Semin Immunopathol
DOI 10.1007/s00281-017-0639-8
REVIEW
Cytokine storm and sepsis disease pathogenesis
Benjamin G. Chousterman 1,2 & Filip K. Swirski 3 & Georg F. Weber 4
Received: 11 December 2016 / Accepted: 5 May 2017
# Springer-Verlag Berlin Heidelberg 2017
Abstract Infectious diseases are a leading cause of death
worldwide. Sepsis is a severe clinical syndrome related to
the host response to infection. The severity of infections is
due to an activation cascade that will lead to an
autoamplifying cytokine production: the cytokine storm.
Cytokines are a broad category of relatively small proteins
(<40 kDa) that are produced and released with the aim of cell
signaling. Our understanding of the processes that trigger this
tremendous amount of cytokine production has made dramat-
ic progress over the last decades, but unfortunately, these find-
ings could not translate yet into effective treatments; so far, all
clinical trials targeting cytokine production or effects failed.
This review aims to summarize the pathophysiology of the
cytokine storm; to describe the type, effects, and kinetics of
cytokine production; and to discuss the therapeutic challenges
of targeting cytokines. New promising therapeutic strategies
focusing on the endothelium, as a source and a target of cyto-
kines, are described.
This article is a contribution to the special issue on Cytokine Storm in
Infectious Diseases - Guest Editor: John Teijaro
* Benjamin G. Chousterman
benjamin.chousterman@aphp.fr
1
Département d’Anesthésie-Réanimation, Hôpitaux Universitaires
Lariboisière–Saint-Louis, AP-HP, Paris, France
2
Inserm U1160, Hôpital Saint-Louis, Paris, France
3
Center for Systems Biology, Department of Imaging, Massachusetts
General Hospital, Harvard Medical School, Boston, MA 02114,
USA
4
Department of Surgery, University of Erlangen-Nürnberg,
Erlangen, Germany
Keywords Cytokine . Inflammation . Innate immunity .
Sepsis . Endothelium
Introduction
The World Health Organization (WHO) identifies infectious
diseases as a major cause of death worldwide, especially in
low-income countries where they represent the main cause of
death in 2012. Since the Italian physician Girolamo Fracastoro
hypothesized in 1546 that some diseases could be caused by
Bgerms^ and the work of Louis Pasteur and Robert Koch on
the transmission of infectious disease, the understanding of
infection pathophysiology has been a major domain of re-
search for scientists and physicians.
The technical and conceptual advances of the last decades
accelerated the discovery of mechanisms involved in the host
response to infection; this has generated a tremendous amount
of data that is still accumulating at an exponential rhythm.
The Bbig picture^ is still blurry, but it appears that infec-
tions cause damage not only because of the virulence of the
germ but also because of the host response after the detection
of molecular patterns that can be found in the microorganisms
(the pathogen-associated molecular patterns (PAMPs)).
Infection will cause a local reaction that will extend to the
entire body mainly through the circulation of immune cells
and soluble mediators.
Host response to infection is therefore not only an imme-
diate and local process but a complex time- and space-
compartmentalized process that involves various cell types
(especially leukocytes and endothelial cells). The release of
Binflammatory^ cytokines will induce new cytokine produc-
tion and release that will in turn cause cell and organ damage.
This Bcytokine storm^ or Bcytokine cascade^ is likely re-
sponsible for the diverse, local and remote, signs associated

with infection (Fig. 1). Eventually, when a threshold is
crossed, a severe clinical syndrome, Bsepsis,^ can be ob-
served. It has a dramatic impact on morbidity and mortality.
The term Bcytokine storm^ was first used by Ferrara et al. in
1993 to describe the effects of graft-versus-host disease
(GVHD) [1]. In 2003, the cytokine storm was shown to be
associated with reaction to influenza [2] and subsequently to
various viral, bacterial, or fungal infections [3]. However,
there is no definition of what a cytokine storm is.
The understanding that the global effects of an infection are
mostly due to the host response was acknowledged in the first
consensual definition of sepsis in the early 1990s as the
Bsystemic response to an infection^ which is characterized
by a widespread inflammation [4]. Thus, the diagnosis of sep-
sis necessitated the presence of a systemic inflammatory re-
sponse syndrome (SIRS) and a proven or suspected infection.
