Review
published: 12 August 2015
doi: 10.3389/fimmu.2015.00401
CD1d- and MR1-restricted T cells in
sepsis
Peter A. Szabo 1†, Ram V. Anantha 1,2†, Christopher R. Shaler 1, John K. McCormick 1,3,4 and
S.M. Mansour Haeryfar 1,3,4,5*
1
Department of Microbiology and Immunology, Western University, London, ON, Canada, 2 Division of General Surgery,
Department of Medicine, Western University, London, ON, Canada, 3 Centre for Human Immunology, Western University,
London, ON, Canada, 4 Lawson Health Research Institute, London, ON, Canada, 5 Division of Clinical Immunology and
Allergy, Department of Medicine, Western University, London, ON, Canada
Edited by:
Nilabh Shastri,
University of California Berkeley, USA
Reviewed by:
Olivier Lantz,
Institut Curie, France
Weiming Yuan,
University of Southern California, USA
*Correspondence:
S. M. Mansour Haeryfar,
Department of Microbiology and
Immunology, Schulich School of
Medicine and Dentistry, Western
University, 1151 Richmond Street,
London, ON N6A 5C1, Canada
mansour.haeryfar@schulich.uwo.ca
Dysregulated immune responses to infection, such as those encountered in sepsis, can
be catastrophic. Sepsis is typically triggered by an overwhelming systemic response to
an infectious agent(s) and is associated with high morbidity and mortality even under
optimal critical care. Recent studies have implicated unconventional, innate-like T lymphocytes, including CD1d- and MR1-restricted T cells as effectors and/or regulators of
inflammatory responses during sepsis. These cell types are typified by invariant natural
killer T (iNKT) cells, variant NKT (vNKT) cells, and mucosa-associated invariant T (MAIT)
cells. iNKT and vNKT cells are CD1d-restricted, lipid-reactive cells with remarkable
immunoregulatory properties. MAIT cells participate in antimicrobial defense, and are
restricted by major histocompatibility complex-related protein 1 (MR1), which displays
microbe-derived vitamin B metabolites. Importantly, NKT and MAIT cells are rapid and
potent producers of immunomodulatory cytokines. Therefore, they may be considered
attractive targets during the early hyperinflammatory phase of sepsis when immediate
interventions are urgently needed, and also in later phases when adjuvant immunotherapies could potentially reverse the dangerous state of immunosuppression. We will
highlight recent findings that point to the significance or the therapeutic potentials of NKT
and MAIT cells in sepsis and will also discuss what lies ahead in research in this area.
Keywords: CD1d, MR1, NKT cell, MAiT cell, LPS, α-galactosylceramide, infection, sepsis
†
Peter A. Szabo and Ram V. Anantha
have contributed equally to this work.
Preamble
Specialty section:
This article was submitted to T Cell
Biology, a section of the journal
Frontiers in Immunology
Received: 05 June 2015
Accepted: 22 July 2015
Published: 12 August 2015
Citation:
Szabo PA, Anantha RV, Shaler CR,
McCormick JK and Haeryfar SMM
(2015) CD1d- and MR1-restricted
T cells in sepsis.
Front. Immunol. 6:401.
doi: 10.3389/fimmu.2015.00401
Sepsis is a life-threatening syndrome typically associated with early hyperinflammation, immunosuppression in its protracted phase, and a continuum of organ dysfunction abnormalities. It is a
significant cause of death across all age groups and in both developed and developing countries. It
also negatively affects the quality of life among survivors. Sepsis is usually a consequence of infection
although sterile tissue damage inflicted by non-infectious causes or conditions, such as pancreatitis,
ischemia–reperfusion injury, and cancer may also lead to sepsis (1). In this article, we will only
focus on the syndrome caused by disproportionate, excessive, or sometimes defective host responses
to infection. We will provide a general overview of sepsis, its epidemiology, prognosis, management, and immunopathogenesis. We will briefly discuss experimental immunotherapeutic strategies tested in animal models of sepsis or used in clinical trials. Many such strategies have targeted
antigen-presenting cells (APCs) and conventional T cells or their products, such as inflammatory
cytokines, albeit with little success. Recent progress in our understanding of natural killer T (NKT)
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NKT and MAIT cells in sepsis
cell and mucosa-associated invariant T (MAIT) cell responses to
infection and their regulatory functions may open a new front
in our fight against sepsis. These unconventional T cells respond
rapidly to infection by secreting large quantities of pro- and/or
anti-inflammatory cytokines, thereby controlling the effector
functions of numerous other cell types belonging to both innate
and adaptive arms of immunity. Also importantly, NKT cells can
be easily manipulated by “disease-tailored” synthetic glycolipids.
Therefore, the quick and wide-ranging actions of NKT cells, and
potentially of MAIT cells, may be exploited to the host’s benefit
in different forms or stages of sepsis. We will review NKT and
MAIT cell functions in antimicrobial immunity and highlight
recent findings on these cell types in the context of sepsis.
synthesize tumor necrosis factor (TNF)-α and interferon (IFN)-γ
upon ex vivo stimulation can be either CD4+ or double negative
(17). Another example is the case of adipose tissue iNKT cells that
secrete IL-10, impart an anti-inflammatory phenotype to macrophages, and control the expansion and suppressor function of
regulatory T (Treg) cells (18). Moreover, adipose tissue iNKT cells
lack promyelocytic leukemia zinc finger (PLZF), a transcription
factor otherwise regarded as a “master regulator” of iNKT cell
effector functions (19).
iNKT cells are armed with cytotoxic effector molecules such
as perforin, granzymes, TNF-α, Fas ligand, and TNF-related
apoptosis-inducing ligand (TRAIL), and may be able to lyse
neoplastic or infected cells directly (20–22). However, they are
best known for their immunomodulatory functions mediated
by the early production of pro- and/or anti-inflammatory
cytokines. iNKT cells can thus transactivate numerous downstream effector cell types including natural killer (NK) cells,
macrophages, dendritic cells (DCs), conventional CD4+
and CD8+ T cells, and B cells. They are rapid producers of
enormous quantities of T helper (Th)1-, Th2-, and Th17-type
cytokines, although Th9- and Th10-like iNKT cells have also
been described (23, 24). The constitutive presence of preformed messenger RNA (mRNA) encoding at least some of
such cytokines in iNKT cells explains the rapidity with which
they are released (25).
The identity of endogenous CD1d ligand(s) that participate in
positive selection and also perhaps in peripheral maintenance of
iNKT cells remains ill-defined and controversial. iNKT cells can
recognize and respond to certain glycolipids present in various
microbes, including but not limited to Novosphingobium spp.,
Ehrlichia spp., Borrelia burgdorferi, Streptococcus pneumoniae,
and Streptococcus agalactiae (26–28). Of note, the latter pathogen,
which is often referred to as group B streptococcus, is a common
cause of neonatal sepsis.
Of all exogenous glycolipid agonists of iNKT cells,
α-galactosylceramide (α-GalCer) has been used most extensively,
not only as a research tool but also in clinical trials for cancer
and viral diseases (29). α-GalCer was initially isolated from an
extract of a marine sponge called Agelas mauritanius (30), and
is believed to have originated from microbes co-existing in a
symbiotic relationship with this sponge. Until recently, α-GalCer
was considered to be a merely exogenous and unnatural glycolipid
given the presence of only one glucosylceramide synthase and one
galactosylceramide synthase in mammalian species, both of which
are β-transferases. However, a recent report has demonstrated
the presence of endogenous α-anomeric glycolipids including α-GalCer in mammals, due perhaps to the operation of an
“unfaithful” enzyme or a novel, as-yet-unidentified pathway (31).
α-GalCer and its analogs possess a lipid tail that can be
buried deep inside the hydrophobic pocket of CD1d, while
their galactose head protrudes out of CD1d to be contacted by
the iTCRα chain (32). The length and composition of acyl and
phytosphingosine chains of synthetic α-GalCer analogs impact
the binding affinity of α-GalCer:CD1d:iTCR interactions
(33), which partially determines the type of cytokines that an
activated iNKT cell will secrete. For example, OCH is a sphingosine-truncated derivative of α-GalCer with Th2-skewing
NKT Cells: A Brief Overview
Natural killer T cells are innate-like T lymphocytes with impressive
immunomodulatory properties. They express glycolipid-reactive
αβ T cell receptors (TCRs) along with several characteristic
markers of NK cells (e.g., mouse NK1.1 and human CD161) (2,
3). NKT cells develop in the thymus where they are positively
selected by CD1d+CD4+CD8+ thymocytes and consequently
become “CD1d-restricted” (4). As such, CD1d-deficient mice
are devoid of NKT cells (5). CD1d is a monomorphic major
histocompatibility complex (MHC) class I-like glycoprotein that
is highly conserved across mammalian species (6). It is a member
of the CD1 family of lipid antigen (Ag)-presenting molecules (7,
8). The CD1 family in human has five members, namely CD1a–e,
while rodents only express CD1d. Murine and human CD1d can
present normal self- and tumor-derived lipids as well as microbial
glycolipids to NKT cells. The discovery of CD1d restriction led
to the invention of glycolipid-loaded CD1d tetramer reagents
enabling accurate tracking, enumeration, and phenotypic and
functional analysis of NKT cells (9–11).
The major subset of NKT cells is defined by the expression
of a canonical or invariant TCR (iTCR) with a unique α chain
rearrangement (Vα14–Jα18 and Vα24–Jα18 in mice and humans,
respectively), which is paired with one of only a limited choices of
β chains (Vβ8.2, Vβ2 or Vβ7 in mice and Vβ11 in humans). These
cells are called type I or invariant NKT (iNKT) cells (2, 3). Two
phenotypically distinct subpopulations of iNKT cells have been
identified in mice, the CD4+CD8− subset and the double-negative
(CD4−CD8−) subset (12). An additional CD8α+ subset exists in
humans (13). iNKT cells constitutively express CD69, CD25, and
CD44 on their surface, which is consistent with their “partiallyactivated” or “memory-like” status even in germ-free mice (14)
and in human cord blood (15).
iNKT cells are present at low frequencies in the circulation
and in various tissues including bone marrow, thymus, spleen,
and lymph nodes. However, they are abundant in the mouse
liver and in the human omentum (16). The prevalence of iNKT
cells varies considerably among different individuals for reasons
that are currently unknown. Also importantly, iNKT cell subsets
found in different anatomical locations exhibit functional or
even transcriptional heterogeneity. For instance, interleukin
(IL)-4- and IL-13-producing human peripheral blood iNKT cells
fall exclusively within the CD4+ subset, whereas iNKT cells that
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characteristics (34). We have successfully used this glycolipid
to delay Th1-mediated cardiac allograft rejection (35), to prevent or cure citrulline-induced autoimmune arthritis (36), and
to reduce the severity of intra-abdominal, polymicrobial sepsis
(37) in mouse models. Another Th2-favoring agonist of iNKT
cells is C20:2, an α-GalCer analog with a short fatty acyl chain
containing two unsaturation sites at carbon-11 and -14 positions (38). C20:2 is reportedly superior to OCH in polarizing
human iNKT cells. Th1-biasing ligands of iNKT cells can be
exemplified by a C-glycoside analog of α-GalCer, also known
as α-C-GalCer, which potentiates IL-12 and IFN-γ production
in mice (39). Therefore, α-C-GalCer may be useful in adjuvant
glycolipid immunotherapy of cancer and infectious diseases.
