, 548–555
30. Bates, P.A. (2018) Revising Leishmania's life cycle. Nat.
Microbiol. 3, 529–530
31. Marayati, B.F. et al. (2015) Attraction and oviposition preferences
of Phlebotomus papatasi (Diptera: Psychodidae), vector of Old-
World cutaneous leishmaniasis, to larval rearing media. Parasit.
Vectors 8, 663
32. Volf, P. and Volfova, V. (2011) Establishment and maintenance of
sand fly colonies. J. Vector Ecol. 36, S1–S9
33. Rogers, M.E. et al. (2008) Leishmania chitinase facilitates coloni-
zation of sand fly vectors and enhances transmission to mice.
Cell. Microbiol. 10, 1363–1372
34. Ashwin, H. et al. (2021) Characterization of a new Leishmania
major strain for use in a controlled human infection model. Nat.
Commun. 12, 215
35. Kimblin, N. et al. (2008) Quantification of the infectious dose of
Leishmania major transmitted to the skin by single sand flies.
Proc. Natl. Acad. Sci. U. S. A. 105, 10125–10130
36. Maia, C. et al. (2011) Experimental transmission of Leishmania
infantum by two major vectors: a comparison between a
viscerotropic and a dermotropic strain. PLoS Negl. Trop. Dis.
5, e1181
37. Doehl, J.S.P. et al. (2017) Skin parasite landscape determines host
infectiousness in visceral leishmaniasis. Nat. Commun. 8, 57
38. Kamhawi, S. and Serafim, T.D. (2017) Patchy parasitized skin
governs Leishmania donovani transmission to sand flies. Trends
Parasitol. 33, 748–750
39. Carmichael, S. et al. (2021) Variable bites and dynamic popula-
tions; new insights in Leishmania transmission. PLoS Negl.
Trop. Dis. 15, e0009033
40. Guimaraes, E.S.A.S. et al. (2017) Leishmania infection and blood
food sources of phlebotomines in an area of Brazil endemic for
visceral and tegumentary leishmaniasis. PLoS One 12,
e0179052
41. Chagas, E. et al. (2018) Composition of sand fly fauna (Diptera:
Psychodidae) and detection of Leishmania DNA (Kinetoplastida:
Trypanosomatidae) in different ecotopes from a rural settlement
in the central Amazon, Brazil. Parasit. Vectors 11, 180
42. Pech-May, A. et al. (2016) Assessing the importance of four
sandfly species (Diptera: Psychodidae) as vectors of Leishmania
mexicana in Campeche, Mexico. Med. Vet. Entomol. 30,
310–320
986 Trends in Parasitology, November 2021, Vol. 37, No. 11
Trends in Parasitology
43. Moraes, C.S. et al. (2018) Second blood meal by female Are DRMs and neutrophils the key cells
Lutzomyia longipalpis: enhancement by oviposition and its ef- to be targeted to prevent leishmaniasis
fects on digestion, longevity, and Leishmania infection. Biomed.
at its most vulnerable state?
Res. Int. 2018, 2472508
44. Armstrong, P.M. et al. (2020) Successive blood meals enhance
virus dissemination within mosquitoes and increase transmission Will studies in human and canine skin
potential. Nat. Microbiol. 5, 239–247 replicate the immunological findings
45. Shaw, W.R. et al. (2020) Multiple blood feeding in mosquitoes observed after vector bites in murine
shortens the Plasmodium falciparum incubation period and in- skin?
creases malaria transmission potential. PLoS Pathog. 16,
e1009131
46. Richmond, J.M. and Harris, J.E. (2014) Immunology and skin in
Is it possible to develop a vaccine
health and disease. Cold Spring Harb. Perspect. Med. 4, against arthropod-borne diseases by
a015339 targeting vector components?
47. Walter, K. et al. (2010) NALP3 is not necessary for early protec-
tion against experimental tuberculosis. Immunobiology 215,
804–811
48. Schroder, K. and Tschopp, J. (2010) The inflammasomes. Cell
140, 821–832
49. Chen, K.W. et al. (2014) The neutrophil NLRC4 inflammasome
selectively promotes IL-1beta maturation without pyroptosis
during acute Salmonella challenge. Cell Rep. 8, 570–582
50. Lestinova, T. et al. (2017) Insights into the sand fly saliva: Blood-
feeding and immune interactions between sand flies, hosts, and
Leishmania. PLoS Negl. Trop. Dis. 11, e0005600
51. Dey, R. et al. (2018) Gut microbes egested during bites
of infected sand flies augment severity of leishmaniasis via
inflammasome-derived IL-1beta. Cell Host Microbe 23, 134–143 e6
52. Peters, N.C. and Sacks, D.L. (2009) The impact of vector-medi-
ated neutrophil recruitment on cutaneous leishmaniasis. Cell.
Microbiol. 11, 1290–1296. https://doi.org/10.1111/j.1462-
5822.2009.01348.x
53. DeSouza-Vieira, T. et al. (2020) Heme oxygenase-1 induction by
blood-feeding arthropods controls skin inflammation and pro-
motes disease tolerance. Cell Rep. 33, 108317
54. Guimaraes-Costa, A.B. et al. (2021) A sand fly salivary protein
acts as a neutrophil chemoattractant. Nat. Commun. 12, 3213
55. Pingen, M. et al. (2016) Host inflammatory response to mosquito
bites enhances the severity of arbovirus infection. Immunity 44,
1455–1469
56. Kayagaki, N. et al. (2013) Noncanonical inflammasome activa-
tion by intracellular LPS independent of TLR4. Science 341,
1246–1249
57. Gurung, P. et al. (2015) An NLRP3 inflammasome-triggered
Th2-biased adaptive immune response promotes leishmaniasis.