In 2016, a new definition of sepsis emerged (sepsis 3.0) and
states that BSepsis is life-threatening organ dysfunction due to
a dysregulated host response to infection^ [5]. The clinical
criteria for the diagnosis of sepsis have evolved to an elevation
of at least 2 points of the sepsis occurrence of failing organs
score (SOFA) and a suspected or proven infection. Recent
data regarding sepsis epidemiology show that the incidence
of sepsis is dramatically high, close to 450 per 100,000 inhab-
itants [6], far more than that of myocardial infarction [7] or the
combination of prostate, breast, and lung cancer [8]. It is esti-
mated that there are between 15 and 30 million of sepsis cases
worldwide every year [6, 9]. With a nearly 20% mortality rate,
it clearly appears that this cytokine storm-induced syndrome is
a major public health issue. Unfortunately, despite immense
progress in the Bomics^ (genomic, transcriptomic, proteomic,
metabolomic) of sepsis, all clinical trials aiming to dampen the
inflammatory response or targeting the cytokines failed. The
translation of basic science findings and clinical data into ef-
fective therapeutics is highly complex and will necessitate an
integrated view of the orchestration of the cytokine storm and
its effects.
In this review, we will detail the mechanisms leading to the
induction of the cytokine storm during sepsis and describe
how and what cytokines are released and how they may con-
tribute to cell or organ damage. Finally, we will discuss the
various therapeutic interventions tried or to be tried to stop or
quell this deleterious process.
Sepsis pathophysiology—activation of innate
immunity
When microorganisms penetrate into the body, they will even-
tually be spotted by the innate immune system. Innate immu-
nity is the ensemble of cellular and humoral mechanisms that
does not need training (i.e., previous exposure to a germ) and
that will act quasi-automatically against an insult and generate
Semin Immunopathol
inflammation [10, 11]. Actually, it appears that innate immu-
nity can be trained and be based on a memory that will last
through epigenetic regulation [12, 13].
Innate immunity is of variable forms but is remarkably
conserved through evolution, and most of the species share a
bundle of common responses to infectious stimuli. The leuko-
cytes that are considered to be the key actors of the innate
response are the monocytes/macrophages, neutrophils, eosin-
ophils, basophils, and natural killers (NKs).
The activation of innate immunity is made through the
pattern recognition receptors (PRRs).
Leukocyte activation by the PRRs will occur after the bind-
ing of a wide variety of molecules originating from the infecting
microorganism, the pathogen-associated molecular patterns
(PAMPs), or from necrotic cells, the damage-associated molec-
ular patterns (DAMPs). The recognition of PAMPs or DAMPs
will lead to a cascade of activation/phosphorylation inducing a
stereotyped inflammatory answer.
To date, four types of PRRs have been identified in verte-
brates: the Toll-like receptors (TLRs), the Nod-like receptors
(NLRs), the RIG-like receptors (RLRs), and the C-type lectin
receptors (CLRs). The most studied PRRs are the TLRs, par-
ticularly TLR4. Pioneer work by Drs. Hoffman, Beutler,
Janeway, and Medzhitov (among others) [14, 15] led to the
discovery of TLRs in both drosophil and man and paved the
way to the discovery of the innate immune system. The TLR4
was identified as the receptor for lipopolysaccharide (LPS)
[16, 17], which is a wall compound of gram-negative bacteria
and is studied in depth to investigate host response after
Bendotoxemia,^ a model aimed to mimic the response to a
bacterium. To date, 10 TLRs have been identified in man
(12 in mouse) [18]. TLRs have a leucin-rich repeat extracel-
lular domain and an intracellular Toll-interleukin-1 receptor
domain (TIR) [19]. The list of the ligands binding to TLRs
is still expanding (Table 1).
The interaction between TLRs and their ligands induces,
through the TIR domain, a cascade of activation via various
mediators such as myeloid differentiation protein 88
(MyD88), TIR domain-containing adaptor protein (TIRAP),
TIR receptor-inducing interferon-beta (TRIF), TRIF-related
adaptor molecule (TRAM), and several tyrosine kinases. As
a result of these signaling cascades, transcription factors such
as NFκB will induce the generation of inflammatory cyto-
kines such as TNF-α, IL-1β, interferon regulatory factor 3
(IRF3), IRF7, or adaptor-protein 1 (AP-1) [18, 20]. These
pathways are tightly modulated by cytoplasmic (IRAK-M,
Tollip, SOCS1) or membrane-bound (SIGIRR, ST2) proteins
[21]. Regulation of TLR signaling is also mediated by a tight
control of TLR expression at the cell membrane. Septic pa-
tients have higher levels of TLR4 mRNA and higher levels of
TLR2 receptors [22, 23].