Cell membrane location of glycolipid loading onto CD1d
and its presentation within or outside lipid rafts (40), the type
of CD1d+ APCs involved (41), the presence and intensity of
costimulatory and danger signals transmitted or exchanged (29),
and the cytokine milieu in which iNKT cell priming occurs are
among other important factors that shape the cytokine profiles of
iNKT cells. Remarkably, mouse iNKT cells can recognize human
CD1d and vice versa (6), and iNKT cells from either species are
responsive to α-GalCer. Therefore, at least some of the findings
obtained in mouse models of CD1d-mediated iNKT cell activation are likely to be translatable to the clinic.
iNKT cells can also be activated in the absence of exogenous glycolipids. During infection, microbial components
may engage pattern recognition receptors (PRRs), such as
Toll-like receptors (TLRs) on APCs, thus resulting in secretion
of IL-12 and IL-18. Together, these cytokines trigger indirect
activation of iNKT cells (42, 43), which is often dependent
upon the presence of CD1d. This indicates an intriguing but
poorly understood role for endogenous lipids in the context
of antimicrobial immunity. A combination of IL-12 and IL-18
can also reportedly induce iNKT cell responses in a truly iTCRindependent fashion (44). We recently reported that group II
bacterial superantigens (SAgs) can directly activate iNKT
cells in a CD1d-independent manner (45). Therefore, iNKT
cells may serve as effectors and/or regulators of early cytokine
responses to bacterial SAgs.
Type II or variant NKT (vNKT) cells are CD1d-restricted
cells with a relatively diverse αβ TCR repertoire (3, 11). They
exhibit reactivity with certain self lipids, but not with α-GalCer
(46). Compared with iNKT cells, vNKT cells are less frequent
in mice but more prevalent in humans (47). A major fraction
of vNKT cells can recognize sulfatide, a self glycolipid that is
highly enriched in the central nervous system, kidney and liver
(48). Several other endogenous lipids, including but not limited
to β-d-glucopyranosylceramide (β-GlcCer), have also been
recently discovered to activate vNKT cells (49). Given the relative
promiscuity of vNKT cell Ag receptors, it is not too far-fetched to
envisage scenarios where vNKT cells recognize microbial lipids
cross-reactive to self components. In addition, infection may lead
to the release of self lipids in sufficient quantities to induce vNKT
cell activation.
Our overall understanding of vNKT cell responses in health
and disease is limited. This is in large part due to a lack of firm
molecular markers, stable reagents and direct methods to detect
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and characterize vNKT cells. Sulfatide-loaded CD1d-tetramer
reagents have been generated (46). However, they are not popular due to their low stability and high background staining. In
addition, there is no mouse model of pure vNKT cell deficiency.
CD1d−/− mice are devoid of both iNKT and vNKT cells since
CD1d is required for the positive selection of both cell types in
the thymus (50). Experimental evidence indicates that vNKT cells
have an activated phenotype and depend on PLZF for their development (49), and that vNKT and iNKT cells may exert opposing
functions with broad implications for antitumor responses (51)
and antimicrobial immunity (52, 53).
MAiT Cells and Their Roles in Microbial
immunity
MAIT cells are another evolutionarily conserved subset of
innate T lymphocytes that have captured the attention of the
immunological community in the past few years (54, 55). MAIT
cells develop in the thymus where they rearrange their semiinvariant TCR with a characteristic Vα19–Jα33 and Vα7.2–Jα33
TCRα chain in mice and humans, respectively (56, 57).
Similar to NKT cells, MAIT cells are positively selected by
CD4+CD8+ thymocytes (58). However, their selection requires
the expression of MHC-related protein 1 (MR1), as opposed to
CD1d, on thymocytes. Accordingly, MR1-deficient mice lack
MAIT cells in their T cell repertoire (59). MR1 is a monomorphic,
non-classical MHC I molecule that is markedly conserved among
various mammals (60–62). There is 90% sequence homology
between mouse and human MR1 ligand-binding domains and a
high degree of functional cross-reactivity, which is highly reminiscent of cross-species CD1d conservation.
MAIT cells are infrequent and immature in the human fetal
thymus (63). Their maturation is accompanied by a gradual, postthymic acquisition of PLZF expression and the ability to secrete
IFN-γ and IL-22 upon exposure to microbes in mucosal layers.
A PLZF-expressing CD161highCD8+ population is detectable in
human cord blood, from which Vα7.2+ MAIT cells emerge in
adults (64).
MAIT cells are severely depleted in B cell-deficient patients
and mice, and are also entirely absent in the peripheral tissues
of germ-free mice (59), indicating that B cells and commensal
microflora are essential for MAIT cell peripheral maintenance/
expansion. Therefore, it is not surprising that MAIT cells preferentially accumulate in the mucosal compartments, such as the gut
lamina propria, hence their denomination. MAIT cells are also
present in other tissues. In human, they are particularly abundant
in peripheral blood and can comprise up to ~50% of all T cells
in the liver (65, 66). There are far fewer MAIT cells in mice than
in humans. This, together with other differences between the
two species (67), indicates that caution needs to be exercised in
extrapolating experimental data from mice to human conditions.
Until recently, there was no single reagent to directly detect
mouse MAIT cells. In addition, human MAIT cells have been
commonly defined as CD3+Vα7.2+CD161+. However, recent
identification of a MAIT cell Ag, namely reduced 6-hydroxymethyl-8-d-ribityllumazine (rRL-6-CH2OH), led to the development of MR1 tetramer reagents loaded with this compound to
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accurately identify mouse and human MAIT cells (68). Once
widely available, these reagents will undoubtedly boost research
in the area of MAIT cell biology. Human peripheral blood MAIT
cells are CD45RA−CD45RO+CD62LlowCD95high, which is consistent with an effector memory phenotype (65). They also express
the receptors for IL-12, -18, and -23. Human hepatic MAIT cells
have a more activated phenotype and express elevated levels of
CD69 in comparison with their blood counterparts (66). They are
also human leukocyte Ag (HLA)-DR+ and CD38+. This may be
due to continuous exposure to microbial Ags accessing the liver
from the gut through the portal system.
MAIT cells bridge innate and adaptive arms of immunity
to microbial intruders. They quickly amass in sites of infection
where they can keep pathogens in check. For instance, in a mouse
model of pulmonary infection with Francisella tularensis, MAIT
cells reduce bacterial burden in the lungs and prevent mortality
from infection even in the absence of conventional T cells (69).
They can produce inflammatory cytokines such as IFN-γ, IL-17,
and TNF-α readily, amply and promptly after TCR stimulation
(54, 55). Human MAIT cells express granzymes A and K, and
are able to kill infected cells (70). They were shown to lyse, in an
MR1-dependent fashion, epithelial cells infected by the intestinal
pathogen Shigella flexneri (71), and THP1 monocytic cells infected
by Escherichia coli (E. coli) (70). MAIT cells are responsive to a
variety of bacteria and yeasts including Lactobacillus acidophilus,
Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus
aureus (S. aureus), Staphylococcus epidermidis, Candida albicans,
Candida galbrata, and Saccharomyces cerevisiae. A limited
number of studies have utilized MR1-deficient mice to explore
the antimicrobial potentials of MAIT cells in vivo. The ability
to control infection with Klebsiella pneumoniae, Mycobacterium
bovis bacillus Calmette–Guérin (BCG), or Francisella tularensis
was found to be impaired in MR1-deficient mice (69, 72, 73).
McCluskey’s and Rossjohn’s research teams discovered that
vitamin B metabolites represent a class of MR1-restricted Ags
(74). A folic acid (vitamin B9) metabolite called 6-formyl pterin
(6-FP) was found to bind MR1 without stimulating MAIT cells.
In contrast, MR1 ligands derived from the riboflavin (vitamin
B2) biosynthesis pathway could activate MAIT cells. Of note, this
pathway is operational in all of the microorganisms that activate
MAIT cells, but not in those that reportedly fail to do so.
To confirm that the riboflavin pathway supplies human MAIT
cell ligands, Corbett et al. mutated various enzymes of the riboflavin operon in the Gram-positive bacterium Lactococcus lactis followed by testing the MAIT cell-activating capacity of the mutants
(75). This approach led to the identification of 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate of the riboflavin
pathway, as a key compound in generating MAIT cell “neoantigens.” Through non-enzymatic interactions, 5-A-RU forms
simple adducts with small molecules arising from other metabolic
pathways (e.g., glycolysis), such as glyoxal and methylglyoxal, thus
giving rise to 5-(2-oxoethylideneamino)-6-d-ribytilaminouracil
(5-OE-RU) and 5-(2-oxopropylideneamino)-6-d-ribytilaminouracil (5-OP-RU), respectively. MR1 in turn captures, stabilizes,
and presents these neo-antigens to MAIT cells. Recent work
from Olivier Lantz’s laboratory demonstrated that most, if not
all, mouse MAIT cell ligands harbored by the Gram-negative
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bacterium E. coli are also related to the riboflavin pathway (76).
MR1-mediated activation of mouse MAIT cells was most robust
upon stimulation with a mixture of 5-A-RU and methylglyoxal,
and also detectable when a combination of 5-A-RU and glyoxal
was used. This study also reported the synthesis of a new 6-FP
variant in which the amine and the formyl group are blocked.
This compound could efficiently inhibit the activation of MAIT
cells by semipurified soluble bacteria (SPB) or by 5-A-RU plus
methylglyoxal, and may therefore represent a new class of inhibitors of MAIT cell activation. Finally and importantly, in vivo
activation of MAIT cells was demonstrated for the first time when
iVα19 transgenic mice on a Cα−/− background, which harbor
many MAIT cells, were directly injected with the SPB fraction
from riboflavin-sufficient E. coli or with a mixture of 5-A-RU and
methylglyoxal. Interestingly, administration of 5-A-RU alone
failed to activate MAIT cells, which may be probably due to its
instability and/or low bioavailability for interaction with small
metabolites and loading onto MR1 (76).
Mammals do not synthesize riboflavin, but host-derived
metabolites could potentially generate adducts with 5-A-RU of
bacterial origin (75). MR1-restricted recognition of the formed
neo-antigens may be considered a new mechanism of self–nonself discrimination, especially in mucosa-associated lymphoid
tissues. MR1 ligands are ubiquitous and present in many bacteria,
including commensals. In addition, they can readily diffuse across
epithelial barriers (55). Therefore, how MAIT cell activation is
controlled in vivo remains enigmatic at this point.
MR1-independent responses can also be mounted by MAIT
cells. The in vitro response of MAIT cells to BCG-infected cells is
an example (73). Moreover, MAIT cells can produce IFN-γ when
cultured with a combination of IL-12 and IL-18 in the absence
of TCR triggering (77). Therefore, bystander activation of MAIT
cells may occur during infection with viral pathogens or other
germs that do not harbor MR1 ligands.
Sepsis
Definitions and epidemiology
Although sepsis is often discussed in the context of intensive
care in modern settings, the syndrome is almost as old as medicine itself. Derived from the Greek sipsi meaning “make rotten,”
the term sepsis was first coined by Hippocrates (460–370 BC) to
describe the unpleasant process of organic matter putrefaction
(78). Avicenna (980–1037 AD), the great Persian physician/
scientist/philosopher, noted the frequent coincidence of blood
putrefaction, what is known today as septicemia, and fever in
the aftermath of surgery (79). The centuries that followed witnessed important discoveries linking germs to a wide array of
disorders including sepsis. However, the germ theory of disease
failed to fully explain the pathogenesis of sepsis since many
patients succumbed to it despite successful eradication of the
microbial intruder(s). Therefore, the host response to the germ,
and not the germ per se, was proposed to drive the pathogenesis
of sepsis (80).