J. Clin. Invest. 125, 1329–1338
58. Oliveira, F. et al. (2013) sand fly saliva-leishmania-man: the
trigger trio. Front. Immunol. 4, 375
59. Martin-Martin, I. et al. (2018) Immunity to LuloHya and Lundep,
the salivary spreading factors from Lutzomyia longipalpis,
protects against Leishmania major infection. PLoS Pathog. 14,
e1007006
60. Atayde, V.D. et al. (2015) Exosome secretion by the parasitic
protozoan Leishmania within the sand fly midgut. Cell Rep. 13,
957–967
61. Rogers, M.E. (2012) The role of leishmania proteophosphoglycans
in sand fly transmission and infection of the mammalian host. Front.
Microbiol. 3, 223
62. Olivier, M. and Fernandez-Prada, C. (2019) Leishmania and its
exosomal pathway: a novel direction for vaccine development.
Future Microbiol. 14, 559–561
63. Perez-Cabezas, B. et al. (2019) More than just exosomes:
distinct Leishmania infantum extracellular products potentiate
the establishment of infection. J. Extracell. Vesicles 8, 1541708
64. Marshall, S. et al. (2018) Extracellular release of virulence factor
major surface protease via exosomes in Leishmania infantum
promastigotes. Parasit. Vectors 11, 355
65. Atayde, V.D. et al. (2019) Exploitation of the Leishmania
exosomal pathway by Leishmania RNA virus 1. Nat. Microbiol.
4, 714–723
66. Rogers, M.E. et al. (2010) Leishmania infantum
proteophosphoglycans regurgitated by the bite of its natural
sand fly vector, Lutzomyia longipalpis, promote parasite establish-
ment in mouse skin and skin-distant tissues. Microbes Infect. 12,
875–879
Trends in Parasitology
67. Rogers, M. et al. (2009) Proteophosophoglycans regurgitated by
Leishmania-infected sand flies target the L-arginine metabolism
of host macrophages to promote parasite survival. PLoS
Pathog. 5, e1000555
68. Rogers, M.E. et al. (2004) Transmission of cutaneous leishman-
iasis by sand flies is enhanced by regurgitation of fPPG. Nature
430, 463–467
69. Giraud, E. et al. (2018) Leishmania proteophosphoglycans re-
gurgitated from infected sand flies accelerate dermal wound re-
pair and exacerbate leishmaniasis via insulin-like growth factor 1-
dependent signalling. PLoS Pathog. 14, e1006794
70. Chaves, M.M. et al. (2020) The role of dermis resident macro-
phages and their interaction with neutrophils in the early estab-
lishment of Leishmania major infection transmitted by sand fly
bite. PLoS Pathog. 16, e1008674
71. Lee, S.H. et al. (2018) Mannose receptor high, M2 dermal mac-
rophages mediate nonhealing Leishmania major infection in a
Th1 immune environment. J. Exp. Med. 215, 357–375
72. Lee, S.H. et al. (2020) M2-like, dermal macrophages are main-
tained via IL-4/CCL24-mediated cooperative interaction with eo-
sinophils in cutaneous leishmaniasis. Sci. Immunol. 5, eaaz4415
73. Teixeira, C.R. et al. (2018) Lutzomyia longipalpis saliva drives
interleukin-17-induced neutrophil recruitment favoring Leishmania
infantum infection. Front. Microbiol. 9, 881
74. Romano, A. et al. (2017) Divergent roles for Ly6C+CCR2+CX3CR1+
inflammatory monocytes during primary or secondary infection of
the skin with the intra-phagosomal pathogen Leishmania major.
PLoS Pathog. 13, e1006479
75. Carneiro, M.B. et al. (2020) Th1-Th2 cross-regulation controls
early Leishmania infection in the skin by modulating the size of
the permissive monocytic host cell reservoir. Cell Host Microbe
27, 752–768 e7
76. Gimblet, C. et al. (2017) Cutaneous leishmaniasis induces
a transmissible dysbiotic skin microbiota that promotes skin
inflammation. Cell Host Microbe 22, 13–24 e4
77. Winn, N.C. et al. (2020) Regulation of tissue iron homeostasis:
the macrophage 'ferrostat'. JCI Insight 5, e132964
78. Luz, N.F. et al. (2018) Lutzomyia longipalpis saliva induces heme
oxygenase-1 expression at bite sites. Front. Immunol. 9, 2779
79. Silva, R. et al. (2020) Heme oxygenase-1 in protozoan infections:
A tale of resistance and disease tolerance. PLoS Pathog. 16,
e1008599
80. Soares, M.P. and Hamza, I. (2016) Macrophages and iron
metabolism. Immunity 44, 492–504
81. Martins, R. et al. (2019) Disease tolerance as an inherent compo-
nent of immunity. Annu. Rev. Immunol. 37, 405–437
82. Medzhitov, R. et al. (2012) Disease tolerance as a defense strategy.
Science 335, 936–941
Trends in Parasitology, November 2021, Vol. 37, No. 11 987