NLRs are soluble cytosolic PRRs; they share a common
nucleotide-binding oligomerization domain (NOD) and have
Semin Immunopathol

Fig. 1 Cytokine cascade during sepsis

a LRR domain (like the TLRs). The two most studied NLRs
are NOD1 and NOD2, which bind bacterial peptidoglycan.
Activation of the NLR pathway is under the control of the
RICK kinase and induces NFκB and AP-1. Other NLRs
(NLRP, NLRC4) will be involved in the constitution of a
protein complex named inflammasome. Inflammasome in-
duces the cleavage of pro-caspase 1 into active caspase 1 that
in turn will cleave pro-IL-1β and pro-IL-18 intro IL-1β and
IL-18, two inflammatory cytokines [24, 25].
The CLR family includes dectins, DC-SIGN, or mannose-
binding lectin. Dectins signal through Src and Syk and induce
ROS production [26–28]. DC-SIGN is involved in the recog-
nition of patterns from viruses (HIV, HCV, dengue virus, etc.),
leishmaniae, and fungi [29, 30]. Its signaling pathway goes
through Raf-1 [31].
There are three RLRs discovered to date (RIG-I,
MDA5, and LGP2), and they bind double-stranded viral
RNA (dsRNA) [32].
As said earlier, PRRs are activated not only by exogenous
PAMPs but also by endogenous DAMPs. Microorganism cell
toxicity and toxins in addition to the effects of released cyto-
kines will induce a large amount of cell death that will release
DAMPs. Thus, an autoamplified activation cascade will be
initiated and will rapidly be independent of any stimuli com-
ing from the invading microorganism.
In recent years, a significant body of literature has been
published to show the very crucial role of the endothelium
during infection and sepsis [33–35]. Endothelial cells should
be considered not only as a victim of the inflammatory acti-
vation but also as a conveyor and amplifier belt for

Table 1 Toll-like receptor (TLR)

ligands TLR1 Triacyl lipoproteins
TLR2 Lipoproteins, gram-positive peptidoglycan, lipotechoic acid, amphotericin B, saturated fatty acids,
glycoproteins gB and gH, zymosan, HA
TLR3 Double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C)
TLR4 Lipopolysaccharide, RSV fusion protein
TLR5 Flagellin
TLR6 Diacyl lipoproteins, lipotechoic acid, zymosan
TLR7 Single-stranded RNA
TLR8 Imidazoquinolines
TLR9 Bacterial DNA, CpG deoxyribonucleotide
TLR10 Unknown

inflammation. Pro-inflammatory cytokines have a deep im-
pact on endothelial cells by promoting (i) expression of adhe-
sion molecules such as ICAM1 or VCAM1 that will allow
activated immune cells to adhere to the vascular wall and enter
inflamed tissues, (ii) the synthesis of chemokines to attract
immune cells, (iii) secretion/exhibition of procoagulant fac-
tors, and also (iv) endothelial cell dysfunction and necrosis
that contribute to increased vascular permeability, a major
feature of severe infection; increased production of NO, a
vasodilatory agent; and organ dysfunction (induced by both
cellular infiltration and ischemia).
Individual response and genetic polymorphisms
Infections lead to a wide variety of clinical signs with a wide
range of intensities. The individual host response to infection
is related to numerous factors such as the microorganism, the
site of infection, the immune status, the balance between acti-
vating factors of the gene network involved in sepsis patho-
physiology, the epigenetic control of gene transcription, and
genetic polymorphisms of sepsis-associated genes. While
most of these factors may vary a lot during a lifetime, the
nuclear DNA is stable; therefore, genetic polymorphisms that
are shown to have an impact on sepsis morbidity or outcome
can be identified and could theoretically lead to a better prog-
nostication of the disease and/or the choice of more adequate
treatments.
Dozens of genetic polymorphisms were identified as hav-
ing an impact on the course of sepsis [36–38], and each new
large-scale, genome wide or not, association study of genetic
polymorphisms in sepsis cohorts brings new players in the
game [39, 40]. Bioinformatic advances help to identify geno-
typic and phenotypic clusters and could improve our under-
standing of pathways involved in the host response and reveal
potential therapeutic targets. Among the identified single nu-
cleotide polymorphisms (SNPs), cytokine genes are well rep-
resented. TNF-α levels vary greatly in septic patients. One
possible explanation lies in the occurrence of a SNP in the
TNF-α gene promoter in the -308 position (G308A). Thus,
two alleles of the gene exist, one named TNF1 and the other
TNF2; TNF2 is associated with high levels of TNF-α and an
increased severity of infections such as meningococcemia or
in a cohort of septic patients [41, 42]. The ILRN*2 allele of the
interleukin-1 receptor antagonist (IL-1Ra) is associated with
high levels of IL-1α [43] and lower levels of IL-1Ra [44]
while the rs315952 SNP is associated with survival [45].