The modern terminology for sepsis and its sequelae was standardized during an American College of Chest Physicians/Society
of Critical Care Medicine Consensus Conference in 1991 (81).
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Accordingly, sepsis is defined as documented or suspected infection accompanied by at least two of the following abnormalities:
(i) a body temperature of >38°C or <36°C; (ii) a heart rate of >90
beats/min; (iii) a respiratory rate of >20 breaths/min or PaCO2
of <32 mm Hg; (iv) a blood leukocyte count of >12,000/mm3
or <4,000/mm3, or detection of >10% immature neutrophils
(aka. band cells) in the leukocyte differential count. The panel of
experts recommended the application of the term “severe sepsis”
when sepsis is further complicated by organ dysfunction, perfusion abnormalities (e.g., lactic acidosis, oliguria, acute alteration
in mental status), or hypotension (a systolic blood pressure of
<90 mm Hg or a reduction of ≥40 mm Hg from the baseline in
the absence of other causes of hypotension). It needs to be noted
that the terms “sepsis” and “severe sepsis” have often been used
interchangeably. Finally, a severely septic patient should be classified as having “septic shock” when her/his hypotensive state is
refractory to fluid resuscitation.
Sepsis is a leading cause of death following hospitalization and
represents a major challenge in the management of critically ill
patients in non-coronary intensive care units (ICUs) (82). It is
estimated that 25% of patients who develop severe sepsis die during hospitalization, and septic shock is associated with mortality
rates approaching 50% (83). Alarmingly, the incidence of severe
sepsis is on the rise (84). Of equal importance, sepsis worsens the
quality of life among survivors and increases their risk of morbidity and early death. In fact, the 5-year mortality rate in the sepsis
survivor pool can be as high as 75% (84).
It cannot be overstated that the prognosis of sepsis is also
determined by the speed with which the diagnosis is established
and proper management strategies implemented. The earlier the
treatment is started, the more favorable the outcome will be.
Clinical Management
Despite advances in our understanding of sepsis at organismal,
cellular and molecular levels, not even a single drug is approved
as a mechanism-based treatment option for sepsis. The clinical
guidelines established by the Surviving Sepsis Campaign (SSC),
an international consortium of professional societies committed to reducing mortality from severe sepsis and septic shock,
are organized into two “bundles,” each comprising a select but
non-specific set of care elements distilled from evidence-based
practice (90). The initial “resuscitation bundle” should be applied
within 6 h after the patient’s presentation to prevent or resolve
cardiorespiratory insufficiency and to combat the immediate
threats posed by uncontrolled infection(s). Hemodynamic resuscitation is achieved by administration of intravenous fluids and
vasopressors while oxygen therapy and mechanical ventilation
can also be supplied as needed. The timely management of infection requires obtaining blood cultures before broad-spectrum
antibiotic therapy is launched as well as source control (e.g., drainage of pus). The subsequent “management bundle” is typically
accomplished in the ICU where the attention is shifted toward
monitoring and supporting vital organ functions and avoiding
complications. In addition, the efficacy of antibiotic therapy is
evaluated for potential de-escalation to prevent the emergence of
microbial resistance and to lower the risk of drug toxicity (90, 91).
A recent meta-analysis of 13 randomized controlled trials has
demonstrated that early goal-directed therapy, which is perhaps
best exemplified by the SSC-recommended resuscitation bundle,
reduces overall mortality from sepsis when initiated within the
first 6 h (92). This should reinforce the notion that there usually
exists a short window of opportunity in which current management strategies or novel future therapies are expected to be most
effective.
Risk Factors and Prognosis
In general, the prognosis of sepsis is dependent upon demographic, socioeconomic, and iatrogenic factors in addition to
the patient’s medical history, immunological, nutritional and
overall health status, and the type of microorganism(s) involved
in triggering or perpetuation of sepsis (79, 85). For instance,
being over 65 years of age, being a male, being a nursing home
resident, being in a poor nutritional state, having low household income, or receiving treatment in a non-teaching hospital
predisposes to sepsis and to its elevated severity. Several studies
have found that age is an independent predictor of mortality
from sepsis (86–88). However, the elderly are vulnerable to sepsis
also due to a higher likelihood of pre- or co-existing morbidities
(e.g., diabetes and cardiovascular problems) requiring medication, malnutrition, repeated and/or prolonged hospitalizations,
decline in immunity, and functional restrictions (89). Some
of the above factors are taken into account in calculation of
Mortality in Emergency Department Sepsis (MEDS) score to
predict 1-year mortality (85).
Adverse iatrogenic factors include steroid therapy and
immunosuppression prior to surgery and a need for multiple
operations (79). Invasive devices such as urinary and intravenous catheters and breathing tubes also increase the risk of
sepsis. The main predisposing factor for urinary tract infections,
which are the most frequent nosocomial infections in surgical
patients, is the usage of an indwelling urinary catheter. Vascular
catheters, especially central venous catheters, are also common
vehicles for nosocomial infections caused by Gram-positive skin
commensals.
Frontiers in Immunology | www.frontiersin.org
immunopathogenesis and immunosuppression
in Sepsis
Disproportionate or dysregulated immune responses to infection
constitute a major culprit in sepsis-related death. Sepsis is no
longer considered a merely or even mainly hyperinflammatory
syndrome. Rather, in “sepsis-prone” individuals and conditions,
infection triggers a highly complex response that is variable in
proportion or in pro- versus anti-inflammatory nature depending upon the pathogen load and virulence, genetic and other
host factors including age and co-morbidities, and the time
point at which the response is evaluated (91). Pro-inflammatory
responses mounted in septic patients help eradicate the inciting
microbe(s) but may cause collateral organ damage. On the other
hand, anti-inflammatory and immunosuppressive mechanisms
contribute to tissue recovery but also make the patient susceptible
to secondary infections and opportunistic pathogens, especially
during protracted sepsis (93).
The pioneering studies of Tracey et al. in the mid-1980s revealed
that many deleterious features of endotoxin administration to
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rats could be simulated by human cachectin (aka. TNF) (94) and
that cachectin-neutralizing antibody F(ab′)2 fragments could
prevent acute and otherwise lethal septic shock in E. coli-infected
baboons (95). We now know that acute septic shock, which occurs
in a relatively small fraction of patients with sepsis, is indeed a
dangerous immunopathology mediated by an overly exuberant TNF response (91). TNF-α and other pro-inflammatory
cytokines including IL-1β and IL-6 and chemokines like IL-8 are
released from activated macrophages and other APCs after they
sense the presence of invading microbes by PRRs (e.g., TLRs) and
phagocytose them. This in turn leads to neutrophil mobilization,
lymphocyte activation, and more pro-inflammatory cytokine
(e.g., IFN-γ) secretion. These cytokines limit microbial infections,
but their elevated levels are associated with a poor outcome in
sepsis (96, 97). The pleotropic cytokine IL-3 was recently found to
be an upstream orchestrator of inflammation in the early phase of
polymicrobial sepsis modeled by the cecal ligation and puncture
(CLP) procedure in mice (98). In addition, retrospective and
prospective analyses of plasma IL-3 in septic patients linked
heightened levels of this cytokine to a poor outcome.
Both pro- and anti-inflammatory processes get underway
promptly after the initiation of sepsis. A hyperinflammatory
“cytokine storm” dominates the initial phase in many patients
and accounts for death within the first 3 days from septic shock
and multiple organ failure in a substantial fraction of patients
(99). However, more than 70% of deaths due to sepsis occur after
the first 3 days, with many occurring weeks later. One needs to
keep in mind that many, if not most, epidemiological studies on
sepsis have been conducted in developed countries with an aging
population and advanced ICU facilities. Therefore, the reported
decline in mortality rates of early sepsis is likely owed to better
management protocols and also perhaps a reflection of immunosenescence in the elderly.
Death during protracted sepsis is sometimes the result of the
family’s decision to withdraw aggressive support measures to
switch to palliative care for patients with severe co-morbidities
and a slim chance of recovery. However, the fact remains that
with or without such decisions, many patients in this phase
succumb to stubborn infections that are difficult to resolve even
with broad-spectrum antimicrobial therapy and infection source
control (100). In a retrospective review of macroscopic autopsy
findings, approximately 77% of surgical ICU patients who had
died from sepsis or septic shock were found to have continuous
septic foci (101), suggesting a failure to clear the inciting pathogen and/or to eradicate nosocomial infections. This is thought
to be a consequence of immunosuppression (99, 100), especially
in patients who survive the early hyperinflammatory phase.
The reported inability of many septic patients to elicit normal
delayed-type hypersensitivity (DTH) skin reactions to standard
recall Ags (102) and the frequent reactivation of latent viruses
(e.g., cytomegalovirus, Epstein–Barr virus, herpes simplex virus,
human herpesvirus-6), sometimes involving multiple viruses at
the same time in prolonged sepsis (103), also point to a profound
state of immunosuppression.
Multiple other findings lend support to the notion of sepsisinduced immunosuppression. In an earlier study, van Dissel et al.
demonstrated that a high ratio of plasma IL-10:TNF-α correlates
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with increased mortality in febrile patients with communityacquired infection and cautioned against the application of proinflammatory cytokine inhibition in sepsis (104). In a separate
study, circulatory levels of IL-10 paralleled the sepsis score, and
its sustained overproduction was deemed a predictor of severity
and fatal outcome (105).
A global cytokine depression has been noted in numerous other studies. After stimulation with lipopolysaccharide
(LPS), whole blood samples from septic patients contained
less IL-1β, TNF-α, and IL-6 in comparison with samples
obtained from non-septic patients admitted for hernia repair
or cholecystectomy (106). Munoz et al. reported a profound
decrease in the ability of freshly isolated monocytes from ICU
patients with sepsis to produce IL-1β, TNF-α, and IL-6 following ex vivo exposure to LPS (107). An important finding
of this investigation was that monocytes from the survivor
subpopulation, but not from those who eventually died from
sepsis, regained their cytokine production capacity. Also interestingly, the blunted pro-inflammatory cytokine response was
most pronounced in patients with Gram-negative infections.
This may be a manifestation of the long-known phenomenon
of “endotoxin tolerance,” according to which LPS-exposed
cells become refractory to subsequent LPS challenges (108).
Endotoxin tolerance arguably serves to protect against uncontrolled inflammation in sepsis, but is also correlated with a
high risk of secondary infection and mortality. In septic
patients, monocytes are also hyporesponsive to CD40 ligation,
which would otherwise result in the upregulation of classic
costimulatory molecules B7-1 (CD80) and B7-2 (CD86) and
enhanced ability of monocytes to activate T lymphocytes
(109). The CD40–CD40L cross-talk does not directly involve
the CD14/TLR-4 pathway governing cellular responses to LPS.
Therefore, endotoxin tolerance may only partially explain
monocyte hyporesponsiveness in sepsis.