The CD14 gene promoter SNP C(-159)T increases suscepti-
bility to gram-positive sepsis [46]; the Asp299Gly allele of the
TLR4 gene also may have a negative impact on sepsis [47]
and the TLR1 (-7202G) SNP induces high NFκB activation
and therefore higher levels of cytokines [48]. This is also
observed with anti-inflammatory cytokines, e.g., the G-
1082A SNP of the IL-10 gene was shown to be associated
Semin Immunopathol
with the severity of pneumonia [49, 50]. Most of the cytokine,
receptor, or transcription factor genes have SNPs that were
studied in the context of severe infections, and these genetic
variations often have an impact on sepsis outcome.
Cytokines in sepsis pathophysiology
The production and release of potential cytotoxic factors by
immune cells was reported in the 1960s by the discovery of
the antitumor effects of the Blymphotoxin^ produced by lym-
phocytes [51, 52]. In 1975, Carswell et al. reported the pro-
duction of another cytotoxic factor by macrophages, the
Btumor necrosis factor^ (TNF) [53], which will later be iden-
tified to be the same molecule as that named Bcachectin^ [54].
Clark et al. then emitted the hypothesis that TNF could be
responsible for the symptoms observed during malaria and
endotoxemia [55–57]. This was the angular stone of the con-
ceptual changes in the pathophysiology of infections.
Infection causes the release of cytotoxic factors, and this phe-
nomenon is observed with different kinds of infections.
Tracey et al. then showed that TNF was able to induce shock
and tissue injury [58] and that anti-TNF antibodies could pre-
vent the lethality induced by bacteremia [59]. In 1989, the
same group proposed that viral disease, especially influenza,
could also have an inflammatory cytokine origin [60].
Cytokines
Cytokines are a broad category of relatively small proteins
(<40 kDa) that are produced and released with the final aim of
cell signaling [61]. They cover autocrine, paracrine, and endo-
crine activities and play an immunomodulating function. After
binding to specific receptors on various types of cells, cytokines
induce activation, proliferation, or migration of target cells.
Cytokine taxonomy is highly complex and is based both on
timing of discovery and on association by structure, target, or
function. The term Bcytokine^ encompasses roughly 100 sep-
arate genes coding for the cytokine of cytokine-like proteins
[61]. This dense cytokine network supports and promotes the
inflammatory process. Cytokines can be divided into several
categories: interleukins, chemokines, interferons, tumor ne-
crosis factor, and growth factors.
Interleukins are the most important group of cytokines re-
leased during infectious processes. They encompass a large
variety of proteins secreted by leukocytes and endothelial cells
(among others) and that contribute to cell signaling and pro-
mote activation, proliferation, death, and/or motility of im-
mune cells. They are artificially divided into pro- and anti-
inflammatory interleukins. Pro-inflammatory interleukins are
supposed to be responsible for cell activation, tissue damage,
and necrosis while anti-inflammatory interleukins aim to
dampen and finally reverse the inflammatory process.