Sepsis-induced immunological shortcomings are not limited
to leukocytes traveling in the bloodstream. Boomer et al. found
that post-mortem splenocytes from septic patients secreted
significantly less TNF-α, IFN-γ, IL-6, and IL-10 in response
to LPS, CD3/CD28 co-ligation, or stimulation with phorbol
12-myristate 13-acetate (PMA) plus ionomycin when compared
with splenocytes from patients who were declared brain dead or
those who underwent emergency splenectomy due to trauma
(110). Moreover, cytofluorimetric analyses of splenic cell populations revealed signs of T cell exhaustion or anergy. For instance,
the frequency of CD4+ T cells displaying the anergy/exhaustion
marker programmed cell death 1 (PD-1) and that of CD8+ T
cells expressing the prototype co-inhibitory molecule cytotoxic
T-lymphocyte antigen-4 (CTLA-4) were higher in septic than in
control patients. Both subsets also expressed low levels of IL-7
receptor α chain (CD127) that promotes cell survival. Consistent
with these observations, splenic APCs from septic patients exhibited decreased B7-2 and HLA-DR and increased PD-ligand 1
(PD-L1) levels. It is noteworthy that weak expression of HLA-DR
is a common abnormality in sepsis. In fact, measuring monocytic
HLA-DR levels has been used to identify an immunosuppressed
state in patients with sepsis and septic shock and to monitor the
efficacy of sepsis immunotherapy (111).
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Boomer et al. also demonstrated that within the post-mortem
lung tissues of septic patients, PD-1 expression on CD4+ cells and
PD-L1 expression on plasmacytoid dendritic cells (pDCs) were
augmented in comparison with control lung tissues obtained
from transplant donors or cancer resections (110). Finally, this
comprehensive study reported two- and three-fold increases in
the frequencies of splenic Treg cells and lung myeloid-derived
suppressor cells (MDSCs), respectively, in sepsis. Treg cells are
relatively resistant to sepsis-induced apoptosis, and their percentage increases also in the circulation of patients with sepsis
(99, 112). Using the CLP mouse model, Delano et al. found
that GR-1+CD11b+ MDSCs that produce IL-10 among other
cytokines and skew T cell responses toward a Th2 phenotype
increase numerically and remain elevated within the spleen,
lymph nodes, and bone marrow (113). Therefore, suppressor cell
function appears to be a significant component of immunosuppression in sepsis.
Apoptotic death of naïve and adaptive cells of the immune
system also contributes to immunosuppression. We detected
widespread apoptosis in the spleen of mice with feces-induced
peritonitis (FIP), which we used as a model of intra-abdominal
sepsis (114). This was due to a profound apoptotic loss of splenic
T cells, B cells, NK cells, and macrophages (37). Hotchkiss et al.
performed rapid tissue harvesting at the bedside of patients dying
from sepsis and demonstrated a marked loss of splenic CD4+ T
cells, B cells and DCs (115, 116). Felmet and coworkers reported
similar depletions, prolonged lymphopenia, and hypocellularity
accompanied by apoptosis in the thymus, spleen and lymph node
autopsies of pediatric ICU patients with nosocomial sepsis and
multiple organ failure (117). Toti et al. found a dramatic depletion
of B and T cells in the spleen of preterm and full-term neonates
who died of early-onset sepsis due, likely, to in utero infection with
Gram-positive or -negative microbes (aka. chorioamnionitis)
(118). These findings indicate that immune effector cell loss during
sepsis is a universal phenomenon across all age groups.
Apoptosis causes immunosuppression through multiple
mechanisms. First, severe depletion of B and T cells creates “holes
in the repertoire” of adaptive lymphocytes. This jeopardizes the
ability of the immune system to launch highly specific responses to
pathogens. Furthermore, immunological memory cannot be built
to protect the survivors at later time points. Apoptosis-mediated
shrinkage of the DC compartment not only weakens innate
immunity but also contributes to functional T cell inadequacies
since naïve T cells can only be primed by DCs. Apoptotic cells
are immunosuppressive by nature and their uptake by phagocytic
cells can stimulate the release of anti-inflammatory cytokines
such as IL-10 (119). In addition, after ingesting apoptotic bodies, DCs may induce death in T cells with which they interact
or render them anergic (120). The importance of immune cell
apoptosis in the pathogenesis of sepsis can be underscored by the
observations that Bcl-2 overexpression or treatment with z-VADfmk, a pan-caspase inhibitor, improves survival in mouse models
of sepsis (121, 122).
agents to date. However, the results have been by and large
disheartening, with many trials yielding no benefits while a few
even aggravated the syndrome, thus leading to their premature
termination.
Most previous trials have employed agents that neutralize
pathogens or their products [e.g., intravenous immunoglobulin
(123) and the anti-endotoxin antibody nebacumab (124)],
interfere with pathogen recognition by the host [e.g., the
TLR4 antagonist eritoran (125)], or target pro-inflammatory
cytokines/mediators [e.g., the anti-TNF-α antibody afelimomab
(126) and the recombinant TNF receptor p55–IgG1 Fc fusion
protein lenercept (127)] or their receptors [e.g., the IL-1
receptor antagonist anakinra (128) and the platelet-activating
factor receptor antagonist lexipafant (129)]. Pro-inflammatory
cytokines sometimes exert redundant functions. Therefore,
therapeutic approaches targeting individual cytokines are often
ineffective. Non-specific corticosteroid therapy has also been
used in sepsis, albeit to little avail (130).
Dampening hyperinflammatory responses may benefit
some patients in the early phase of sepsis. However, it is now
recognized that many others have a global cytokine depression
or even a predominance of anti-inflammatory cytokines. Equally
important is the fact that most patients rapidly progress to an
immunosuppressed state associated with a higher susceptibility to secondary and opportunistic infections, in which case
weakening the immune system may be counterintuitive. This
may explain, at least partially, the failure of the vast majority
of previous trials designed to block inflammatory mediators in
sepsis. In fact, apart from prophylactic measures and antibiotic
administration, adjuvant therapy to restore immune competence
in immunosuppressed septic patients may prove beneficial or
even lifesaving (99). In an earlier application of such approaches,
Döcke et al. administered IFN-γ to a small cohort of septic
patients whose monocytes had reduced HLA-DR expression
and whose whole blood cells produced only minute amounts
of TNF-α in response to LPS stimulation (131). Treatment with
IFN-γ reversed these deficits and also importantly resulted in
resolution of sepsis in most cases. In a more recent case report by
Nalos and coworkers, successful IFN-γ therapy in a male patient
with type-2 diabetes and prolonged, disseminated S. aureus
sepsis was documented (132).
Granulocyte-macrophage colony-stimulating factor (GMCSF), a hematopoietic growth factor that stimulates the production of neutrophils and monocytes from bone marrow stem cells,
has also been used and shown promise in immunosuppressed
septic patients. In a relatively small-scale clinical trial, GM-CSF
administration was safe and normalized the expression of
monocytic HLA-DR and shortened the duration of mechanical ventilation and hospital/ICU stay due to sepsis (111). In a
subsequent study, GM-CSF restored the ex vivo TNF-α production capacity of whole blood cells and prevented nosocomial
infections in pediatric patients with multiple organ dysfunction
syndrome (133).
IL-7 and IL-15 are two other immune-enhancing cytokines
with enormous therapeutic potentials. Dubbed as the “maestro
of the immune system” (134), IL-7 is a pleiotropic cytokine
with diverse biological properties, some of which may correct
immunotherapy for Sepsis
Advances in our understanding of the pathogenesis of sepsis have
prompted more than 40 clinical trials of immunotherapeutic
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NKT and MAIT cells in sepsis
immunological abnormalities linked to sepsis. Clinical trials of
IL-7 in other conditions (e.g., metastatic cancer, HIV-1 infection,
and progressive multifocal leukoencephalopathy) have demonstrated that its systemic administration is safe and well tolerated
(135–137). Furthermore, it seldom causes fever or significant
pro-inflammatory cytokine production. IL-7 induces naïve and
memory T cell proliferation without a predilection for Treg cell
expansion (138). Therefore, its administration could potentially
replenish the T cell pool following drastic lymphocyte depletion
in sepsis. IL-7 is known to upregulate the expression of the antiapoptotic molecule Bcl-2 in T cells, thus promoting their survival
(139) and that of cell adhesion molecules (140), thus potentiating
leukocyte trafficking into the site(s) of infection. In addition, treatment with IL-7 increases the diversity of the TCR repertoire (138,
139), which in turn improves the breadth of pathogen-specific T
cell responses. Together, these activities can immensely help combat pathogens during sepsis. The therapeutic benefit of IL-7 has
been validated in CLP. Using this animal model, Unsinger et al.
found that recombinant human IL-7 (rhIL-7) can normalize the
DTH reaction, block T cell apoptosis, restore IFN-γ production,
and improve host survival (140). Similar results were obtained
in a “two-hit” model of fungal sepsis in which mice underwent
CLP to induce peritonitis followed by an intravenous injection of
Candida albicans (141) to mimic delayed secondary infections
in ICU patients. Venet et al. reported that IL-7 plasma levels and
CD127 expression by T lymphocytes remain unaltered in septic
shock (142). More importantly, T cells from septic patients and
healthy volunteers exhibited comparable signal transducer and
activator of transcription 5 (STAT5) phosphorylation and Bcl-2
upregulation when exposed to rh-IL-7. In addition, rh-IL-7
augmented T cell proliferation and IFN-γ production by CD8+ T
cells in response to anti-CD2/CD3/CD28-coated beads that were
used ex vivo as artificial APCs. Therefore, the IL-7:IL-7 receptor
machinery appears to be fully operative in septic patients and
may thus be utilized to reverse their immunological impairments.
IL-15 is another pleotropic cytokine involved in the development, maintenance, and proliferative responses of multiple lymphocyte lineages. It optimizes effector and memory CD8+ T cell
functions under normal conditions and also reportedly controls
the homeostatic recovery of naïve CD8+ T cells after CLP-induced
sepsis (143). Unlike IL-7, IL-15 is a potent promoter of NK cell
and DC functions, which can be defective in sepsis. In fact, IL-15
therapy was demonstrated to block NK cell, DC, and CD8+ T
cell apoptosis, to increase IFN-γ levels in the circulation, and to
improve survival of mice rendered septic by the CLP procedure
or Pseudomonas aeruginosa pneumonia (144). In a recent study,
septic patients with severe lymphopenia had low expression
of Bcl-2 mRNA in their peripheral blood mononuclear cells
despite moderately increased plasma IL-15 concentrations (145).
Whether such IL-15 quantities are still insufficient and whether
treatment with exogenous IL-15 may help correct immunological
incompetence in sepsis warrant further investigation.
Several other studies have focused on blockade of co-inhibitory
receptors (e.g., PD-1) to alleviate sepsis-induced immunosuppression. Since the induced expression of PD-1 on T cells was first
linked to their exhaustion in the context of chronic viral infection
(146), interfering with PD-1:PD-L1 interactions has been viewed
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as a tempting therapeutic approach to rejuvenating T cells in various conditions including sepsis. Administration of an antagonistic monoclonal antibody (mAb) to PD-1 after the CLP procedure
rescued the DTH response and prevented the expression loss of
the pro-survival protein Bcl-xL in splenic T cells (147). This was
accompanied by a reduction in depletion of lymphocytes and
DCs and mortality. In a separate study, treatment with an antiPD-L1 Ab either before or after CLP led to improved survival of
septic mice (148). In addition, PD-L1 blockade prevented the loss
of B and T cells, increased blood levels of IL-6 and TNF-α while
decreasing IL-10, and lowered bacterial burden in the circulation
and within the peritoneal cavity. Therefore, the PD-1:PD-L1 axis
is an attractive target for sepsis immunotherapy.
Tailoring immune intervention strategies to patients’ factors
and conditions (e.g., age, cytokine profiles, immune competence,
co-morbidities) and to the phase of sepsis (i.e., early versus protracted) will improve the likelihood of success (99). Biomarkerguided, personalized therapies that are carefully timed and
sufficiently monitored using laboratory and/or clinical measures
should prevent short- and long-term, adverse consequences of
sepsis. Agents that block inflammatory cytokines need to be
short-acting, used in early sepsis, and reserved for a group of
patients with drastically elevated pro-inflammatory cytokine
levels. On the contrary, adjuvant immunotherapy will benefit
septic patients who are in an immunosuppressed state. Failure of
leukocytes to produce TNF-α in response to LPS stimulation ex
vivo, subnormal expression of monocytic HLA-DR, upregulated
expression of PD-1 or PD-L1 on circulating leukocytes, infections caused by opportunistic pathogens (e.g., Candida spp.) and
reactivation of otherwise latent viruses, such as cytomegalovirus
and herpes simplex virus, can help identify such patients.
Animal Models of Sepsis
Using preclinical models that reliably replicate human sepsis is
essential for the development of novel diagnostic biomarkers,
prognostic indicators and therapeutic modalities that can be
truly translatable from the benchtop to the bedside. Common
animal models of sepsis, which are summarized in Table 1, utilize a variety of septic triggers or insults including LPS injection,
systemic administration of microbes, surgical disruption of the
intestinal barrier integrity, and direct introduction of feces into
the peritoneal cavity.
Clinical and paraclinical (e.g., biochemical) features of sepsis
serve as guiding principles for the development of bona fide
animal models and for validation of their relevance to the human
syndrome. Such models should take into consideration both the
early hyperinflammatory state, which is characterized by massive
pro-inflammatory cytokine production and its consequences
(e.g., fever), and the concurrent or subsequent anti-inflammatory
responses that contribute to anergy, immunosuppression
and susceptibility to secondary and opportunistic infections.
Hemodynamic changes sometimes requiring fluid resuscitation,
organ damage, apoptotic death of immunocytes, and mortality
from sepsis also need to be simulated. Animal models should
also ideally permit therapeutic intervention at defined stages
of sepsis and efficacy testing of such treatments. Accordingly,
gross outcome measurements, such as weight loss and death
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Species Model
Mouse
Endotoxicosis
Systemic
bacterial
administration
Host barrier
disruption
(CLP/CASP)
Feces-induced
peritonitis (FIP)
Rat
Endotoxicosis
Systemic
bacterial
administration
9
Host barrier
disruption
(CLP/CASP)
Rabbit
Endotoxicosis
Systemic
bacterial
administration
Pig
Endotoxicosis
Systemic
bacterial
administration
NonHuman
Primate
Endotoxicosis
Systemic
bacterial
administration
Rapid but transient inflammatory cytokine response, hypotension (149); leukopenia
(150) hypodynamic cardiovascular changes (151); multi-organ injury, mortality within
days
Advantages
Simple and reproducible
Disadvantages
Lack of infectious focus; cytokine response magnitude may
not represent human sepsis (149); poor reflection of complex
physiological/immunological changes of human sepsis
Rapid but transient inflammatory cytokine response when given i.v., slow and
sustained inflammatory cytokine response when given i.p. (180); bacteremia,
hypotension, hypodynamic cardiovascular changes with infected fibrin clot (181);
multi-organ injury, mortality within hours to days
Rapid pro/anti-inflammatory cytokine response (187) that is more severe in CASP
vs CLP (201); polymicrobial bacteremia, hypotension, hyperdynamic cardiovascular
changes (188); T and B cell apoptosis, immunosuppression (189); multi-organ injury,
mortality within hours to days
Simple and reproducible
Rapid pro/anti-inflammatory cytokine response, systemic bacterial dissemination,
splenocyte apoptosis (114); hypothermia, impaired metabolism, hypodynamic
cardiovascular changes (203); mortality within days
Rapid pro/anti-inflammatory cytokine response (152); hypermetabolism, hypotension,
hypodynamic cardiovascular changes with lethal dose i.v. (153); hyperdynamic
cardiovascular changes with non-lethal dose i.p. (165); multi-organ injury, mortality
within hours to days
Simple, controlled inoculum;
reflects polymicrobial peritonitis
Rapid pro/anti-inflammatory cytokine response (170); hypotension, bacteremia,
hypodynamic cardiovascular changes with high dose (171); hyperdynamic
cardiovascular changes with low dose (177) and infected fibrin clot (185); mortality
within hours to days
Rapid pro/anti-inflammatory cytokine response (190), hyperdynamic cardiovascular
changes (191); polymicrobial bacteremia, leukopenia, thrombocytopenia (192); multiorgan injury, mortality within hours to days
Simple and reproducible, can
reproduce hyperdynamic
changes in human sepsis
Rapid inflammatory cytokine response, hypotension, hypodynamic cardiovascular
changes with high dose (154); hyperdynamic cardiovascular changes with low dose
(164); hypothermia, leukopenia (155); multi-organ injury, mortality within hours to days
Rapid inflammatory cytokine response, hypotension, leukopenia, thrombocytopenia
(172); bacteremia, hypothermia, neutrophil apoptosis (182); multi-organ injury,
mortality within hours
Simple and reproducible,
increased sensitivity to LPS
compared to rodents
Rapid pro/anti-inflammatory cytokine response, neutropenia, lymphopenia (156);
hypotension, DIC, hypodynamic cardiovascular changes (157); hyperdynamic
cardiovascular changes with fluid resuscitation (160); mortality within hours
Simple, reproducible, porcine
physiology and LPS sensitivity
similar to humans
Expensive housing and care costs; lack of infectious focus;
poor reflection of complex physiological/immunological
changes of human sepsis
Simple, reproducible, porcine
physiology similar to humans
Expensive housing and care costs; large bolus of bacteria
may not reproduce changes of human sepsis; may reflect
endotoxicosis in the case of Gram-negative bacteria
Rapid pro/anti-inflammatory cytokine response, bacteremia, DIC (173); hypotension,
hypodynamic cardiovascular changes (174); multi-organ injury, mortality within hours
to days
Inflammatory cytokine response, hypotension, hyperdynamic cardiovascular
changes with fluid resuscitation (204, 205); leukocytosis, endotoxemia (174); multiorgan injury, mortality
Rapid but transient pro-inflammatory cytokine response, hypotension, hypodynamic
cardiovascular changes (158); thrombocytopenia, leukopenia (159)
Rapid pro/anti-inflammatory cytokine response, hypotension, leukopenia,
thrombocytopenia, DIC (175); hypodynamic cardiovascular changes that become
hyperdynamic with fluid resuscitation (176); multi-organ injury, mortality within
hours to days
Polymicrobial, severity
controlled by size of puncture/
stent diameter; CLP reproduces
immunosuppressive phase
Simple and reproducible
Polymicrobial, severity
controlled by size of puncture/
stent diameter
Simple and reproducible
Variability introduced by the choice of bacterial strain
and administration route; large bolus of bacteria may
not reproduce changes of human sepsis; may reflect
endotoxicosis in the case of Gram-negative bacteria
Requires surgical techniques; high experimental variability;
abscess formation may prevent disease development (201)
Microbial dose and composition of the fecal inoculum often
unknown; cytokine response magnitude more severe vs.
CLP (206)
Lack of infectious focus, poor reflection of complex
physiological/immunological changes of human sepsis
Large bolus of bacteria may not reproduce changes of
human sepsis; may reflect endotoxicosis in the case of
Gram-negative bacteria
Requires surgical techniques; high experimental variability
More expensive than rodent models; lack of infectious focus,
poor reflection of complex physiological/immunological
changes of human sepsis
More expensive than rodent models; less wellcharacterized; may reflect endotoxicosis in the case of
Gram-negative bacteria
Porcine physiology similar to
humans; reflects polymicrobial
peritonitis
Cross-reactivity with human
thera-peutics and diagnostic
tools, most comparable to
human physiology
Expensive housing and care costs; microbial dose and
composition of the fecal inoculum often unknown
Cross-reactivity with human
therapeutics and diagnostic tools,
most comparable to human
physiology
Most expensive housing and care costs; ethical concerns;
may reflect endotoxicosis in the case of Gram-negative
bacteria
Most expensive housing and care costs; ethical concerns;
more accurately reflects human endotoxicosis rather than
sepsis
CASP, colon ascendens stent peritonitis; CLP, cecal ligation and puncture; DIC, disseminated intravascular coagulation; FIP, feces-induced peritonitis; i.p., intraperitoneal; i.v., intravenous; LPS, lipopolysaccharide.
NKT and MAIT cells in sepsis
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Feces-induced
peritonitis (FIP)
immunopathology and reported manifestations
Szabo et al.
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TABLe 1 | Common Animal Models of Sepsis.
Szabo et al.
NKT and MAIT cells in sepsis
rates, should be complemented with laboratory assessments of
immune competence or incompetence (e.g., cytokine production
and anergy/exhaustion marker expression).
Findings from models in which young, adult and otherwise
healthy animals are utilized may not accurately represent the
“real-life” features of sepsis in the rising elderly populations. This
is a major limitation of animal models that place a disproportionate emphasis on sepsis-induced hyperinflammation, which
no longer accounts for most deaths due to sepsis at least when
advanced ICU facilities and robust practice of critical care are in
place. Therefore, experimentation with older animals and those
with co-morbidities may provide a more realistic picture of sepsis
in vulnerable populations.
One of the most routinely utilized agents to induce sepsis in
small and large animals is LPS (149–160), a glycolipid found
abundantly in the outer membrane of Gram-negative bacteria.
Following intravenous (i.v.) or intraperitoneal (i.p.) injection,
LPS binds to the glycosylphosphatidylinositol (GPI)-anchored
protein CD14 and signals through TLR-4 to provoke a systemic
inflammatory response often referred to as “endotoxicosis”
(161, 162). This response is characterized by pro-inflammatory
cytokine production, multiple organ injury and hypotensive
shock, which are hallmarks of early sepsis. LPS administration
is simple and does not require advanced surgical techniques.
In addition, its dosage can be easily controlled. However, one
should keep in mind that exposure to large amounts of LPS may
result in an immediate hypodynamic cardiovascular state that
does not represent human sepsis (163). Several groups have
overcome this problem by developing models that use sublethal
doses of LPS (164, 165) or aggressive fluid resuscitation (166).
Also importantly, bolus injection of LPS into laboratory animals
triggers a severe inflammatory cytokine response that differs
in magnitude and sustenance from what is observed in clinical
sepsis (163, 167).