Semin Immunopathol
Among the numerous pro-inflammatory cytokines studied
during sepsis, IL-1β, IL-6, IL-12, and IL-17 are of crucial
importance. IL-1β is also referred to as catabolin. It is a mem-
ber of the IL-1 family (11 genes) that is produced after acti-
vation of the inflammasome (especially NLRP3). Activation
of the IL-1 receptor leads to the activation of the JNK and p38-
MAPK pathways and increases the activity of NFκB. IL-1β
promotes the amplification cascade and induces the synthesis
of various inflammatory genes such as IL-6, IL-8, MCP-1,
COX-2, IκBα, IL-1α, IL-1β, and MKP-1. It is interesting to
note that the IL-1 pathway works in parallel with the IL-18
and IL-33 pathways [62]. During sepsis, levels of IL-1β were
shown to be higher in patients who died than in survivors,
suggesting an association of high levels of IL-1β and sepsis
outcome [63]. IL-6 (also known as interferon-ß2, B cell stim-
ulatory factor 2 (BSF-2)) is a pleiotropic interleukin; its role
during the cytokine storm is complex and controversial. IL-6
is actually not a single protein but includes a family of mole-
cules like IL-11, oncostatin M, ciliary neutrophilic factor, or
cardiothrophin-like cytokine. IL-6 cytokine receptors are
made of two subunits: CD126 and CD130 (gp130) [64]. The
main source of IL-6 is tissue macrophages. High levels of
IL-6 were observed in many inflammatory diseases such
as cardiovascular disease, autoimmune diseases, or can-
cer. Similarly to IL-1β, high levels of IL-6 were con-
sistently shown to be associated with the severity of
sepsis and the highest levels associated with the worse
outcome [65–67]. The exact contribution of IL-6 to sep-
sis is still under investigation and is possibly linked to
the activation of the complement pathway [68, 69] and
capillary leakage. Modulation of IL-6 is also made
through the release of a soluble receptor [70].
IL-12 is also elevated during sepsis. This cytokine, pro-
duced by dendritic cells, macrophages, and lymphoblastoid
cells, induces the differentiation of naïve T cells into type 1
helper T cells (TH1) and activates NK cells; as a result, these
cells will produce a high amount of INFγ [71]. IL-17 is pro-
duced by the so-called TH17 T cells after stimulation by IL-23
and induces the synthesis of a wide variety of cytokines: GM-
CSF, pro-inflammatory interleukins (IL-1β, IL-6), TNF-α,
and chemokines (IL-8, Gro-α, CCR2/MCP-1), by many cell
types such as endothelial cells, epithelial cells, fibroblasts, or
macrophages [72, 73]. Generation of IL-17-producing TH17
cells is finely tuned. TGFβ, IL-1β, IL-6, IL-21, and IL-23
were shown to be sufficient to induce TH17 cells from naïve
CD4+ cells. However, several, and sometimes opposed, pro-
cesses occur. TGFβ can induce both regulatory T cells (Tregs)
and TH17 [74]; addition of IL-6 restricts the development of
Tregs. Interestingly, IL-6 can also induce the production of IL-
10, which restrains the expression of IL-17 [75]. Therefore,
there is a balance of pro-inflammatory mediators that will
favor TH17 generation. As a proof of the crucial role of IL-6,
mice lacking IL-6 cannot develop TH17 cells. In IL-6−/− mice,
deletion of Tregs and secretion of IL-21 can restore the produc-
tion of IL-17 [76]. TGFβ, through the nuclear receptor
RORγt, regulates IL-17 in a complex fashion. TGFβ induces
both RORγt expression and reduces its ability to induce IL-17
production. When inflammatory mediators are added,
RORγt-directed IL-17 production is restored [77].
Interferons are classified into three major types according
to their receptor specificity. IFNAR1/IFNAR2 is the heterodi-
meric receptor for type I interferon (IFN-α and IFN-β).
IFN-γR1/IFN-γR2 is the receptor for type II IFN (IFN-γ),
and interferon-λ is the third type of IFN, mainly studied during
viral infections. IFN-γ is mainly produced by CD4 and CD8 T
cells. NK cells also produce IFN-γ but to a lesser extent [78,
79]. IFN is usually used to define TH1-type cells. The IFN-γ
signaling pathway relies mainly on JAK1, JAK2, p38-MAPK,
and STAT1 and induces factors such as IRF1 or SMAD7.
INFγ promotes the inflammatory response during sepsis, but
INFγ production is dampened during sepsis, probably due to
the hyporesponsiveness of lymphocytes during the immuno-
suppressed state [80, 81].
Chemokines (CKs) are small molecules (8–12 kDa), usu-
ally soluble, not only specialized in the recruitment but also
activation of immune cells. CKs need specific receptors
coupled to G proteins. Chemoattraction of cells requires a
gradient of concentration of chemokines. There are four types
of CKs that are classified according to their amino acid se-
quence and the spacing between their two cysteine residues:
CC, CXC, CX3C, and XC CKs. The most studied CKs in the
context of infectious disease are MCP-1/CCL2, CXCL8/IL-8,
CXCL1/GRO-a, and CXCL2/MIP2a. In contrast to other cy-
tokines which have broad effects on many cells, CKs are usu-
ally cell specific. For instance, MCP-1 or CX3CL1 are nearly
specific for monocytes, CXCL1 and CXCL2 for neutrophils,
and CXCR3 for T lymphocytes. Their role in sepsis patho-
physiology was deeply investigated, and they were found to
play a major orchestrating role during the host response to
infection. CKs induce not only leukocyte recruitment to the
site of infection but also the release of immune cells from the
bone marrow or spleen. Once recruited, leukocytes will clear
bacteria and dead cells and generate inflammation, but they
will also be activated and then directly propagate inflamma-
tion throughout the entire organism. Lack of CKs or their
receptors leads to a quasi-immunosuppressed state and makes
the organism more susceptible to infection-induced lethality
[82–85].