Lethal shock and disseminated intravascular coagulation
(DIC) can be induced in mice by two consecutive injections of
LPS separated by a 24-h interval (168). In this model, which is
known as “generalized Shwartzman reaction,” a “super low” dose
of LPS is injected followed by a larger systemic dose that elicits
rapid pro- and anti-inflammatory responses, coagulopathy and
multi-organ damage. It is noteworthy that the initial priming dose in Shwartzman reaction is smaller than that causing
endotoxin tolerance (169). It is believed that tolerizing doses of
LPS activate the canonical nuclear factor-κB (NF-κB) pathway
leading to robust expression of pro-inflammatory mediators as
well as a myriad of suppressive elements designed to prevent
progressive inflammation (169). In contrast, super low doses of
LPS, such as those used in the priming phase of Shwartzman
reaction, fail to activate the NF-κB pathway. Instead, they trigger
the activation of CCAAT/enhancer binding protein δ (C/EBPδ)
in an IL-1 receptor-associated kinase 1 (IRAK1)-dependent
manner resulting in mild but persistent expression of inflammatory mediators (169).
All animal models of endotoxicosis lack an infectious focus.
In addition, since LPS is only present in Gram-negative bacteria,
these models do not represent polymicrobial sepsis caused by
mixed Gram-positive and Gram-negative microbes.
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Systemic administration of a large number of bacteria, typically E. coli, instigates a massive inflammatory cytokine response,
cardiovascular collapse and rapid mortality (170–175). Fluid
resuscitation or sublethal dosages of bacteria can be used to better
mimic the septic response and its hemodynamic manifestations
in humans (176–178). These models allow for bacterial strains
and numbers to be carefully chosen and for host responses to
develop against intact microbial pathogens. However, they are
more similar to models of endotoxicosis than full-blown infections when Gram-negative bacteria are used. Many bacterial
strains are complement-sensitive and lysed shortly after they
enter the circulation, thus releasing their endotoxin content (179).
Moreover, systemic bacterial infusion gives rise to serum TNF-α
concentrations that are orders of magnitude larger than those
found in septic patients or in peritonitis models (179). Lastly,
the route of administration can impact the vigor of the septic
response. For instance, a robust but transient TNF-α response is
elicited following an i.v. challenge of mice with live E. coli O111,
whereas an i.p. challenge leads to much lower but more sustained
blood levels of TNF-α (180).
Surgical implantation of bacteria (e.g., E. coli)-laden fibrin
clots into the peritoneum has also been used to induce sepsis in
several species (181–185). Some of these models more accurately
reproduce the hyperdynamic state and slow, sustained release of
cytokines associated with human sepsis.
Cecal ligation and puncture (CLP) is considered by many
as the “gold standard” of intra-abdominal sepsis models. This
relatively simple surgical procedure involves a laparotomy and
ligation of the cecum in a non-obstructing manner followed
by puncturing the ligated portion to allow fecal content to leak
into the otherwise sterile peritoneal cavity (186). Therefore, a
source of necrotic tissue combined with an infectious focus that
persistently challenges the host with enteric microbes causes
polymicrobial sepsis. CLP-inflicted sepsis resembles the clinical
syndrome since it can set in motion a systemic pro-inflammatory
cytokine response as well as a compensatory anti-inflammatory
response and a hyperdynamic cardiovascular state (162, 187–192).
Furthermore, CLP is particularly useful for studying the delayed
phase of sepsis in which immune responses are impaired. This is
possible by the “two-hit” versions of the model, in which mice
undergo CLP and are subsequently challenged with a secondary/
opportunistic pathogen, such as Streptococcus pneumoniae (193),
Pseudomonas aeruginosa (193–195), Candida albicans (141) or
Aspergillus fumigatus (196). Logistically, the CLP procedure is
quick to perform by an experienced experimentalist. It can also
be readily modified to investigate varying degrees of inflammation and different survival intervals. The length of ligated cecum
(197), the size of the needle used for the perforation (198), and
the number of punctures made (199) can all determine the severity of sepsis and the speed with which death occurs. It needs to be
noted that the CLP outcome may vary considerably among different laboratories and animals depending upon the experimentalist’s surgical expertise and the animals’ sex, age, strain, housing
conditions, cecal content, and even cecal fullness when CLP is
performed (167). Another disadvantage of the CLP model lies in
the host’s natural ability to form an abscess in order to contain
infection (200, 201). Therefore, treatments that promote abscess
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formation may improve survival in CLP, which may introduce
bias by adding a confounding variable in the experiment.
Another model of host barrier disruption leading to sepsis
is colon ascendens stent peritonitis (CASP), in which a stent is
inserted into the ascending colon to allow for leakage of fecal
matter into the peritoneal cavity (201, 202). Although similar to
CLP in principle, CASP represents persistent peritoneal infection rather than abscess formation and causes a more robust
cytokine response and higher bacterial loads within several
organs. The severity of and mortality from CASP are influenced
by the diameter of the stent and also by its removal at defined
time points. This mimics surgical interventions to eliminate
infectious foci in humans.
Host barrier disruption models are heavily reliant on surgical
techniques and relatively difficult to standardize. An alternative
approach is to simply inject animals with a given amount of fecal
solution i.p. (114, 174, 203–205). This is called the feces-induced
peritonitis (FIP) model of polymicrobial sepsis, for which we
recently developed a robust scoring system (114). Early inflammatory cytokine production in FIP is typically much more intense
than that caused by CLP (206). The amount of feces to be injected
i.p. can be adjusted to alter the severity and outcome of sepsis. An
additional advantage of FIP is that fecal solutions with identical
microbial loads and composition can be injected into multiple
recipient cohorts. This is in contrast with barrier disruption
models requiring the leakage of each animal’s intestinal content
into the peritoneal cavity, which is an inevitable source of variation. A limitation of the FIP model is that the dosage and species
of bacteria introduced into the recipients are usually unknown
given that intestinal flora vary according to the animal strain,
commercial source and housing conditions. Finally, the state of
immunosuppression that follows the hyperinflammatory phase
of sepsis has not been fully characterized in FIP.
Despite the abundance of animal models for sepsis, there is
currently no one truly clinically relevant model that fully recapitulates all the complex immunological, hemodynamic, and
pathophysiological responses seen in human sepsis. The reason
for outcome discrepancies between animal models and clinical
sepsis is multifactorial, but partially stems from the heterogeneity
of patient populations. Nevertheless, we continue to rely on current animal models and strive to come up with improved models
in order to better understand the pathogenesis of sepsis and to
design and test novel treatments for this fatal syndrome.
may also potentially activate CD1d+ APCs (208). Therefore, the
mechanism of action of this mAb could not be definitively determined. More importantly, both iNKT and vNKT cells interact with
CD1d (50). We now know that there are other CD1d-restricted T
cell types such as a subpopulation of γδ T cells (209) that can be
affected by anti-CD1d treatment. Nevertheless, the study of Rhee
et al. indicated a role for CD1d-restricted T cells in sepsis and set
the stage for subsequent important investigations.
Hu et al. extended the above study to other mouse strains
(210). They demonstrated that pre-treatment of BALB/c mice
with 1B1 before CLP confers upon them a survival advantage.
This treatment also prevented the rise in circulating levels of
TNF-α, IL-6, monocyte chemotactic protein (MCP)-1 and IL-10.
Within the liver, mice receiving 1B1 had lower frequencies of NKT
cells capable of producing TNF-α, IL-6, IL-4 or IL-10, indicating
no bias toward either a pro- or anti-inflammatory phenotype.
Interestingly, however, the percentage of IL-6-producing hepatic
macrophages declined whereas that of IL-10-producing cells
increased upon anti-CD1d treatment. How CD1d contributes
to the immunopathology of sepsis is not clear. It is possible that
lipid antigens derived from bacterial pathogens are loaded into
CD1d and presented to NKT cells. Alternatively or in addition,
recognition of pathogen-associated molecular patterns (PAMPs)
by PRRs such as TLRs may lead to the production of IL-12 and
IL-18 by APCs during sepsis. Once coupled with CD1d-mediated
presentation of endogenous lipids, these cytokines can induce
NKT cell activation (42, 43). Sepsis-induced tissue injury may
also increase, release and/or modify endogenous lipids that can
be displayed by CD1d to trigger NKT cell responses (Figure 1).
Consistent with this hypothesis, a previous study reported that
serial injections of apolipoprotein E (ApoE), a component of
plasma lipoproteins, alters NKT cell compartments and increases
CLP-induced mortality in rats (211).
Hu et al. also used the CLP model to examine the contribution of the invariant subset of NKT cells to sepsis (210). They
found a marked decline in the frequency of hepatic iNKT cells,
defined by their reactivity with α-GalCer-loaded CD1d tetramer,
in both C57BL/6 and BALB/c mice. This was accompanied by
upregulation of CD69 and CD25 on the surface of iNKT cells
indicating their enhanced activation on a per cell basis. There
exist several possibilities to explain the lower percentage of
detectable iNKT cells in the liver of septic mice. These include
iTCR internalization, which is a well-known phenomenon in the
context of iNKT cell activation by synthetic glycolipids (212), cell
death in situ, or migration to other locations. To address these
possibilities experimentally, one could assay for intracellular
iTCRs or quantify mRNA corresponding to the Vα14–Jα18 TCR
rearrangement in hepatic non-parenchymal cells, or track iNKT
cell movements in the body.
To ascertain whether iNKT cells play a pathogenic or
protective role in sepsis, Hu and coworkers used Jα18−/−
mice that lack iNKT cells (210). These animals exhibited
reduced mortality due to CLP as well as ablated TNF-α,
IL-6, MCP-1 and IL-10 systemic responses in comparison
with wild-type C57BL/6 mice. It was recently found that the
TCRα repertoire of Jα18−/− mice that have been widely available to the research community is shrunk by ~60% (213).
iNKT Cells and Sepsis
Several groups including ours have explored the effector or
regulatory capacities of iNKT cells and their synthetic glycolipid
agonists in sepsis and endotoxic shock.
Rhee et al. from Alfred Ayala’s laboratory first reported that
treating 129S1/SvImJ mice with an anti-CD1d mAb (clone 1B1)
before the CLP surgery could reduce plasma and splenic IL-6
and IL-10 levels and prevented sepsis-induced mortality in some
of the treated mice (207). They also noted a significant increase
in the frequency of cell populations co-expressing T and NK
cell markers, which could be reversed by anti-CD1d treatment.
It needs to be noted that although the 1B1 mAb has been used
extensively to block CD1d interactions with NKT cell TCRs, it
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FiGURe 1 | iNKT cell activation pathways in sepsis. Early in the course of
the host response to bacterial pathogens involved in sepsis, the engulfment of
these microbes by phagocytic cells generates pathogen-derived glycolipids that
can be displayed by CD1d to induce iNKT cell activation. Phagocytic cells that
have taken up bacteria and/or sensed PAMPs (e.g., LPS) through PRRs (e.g.,
TLR-4) secrete inflammatory cytokines. Some of these cytokines (e.g., TNF-α,
IL-1, IL-6) are responsible for clinical manifestations of sepsis, while others (i.e.,
IL-12 and IL-18) can activate iNKT cells. The latter pathway often, but not
always, requires CD1d-mediated presentation of endogenous glycolipids to
iNKT cells. SAg-secreting bacteria, such as Staphylococcus spp. and
Streptococcus spp. participating in Gram-positive bacterial sepsis, can directly
Frontiers in Immunology | www.frontiersin.org
activate iNKT cells. It is possible that bacterial PAMPs may be detected by iNKT
cells. Finally, during or as a result of the septic insult, host cell damage leads to
release and/or modification of endogenous glycolipids that can be potentially
presented by CD1d to trigger iNKT cell activation in an iTCR-dependent
manner. Once activated, iNKT cells produce pro-inflammatory cytokines, most
notably IFN-γ that plays a pivotal role in sepsis-inflicted immunopathology. APC,
antigen-presenting cell; CD, cluster of differentiation; DC, dendritic cell; IFN,
interferon; IL, interleukin; iNKT, invariant natural killer T cell; iTCR, invariant T cell
receptor; LPS, lipopolysaccharide; MHC, major histocompatibility complex;
PAMP, pathogen-associated molecular pattern; PRR, pattern recognition
receptor; TLR, Toll-like receptor.