Growth factors are deeply involved in sepsis pathogenesis.
Among the different types of growth factors secreted during
sepsis, the most involved in the generation of the cytokine
storm are the hematopoietic targeted colony-stimulating fac-
tors (CSFs): granulocyte-macrophage colony-stimulating fac-
tor (GM-CSF), macrophage colony-stimulating factor (M-
CSF), and granulocyte colony-stimulating factor (G-CSF).
In addition, with other factors such as IL-3, CSFs will induce

myeloid cell differentiation and proliferation; thus, a larger
number of activated cells will be put into movement and con-
tribute to the synthesis of more cytokines and therefore pro-
mote the cytokine storm. It is also suggested that CSFs not
only play a numerical role but also a functional one by en-
hancing response to early-phase molecules such as IL-1β or
TNF-α [86]. A recent report shows that during sepsis, a major
source of GM-CSF is the newly discovered IRA B cells [87],
which are innate-like B cells derived from B1 cells and that
relocate into infected tissues.
The most studied anti-inflammatory interleukins are IL-
1RA, IL-4, and IL-10. IL-1RA is secreted by immune cells
or epithelial cells and binds to IL-1R, thus blocking the action
of IL-1α or IL-1β inflammatory signals [88, 89]. Treatment
with IL-1RA improved sepsis induced by polymicrobial peri-
tonitis or gram-negative pneumonia [90, 91]. IL-4 is mainly
produced by T cells and promotes both proliferation of B and
T cells and a shift of T cells towards a TH2 profile [92, 93].
The role of IL-4 in sepsis is under debate; a possible role of IL-
4 in its pathophysiology was inconstantly reported. IL-10 is
the central anti-inflammatory cytokine [94]. It has the ability
to block the production of inflammatory cytokines by myeloid
cells, NK cells, and T cells [95]. The levels of IL-10, even as a
marker of anti-inflammation, are directly related to the degree
of inflammation (as reflected by the levels of IL-6) demon-
strating that pro- and anti-inflammation are closely related.
High IL-10 levels are also associated with more important
features of sepsis-induced immunosuppression [96].
IL-3 is a cytokine discovered in the 1980s and also referred
to as multi-CSF [97]. It is a pleiotropic growth factor that acti-
vates proliferation of hematopoietic stem cells and progenitors.
Until recently, the role of IL-3 during sepsis was unknown. We
identified IL-3 as a key regulator of the cytokine storm during
sepsis [98]. IL-3 is produced by IRA B cells and fuels the
inflammatory cascade by promoting emergency myelopoiesis.
Injection promotes high levels of pro-inflammatory cytokines
through proliferation of myeloid cells but not their activation. A
recent study confirms the role of IL-3 in the immune regulation
and potential response to corticosteroids during sepsis [99].
Cytokine kinetics
The endotoxemia model was widely used to study the type
and the kinetics of the cytokine response. Sensitivity to endo-
toxin varies greatly among species. For example, to generate
an IL-6 response around 1500 pg/mL, several milligrams per
kilogram of LPS are needed in mice while only few nano-
grams per kilogram are necessary in humans. Clinically, after
LPS injection, the heart rate increases within 30 min to reach a
peak at 2–4 h in humans while it will be roughly stable in
mice. Body temperature increases in both species between 1
and 2 °C, but the peak time is slightly shifted from 1 h in mice
to 2–4 h in humans.
Semin Immunopathol
In humans, the white blood cell count will increase until it
reaches a peak at 6 h. Leukocytosis is highly dose dependent
and reflects both cell release from the bone marrow and cell
adhesion and recruitment to the inflamed tissues. Leukocyte
response is also mainly a neutrophilia while the lymphocyte
number is usually decreasing.
Regarding cytokines, the main inflammatory molecules
(TNF-α, IL-6, IL-1β) will have more or less the same kinetics
with a rapid peak between 1.5 and 2 h and a rapid decrease to
the basal levels (nearly undetectable) after 6 h. Anti-
inflammatory molecules (IL-10, IL-1RA, TNF-SRI) and
chemokines (IL-8, KC, MIP-2, GRO-α) will have a parallel
evolution [100–103].
A major work by Calvano et al. in 2005 consisted in
the study of the transcriptome response of white blood
cells after endotoxin injection in humans [104]. Using
serial blood sampling after the onset of endotoxemia,
the authors showed that pro-inflammatory gene expres-
sions such as TNFSF2 (TNF), IL-1α, IL-1β, CXCL1
(GRO-α), CXCL2 (GRO-b), CCL2 (MCP-1), CXCL8
(IL-8), and CXCL10 peaked at 2–4 h with a subsequent
increase in transcriptions factors NFκB1, NFκB2, RelA,
and RelB. The 4–6-h time period appeared to be critical
because of the increase in both initial response genes
and regulation genes of the transcription factor family:
STAT genes, c-AMP-response element binding protein
(CREB), SOCS3, and IKKB, or anti-inflammatory cyto-
kines: IL1RAP, IL1R1, IL-10, and TNFRSF1A. The
global response to endotoxin comprises a network of
1556 genes. More recently, Shalova et al. performed a
transcriptomic characterization of human blood mono-
cytes isolated from septic patients [105]; 1170 genes
were found to be affected by sepsis (561 upregulated
and 609 downregulated), and immune response genes
were found to be the more significantly altered. Sepsis
monocytes have a clearly different transcriptomic pro-
gram compared to basal monocytes. Monocytes upregu-
late a large number of cytokines such as IL-1β, IL-6,
CCL3, CCL5, and IL-10 and transcription factors such
as NFκb and the regulatory phospho-IκBa during sepsis.
Therefore, findings from endotoxemia trials are con-
firmed in septic patients. The genomic response of sep-
tic shock patients was also evaluated in a recent work
by Cazalis et al. [106]. The authors found that both pro-
and anti-inflammatory transcriptome responses were ac-
tivated since the admission of the patients and rapidly
evolved over the first 48 h. Payen et al. studied the
evolution of the transcriptome over the first week and
the first month after sepsis [107]. Among the genes
whose expression was modified over time (in survivors),
some were cytokine related like IL-3 (IL3RA) or
ISTF3G (interferon-stimulated transcription factor 3
gamma).
Semin Immunopathol
Targeting cytokines during sepsis: a therapeutic
challenge
Despite the increasing amount of evidence on the key patho-
physiological role of cytokines during infection, there is no
specific treatment targeting inflammation that was shown to
be effective in sepsis.
As recently reviewed by Opal et al. [108], nearly 30 years
of clinical trials were unsuccessful despite promising preclin-
ical and single-center results.
The first immunomodulatory attempt in sepsis was with the
use of corticosteroids [109]. Corticosteroids are well known to
dampen inflammation and inhibit the activation of the NFκb
and AP-1 pathways [110]. Annane et al. showed that low-dose
corticosteroids could reduce mortality in severe sepsis and
septic shock [111]. Unfortunately, a large-scale clinical trial,
the CORTICUS study [112], showed no clinical benefits of
the systematic use of corticosteroids during sepsis.
More targeted therapies were also tried. Antibodies and cap-
ture of molecules such as TNF-α, IL-1, and LPS had great
preclinical results, in mice models, but these treatments failed
to reduce overall sepsis mortality [113–117]. However, a recent
meta-analysis showed that anti-TNF treatment was associated
with a modest but significant reduction in sepsis mortality
[118]. This could justify a new trial with more selected patients.
The blocking of the TLR4 pathway also faced clinical failure
[114]. The interaction between coagulation and inflammation
was shown in numerous experimental and clinical studies
[119]. The coagulation cascade induces blood clot and endo-
thelial and immune cell activation and amplifies inflammation.
Therefore, targeting this coagulation disorder could be effective
in reducing inflammation. These statements led to the use of
activated protein C (APC) (drotrecogin alfa) in sepsis. The first
large-scale study, the PROWESS trial [120], showed great po-
tential for APC during sepsis. Twenty-eight-day mortality was
assessed in a randomized study including nearly 1700 patients.
The use of APC led to a significant reduction in mortality
(30.8% in the placebo group vs 24.7% in the treatment group,
a nearly 20% relative risk reduction). Nevertheless, when a
larger study attempted to replicate these results, the use of
APC was not associated with mortality reduction [121]. After
publication of these results, the molecule was withdrawn from
the market by the pharmaceutical company producing it.