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Nga liver sections. NC/Nga mice injected with d-GalN and LPS
had negligible levels of IFN-γ protein in their serum or IFN-γ
mRNA in their liver. This was accompanied by a 10-fold reduction in the size of CD3ϵ+DX5+ NKT cell compartment in the
liver although NKT cells were capable of producing ample IFN-γ
on a per cell basis. Finally, administration of recombinant IFN-γ
to d-GalN-sensitized NC/Nga mice rendered them susceptible
to LPS-induced mortality. In this body of work, frequency analyses were performed on NKT cells co-expressing T and NK cell
markers. However, iNKT cells are the likely culprits and the early
triggers of pathology in d-GalN/LPS-prone mice. This is because
iNKT cells comprise the vast majority of hepatic NKT cells in
mice (217). Second, when Koide and coworkers injected NC/
Nga mice with the iNKT cell superagonist α-GalCer a few hours
before the d-GalN/LPS challenge, endogenous IFN-γ production
was restored leading to increased expression of inducible nitric
oxide synthase (iNOS), appearance of apoptotic cells in the liver,
and 100% mortality (215). It was therefore concluded that the
resistance of NC/Nga mice to the LPS-mediated lethality with
d-GalN sensitization is due to impaired IFN-γ production caused
by a shortage of iNKT cells and reduced nitric oxide production
in these animals. An additional confirmatory approach would
have been to adoptively transfer a large number of syngeneic
iNKT cells into NC/Nga mice to increase their frequency before
testing the susceptibility of the recipients to d-GalN/LPS.
In a different model of endotoxic shock, α-GalCer injection
sensitized wild-type mice, but not Jα18−/− mice, to LPS-mediated
lethality (218). Interestingly, shock in these animals was accompanied by severe lesions and hemorrhage, marked accumulation
of polymorphonuclear leukocytes and mononuclear cells, and
significant cell death almost exclusively in the lungs. Although
serum ALT levels were elevated, hepatic lesions were focal and
mild, and other organs showed no signs of overt injury or other
changes except for congestion. Pulmonary manifestations and
lethal shock in this model could not be induced by simultaneous
administration of α-GalCer and LPS, and required an interval
period of 3–24 h between α-GalCer sensitization and the LPS
challenge. This is consistent with the kinetics of IFN-γ secretion
in response to α-GalCer, which is potentiated by iNKT cells and
largely contributed by transactivated NK cells (219). Ito et al.
found that α-GalCer injection gives rise to high blood levels of
IFN-γ within the above timeframe and augments the subsequent
production of TNF-α, a major mediator of endotoxic shock, in
response to LPS (218). They further demonstrated that neutralizing IFN-γ or genetic deficiency of TNF-α abolishes the systemic
lethal shock in this model. Therefore, it was proposed that IFN-γ
and TNF-α play pivotal roles in preparation and execution of
LPS-mediated lethality, respectively, in α-GalCer-primed mice.
Following up on these findings, Tumurkhuu et al. found that
priming with α-GalCer increases the frequency of NKT cells
among pulmonary non-parenchymal leukocytes and induces
local IFN-γ production (220). This resulted in expression of
several adhesion molecules, most notably vascular cell adhesion
molecule-1 (VCAM-1), on vascular endothelial cells of the lungs,
which in turn promoted the accumulation of very late activating antigen-4 (VLA-4)+ cells among inflammatory cell recruits
in the lungs. This was significant because an anti-VCAM-1
Therefore, the cellular deficiency in Jα18−/− mice is not exclusive
to iNKT cells, which necessitates iNKT cell reconstitution
experiments to validate results obtained using these animals.
More recently, Heffernan et al. demonstrated that while CLP
causes a drop in the frequency of iNKT cells in the liver, both
the absolute number of iNKT cells and their frequency among
T lymphocytes are elevated in the circulation and within the
peritoneal cavity, which is considered the site of polymicrobial
infection in this model (214). Furthermore, a much bigger fraction of peritoneal iNKT cells expressed CD69 in septic mice in
comparison with the sham laparotomy control group. Although
iNKT cell mobilization by CLP was not directly monitored,
these results support the scenario in which iNKT cells migrate
out of the liver and toward the source of infection, which should
account for their decline in the liver. Intriguingly, this migration was mediated by PD-1, which is well known as an anergy/
exhaustion marker. Following sepsis, PD-1−/− mice exhibited a
numerical increase in activated hepatic iNKT cell populations but
intact peripheral blood or peritoneal iNKT cell compartments
when they were compared with the sham controls. Whether the
role of PD-1 in iNKT cell migration is intrinsic to these cells was
not studied. This question could be addressed by reconstitution of
Jα18−/− mice with PD-1-sufficient or -deficient iNKT cells prior to
the CLP procedure. These investigators also found that once accumulated in the peritoneal cavity, iNKT cells influence the ability
of local macrophages to phagocytose bacteria and clear infection.
Bacterial load in the cavity was lower in septic Jα18−/− mice than in
wild-type controls. In addition, peritoneal macrophages derived
from septic Jα18−/− mice were more potent than those from septic
wild-type animals in engulfing E. coli. Collectively, the work of
Heffernan and coworkers reveals an interesting interplay between
migrant iNKT cells and macrophages residing within foci of
infection during sepsis. It also suggests that blockade of PD-1
may not only reverse T cell exhaustion to relieve sepsis-induced
immunosuppression but also likely benefits the host by modulating the migration capacity of iNKT cells to further facilitate
microbial clearance.
Taken together, the above studies indicate a pathogenic role
for iNKT cells in CLP-induced sepsis. Several groups have
reached the same conclusion using animal models of LPSinflicted pathology or lethality. Koide et al. established a link
between the resistance of d-galactosamine (d-GalN)-sensitized
NC/Nga mice to LPS and the presence of fewer NKT cells in
these animals (215). This old protocol utilizes the hepatotoxic
agent d-GalN to sensitize laboratory animals to very low doses
of LPS, and has been used extensively as a model of endotoxic
shock and Gram-negative microbial sepsis. We are of the opinion that the d-GalN sensitization model simulates acute hepatic
failure more closely. Nevertheless, participation of inflammatory
mediators is evident in its immunopathology amid severe liver
damage. While co-administration of d-GalN and LPS led to
100% mortality in C57BL/6 mice within 12 h, it failed to kill
NC/Nga mice even at 24 h (215). It also raised the activity
level of alanine aminotransferase (ALT) in the serum and that
of caspase-3 in the liver extract of C57BL/6 mice but not NC/
Nga mice (216). Moreover, drastic lesions with hemorrhage and
many apoptotic cells were observed in C57BL/6 but not in NC/
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thus hampering their IFN-γ production capacity. It would be
interesting to examine the expression level of PD-1 on iNKT cells
obtained from α-GalCer-pretreated mice or to test whether blockade of the PD-1:PD-L1 interaction restores Shwartzman reaction.
In a separate study, Sierci et al. found that α-GalCer administered
within 2 h before or after the LPS challenge rescues the mice
(225). This timeframe is consistent with the period in which IL-4
production by α-GalCer-stimulated iNKT cells reaches its peak
while only minute amounts of IFN-γ are detectable in the serum.
Accordingly, Sierci and coworkers noted increased IL-4 and IL-10
responses and decreased levels of IFN-γ and TNF-α in protected
mice. In addition, blood levels of ALT and aspartate aminotransferase (AST) were lower in these animals indicating milder injury
to the liver. The beneficial effect(s) of Th2-type cytokines were
confirmed when mice receiving either an anti-IL-4 or an antiIL-10 mAb succumbed to endotoxic shock. Therefore, inducing
Th2-skewed iNKT cell responses may have potential therapeutic
applications in sepsis. We recently put this hypothesis to the test
by using Th2-promoting iNKT cell agonists in the FIP model of
sepsis (read below).
In a prospective study, we demonstrated that patients with
sepsis have a significantly elevated ratio of peripheral blood
iNKT:T cells in comparison with non-septic trauma patients
(37). The patient cohorts were similar in age and in severity of
illness that was calculated based on their Acute Physiology and
Chronic Health Evaluation II (APACHE II) scores in the initial
24-h period post-diagnosis (227). Next, we compared wild-type
and Jα18−/− mice receiving a fecal slurry i.p. for severity of FIP
using a murine sepsis score (MSS) that we recently developed
(114) and also for mortality from sepsis. The severity of sepsis
was significantly lower in Jα18−/− mice than in wild-type controls.
In addition, intra-abdominal fecal challenge resulted in 100%
mortality in wild-type animals but no death in septic Jα18−/− mice
within 24 h. Importantly, reconstitution of Jα18−/− mice with
iNKT cells before the septic insult increased the severity of their
symptoms. Together, these results confirm the pathogenic nature
of iNKT cells in the FIP model. In the next series of experiments,
we explored the therapeutic potentials of OCH, a Th2-polarizing
analog of α-GalCer (34), in FIP. We found that a single i.p. injection of OCH within 20 min after the fecal challenge reduced the
MSS scores and prolonged the survival of septic mice compared
with vehicle- or α-GalCer-treated animals. These changes were
associated with elevated blood levels of the Th2-type cytokine
IL-13 and reduced levels of the pro-inflammatory cytokine IL-17.
Furthermore, OCH treatment decreased the number of apoptotic
T cells, B cells and macrophages in the spleen. Anti-inflammatory
mechanisms are known to contribute to sepsis-induced immunosuppression, which may make a septic individual susceptible
to opportunistic infections (93). Therefore, we asked whether
OCH treatment worsens the microbial load in septic mice. Much
to our satisfaction, this was not the case, and blood and tissue
homogenates prepared from the heart, lungs, kidneys, liver and
spleen of vehicle-, OCH- and α-GalCer-treated septic mice
had comparable numbers of microbial colony-forming units.
Finally, administration of C20:2, another glycolipid that is even
more potent than OCH in inducing a Th2 bias (228, 229) and
that additionally suppresses downstream NK cell function (230),
mAb partially averted LPS-mediated lethal shock in α-GalCersensitized mice.
In the above studies, the relative contributions of iNKT and
NK cells to IFN-γ production was not determined. There currently exists no commercially available antibody for selective
depletion of iNKT cells although online literature search through
the World Wide Web indicates that a mAb called NKT14 may
serve this purpose in the future. Until this or similar antibodies
become available, one could employ anti-asialo GM1 antiserum
and an anti-NK1.1 mAb (clone PK136) in parallel cohorts of mice
to address this question. The former depletes NK cells without
affecting the NKT cell population, and the latter depletes both
NK and NKT cells (45, 221).