Failure of sepsis therapy could have several explanations.
The appropriateness of the preclinical models, especially mice
models or in vitro experiments, is a major source of concern.
Immune response between species varies greatly; the transpo-
sition of results obtained in animal experiments into effective
treatment for humans is not straightforward. Another issue
linked to animal models is that they are Bcalibrated,^ meaning
that treatments are administered at a precise time point before
or after the onset of the disease. In clinical practice, it is nearly
impossible to date precisely the beginning of sepsis; therefore,
treatments are given without the exact same timing. This leads
to the more important potential cause. Septic patients do not
represent a homogeneous category of patients. Sepsis presen-
tations are diverse, and patients included in the study have
different sources of sepsis, with different evolutions, different
levels of inflammation, and therefore different responses to
treatment. Applying the same treatment for sepsis is unlikely
to show any beneficial effects. Patients included in studies
need to be selected based on specific parameters or bio-
markers. For instance, patients with levels of pro-
inflammatory cytokines are more likely to respond to the
anti-cytokine treatments.
Another approach to treat the effects of the cytokine storm
during infectious diseases is to target the direct tissue conse-
quences of the inflammation cascade. Blood vessels’ in-
creased permeability is a hallmark feature of serious infec-
tions; thus, London et al. [122] decided to target an
endothelium-specific pathway: Slit-Robo4. Activation of the
Slit-Robo4 pathway (Robo4 is the membrane receptor for Slit)
is associated with a dramatic reduction in vascular permeabil-
ity and organ injury in various models of infections such as
polymicrobial sepsis or H5N1 influenza. In these two models,
Slit treatment does not seem to reduce the amount of circulat-
ing inflammatory cytokines while it appears to be effective in
this task in the endotoxemia model [123]. This innovative
approach is a potential effective treatment of serious infections
that do not aim to block the already self-sustaining inflamma-
tory cascade but to limit what is eventually killing patients,
vascular-induced tissue damage.
Sphingosine-1-phosphate (S1P) was identified as a good
candidate to target the infection-associated cytokine storm.
S1P is a signaling lysophospholipid that promotes cytokine
synthesis and secretion [124]. Ceramides are a component
of the cell membrane; once deacetylated into sphingosine
and a fatty acid, sphingosine is phosphorylated by sphingo-
sine kinase 1 or 2 into its active form, S1P. S1P actions are
mediated by five G protein-coupled receptors expressed on
various cell types like immune cells (macrophages, dendrit-
ic cells, T cells, B cells, NK cells), endothelial cells, or
epithelial cells. In 2011, Teijaro et al. showed that the
S1P1 receptor expressed on endothelial cells and lympho-
cytes was a key regulator in immune and vascular response
after influenza infection [125]. Activation of the S1P1 path-
way with chemical agonists named AAL-R or CYM-5442
dramatically reduced cytokine production and cell recruit-
ment and improved survival after influenza infection. These
effects were mediated by a reduction in chemokine produc-
tion by endothelial cells.
The study of these two pathways, Slit-Robo4 and S1P-
S1P1, confirm that using endothelial activation as a therapeu-
tic target to quell the cytokine storm after infection is highly
effective and is a valid alternative to direct cytokine
antagonism.

Conclusion
The cytokine storm induced by infectious diseases is the result
of an autoamplifying phenomenon aiming to destroy the in-
vader and restore homeostasis that will eventually lead to sep-
sis. While the understanding of the orchestration of the
cytokine-induced cell response during sepsis is in constant
progress, we still lack a global integrative view, at the cell
level, in time and space. The organization of the host response
to infection, locally and remotely, is still incompletely under-
stood. Despite the identification of key molecules, such as the
emblematic tumor necrosis factor alpha, every attempt aiming
to dampen this cytokine storm activation or consequences
failed in clinical trials. While the exact triggers or connectors
that should be targeted are still to be identified, the endothelium
as a node of immune response appears to be a valid and prom-
ising target. The next decade will probably see a more precise
depiction of in-cell, in-organ, and global response to infection;
there is no doubt that future discoveries will challenge our ac-
tual concepts and will make us revisit our therapeutic targets;
this makes the fight against this dramatic response to infection
an exciting challenge.
Acknowledgements This work is supported by a public grant overseen
by the French National Research Agency (ANR) (Project BCMOS,^
ANR-15-CE15-0019).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
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