Another important question is why LPS-induced pathology
in α-GalCer-sensitized mice is restricted to the lungs while the
liver is largely spared. This is particularly interesting in light of
the observation that α-GalCer induces IFN-γ production by
both hepatic and pulmonary iNKT cells and that IFN-γ is readily detectable at mRNA and protein levels in both organs. It has
been argued that IFN-γ signaling is fully operational in the lungs
but not in the liver of α-GalCer-primed mice (222). Augmented
expression of phosphorylated STAT1 was more sustained in the
lungs than in the liver. In addition, IFN regulatory factor 1 (IRF1)
was upregulated in the lungs but not in the liver of α-GalCertreated mice. Second, pulmonary NKT cells reportedly produce
IFN-γ as their main cytokine, whereas hepatic NKT cells produce
IFN-γ, IL-4 and IL-10. Neutralization of IL-4 enhances STAT1
activation, exacerbates the hepatic injury, and increases the
number of apoptotic cells in the liver. Therefore, IL-4 has been
proposed to inhibit IFN-γ signaling in the liver while its absence
promotes IFN-γ-mediated pathology in the lungs (222). Finally,
one might wonder why a potentially similar mechanism is not at
play to protect NC/Nga livers in the d-GalN/LPS model (215).
It is possible that the cytokine profile of α-GalCer-primed NC/
Nga mice differs from that of C57BL/6 and BALB/c mice. The
hepatotoxic nature of the d-GalN insult may also mask the influence of other factors involved. These possibilities are not mutually
exclusive.
Several studies have focused on the role of iNKT cells in systemic Shwartzman reaction. IFN-γ is considered a key cytokine
in the pathogenesis of Shwartzman reaction because it induces
massive production of TNF-α, IL-1 and other inflammatory
mediators. Dieli et al. found Jα18−/− mice on either C57BL/6 or
BALB/c background to be resistant to the LPS-induced mortality
of Shwartzman reaction (223). Jα18−/− mice had lower serum
levels of IFN-γ and TNF-α in comparison with wild-type animals, and administration of recombinant IFN-γ was sufficient
to prime these animals. In two more recent studies, Sierci et al.
tested the effect of α-GalCer treatment at different time points
before or after LPS priming (224, 225). When α-GalCer was
given approximately 6, 9, or 12 days prior to the first injection
of LPS, mice survived the subsequent LPS challenge and their
protection was associated with reduced serum levels of IFN-γ
and TNF-α and hepatic level of MCP-1 (224). In stark contrast,
when administered 1 or 3 days before priming, α-GalCer failed
to protect the mice from lethal endotoxic shock. It appears likely
that earlier α-GalCer injection induces iNKT cell anergy (226),
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also mitigated the severity of FIP-induced sepsis. However, this
effect was only transient, which may be explained by the relatively
short half-life of C20:2 compared with OCH (228, 230). Based
on these results, we propose that Th2-skewing agonists of iNKT
cells may be employed to treat the hyperinflammatory phase of
sepsis without compromising the patient’s immunity to microbial
pathogens.
Finally, during polymicrobial sepsis, common bacterial pathogens, such as Staphylococcus spp. and Streptococcus spp., are likely
to release the SAgs they harbor (Figure 1). We recently discovered that staphylococcal and streptococcal exotoxins belonging
to phylogenetic group II SAgs can directly activate mouse and
human iNKT cells leading to IFN-γ production (45). However,
anticipating the net effect is not simple because: (i) the type of
microbial pathogens involved in sepsis may vary substantially
among different individuals; (ii) how multiple SAgs released by
multiple bacterial pathogens may cross-regulate the response
to each other is far from clear; (iii) host responses to SAgs may
be modulated by cell wall components of the very bacteria that
release SAgs as we previously described (231).
To summarize, the studies highlighted in this section all point
to a pathogenic role for iNKT cells in sepsis regardless of the
experimental model employed, and IFN-γ is a major mediator of
iNKT cell-inflicted damage in this context.
of vNKT cells, have an activated phenotype as judged by their
upregulated expression of CD69 in infected mice. They also
showed that treating wild-type C57BL/6 mice with porcine
sulfatide before bacterial inoculation lowers their blood levels of
TNF-α and IL-6 without altering the staphylococcal burden in
blood, liver or kidneys. Therefore, sulfatide treatment does not
impede the ability of the immune system to combat this pathogen.
In fact, wild-type mice receiving sulfatide 1 h before and 3 days
after bacterial inoculation were partially protected. Importantly,
the survival advantage conferred by sulfatide treatment could be
recapitulated in Jα18−/− (vNKT-sufficient, iNKT-deficient) mice
but was missing in CD1d−/− (vNKT- and iNKT-deficient) mice.
The significance of this finding is two-fold. First, it strongly suggests that activated vNKT cells mediate the protective effect of
sulfatide in septic wild-type mice. Second, unlike in other models
where sulfatide treatment induces iNKT cell anergy to ameliorate
inflammation and injury (235, 236), its beneficial effect in S. aureus
sepsis does not require the presence of iNKT cells. Of note, late
injection of sulfatide in this model (i.e., on day 3 post-bacterial
inoculation) failed to improve survival. This may be viewed as an
impediment to the possibility of sulfatide therapy in staphylococcal sepsis once the symptoms have developed. However, more
comprehensive studies are warranted to possibly find a window
of opportunity during which sulfatide-based interventions may
be effective in staphylococcal and other forms of sepsis.
vNKT Cells and Sepsis
MAiT Cells and Sepsis
The extent to which vNKT cells contribute to or regulate sepsis
is unknown. However, in vivo treatment with sulfatide, a CD1drestricted ligand of vNKT cells, has been demonstrated to attenuate the magnitude of the septic response, thus providing indirect
evidence for a protective role of activated vNKT cells in sepsis.
The beneficial effect of sulfatide was initially noted in two
relatively old studies on LPS-induced sepsis with a focus on
how this glycolipid influences leukocyte adhesive properties and
transendothelial migration as opposed to NKT cell functions
(232, 233). Higashi et al. reported that while 75% of C3H/HeN
mice died within 2 days of injection with a large dose of LPS, only
20% of mice that were pretreated with bovine brain-extracted
sulfatide succumbed even after 7 days (232). Administration
of sulfatide to either C3H/HeN or C57BL/6 mice also partially
inhibited their TNF-α response to a sublethal dose of LPS. Finally,
using a mouse model of endotoxin-induced hypotension, these
investigators demonstrated that treatment with sulfatide prior to
the LPS challenge prevents an otherwise progressive decline in
systolic blood pressure. Squadrito and coworkers explored the
effect of sulfatide on acute lung injury in a rat model of endotoxic
shock (233). When administered shortly after the LPS injection,
sulfatide was able to partially offset hypotension, revert leukopenia, and diminish myeloperoxidase activity in the lungs that was
used as an indication of neutrophil accumulation in this tissue.
Also importantly, sulfatide treatment caused a near complete
prevention of LPS-induced lethality.
The only published report to date addressing a link between
CD1d-restricted, sulfatide-reactive T cells and sepsis has utilized
an experimental mouse model of S. aureus infection (234). In
this work, Kwiecinski et al. found that TCRβ+NK1.1+α-GalCer/
CD1d tetramer− cells, which should contain a sizeable population
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MAIT cells are relatively frequent among human innate-like T
cells and capable of responding to a wide variety of bacterial and
fungal pathogens. The ability of MAIT cells to rapidly produce
inflammatory cytokines, together with their strategic positioning
at the host–pathogen interface, makes them an ideal candidate
to fulfill the role of emergency responders to infection and/or
regulators of the septic response.
Grimaldi et al. recently explored how sepsis may change the
frequency and absolute number of MAIT cells in the circulation
(237). In a prospective study, they recruited a relatively large
number of patients with severe sepsis or septic shock and compared their peripheral blood MAIT, iNKT, and γδ T cell compartments with those of critically ill patients with non-septic (mostly
cardiogenic) shock, and age-matched healthy volunteers. Septic
patients exhibited an early and dramatic decrease in their MAIT
cell count compared with non-infected critically ill patients or
healthy controls. This was unlike iNKT or γδ T cell counts that
remained unaltered in different groups. Also interestingly, there
was no association between MAIT cell and total lymphocyte
counts, suggesting that MAIT cells follow an independent kinetic
pattern in sepsis. By the same token, the frequency of MAIT cells
among CD3+TCRγδ− conventional T cells was significantly lower
in septic patients than in healthy subjects.
The above investigation also led to other potentially important
observations. First, the early drop in MAIT cell frequencies was
more pronounced in septic patients with non-streptococcal
infections than in those with streptococcal infections. In addition, in a small cohort of patients with severe viral infections in
the absence of concomitant bacterial infections, MAIT cell values
were similar to those of healthy controls. Streptococcus spp. and
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viruses are known not to activate MAIT cells (238). Therefore,
the above findings are consistent with the hypothesis that the
observed numerical change in the MAIT cell compartment of
septic patients is dictated by the type of pathogen(s) encountered.
Second, Grimaldi et al. found a higher cumulative incidence of
ICU-acquired infections in patients with a persistent decline
in peripheral blood MAIT cells. In fact, patients who did not
develop secondary infections showed a gradual return to normal
MAIT cell values. Therefore, sepsis-induced changes in the MAIT
cell compartment seem to be reversible by nature.
It is not clear why MAIT cell numbers drop early in sepsis.
Apoptotic cell death, TCR internalization, and migration to
peripheral tissues, for instance toward the infectious focus/foci,
may provide an explanation for this phenomenon. The latter
possibility is supported by the observation that in a few patients
registered in the above study, a higher proportion of MAIT cells
was detectable in unspecified biological fluids than in blood (238).
This is reminiscent of previous reports that MAIT cell numbers
drop in the peripheral blood but increase in the lungs of patients
with tuberculosis (238, 239). Monitoring MAIT cell frequencies
in individual septic patients and future mechanistic studies to
uncover the cause of MAIT cell decline in the blood circulation
will be informative.
Evolutionary conservation of CD1d and MR1 recognition
across mammalian species makes animal models of sepsis particularly useful for studying NKT and MAIT cells. There is an
urgent need for more investigations in models that mirror aging
and various co-morbidities. Humanized mouse models should
also shed mechanistic light on how clinical sepsis is initiated,
perpetuated, or regulated. Such models could also potentially
address some of the discrepancies noted between the results of
rodent and human studies on NKT/MAIT cell frequencies, effector functions and homing properties.
Currently available Jα18−/− mice that have been used extensively by many investigators including us were recently found
to lack T cells other than iNKT cells (213). Therefore, if based
merely on Jα18−/− mice, the findings of preclinical studies on
sepsis need to be revisited. Antibody-mediated depletion of NKT
and NK cells in parallel (45, 221) and functional inactivation
of iNKT cells by carefully timed α-GalCer treatment (226) are
among other experimental options to study these cells in vivo.
Using Jα18−/− and CD1d−/− mice reconstituted with iNKT cells
should help solidify our knowledge of the roles that these cells
play in sepsis. Reconstitution with CD4+ or double-negative
subsets or with iNKT cells isolated from different tissues will
enable functional studies on these cells in the context of sepsis.
This is particularly important in light of reported functional
differences between various iNKT cell subpopulations in other
conditions, such as cancer (17). Even if/when mouse models of
iNKT cell deficiency allow for relatively convincing conclusions
to be drawn, one has to remain cognizant of the possibility that
iNKT cells may behave differently in their steady and activated
states (241). Much remains to be learned about direct activation
of iNKT cells by microbial glycolipids and SAgs likely secreted
during polymicrobial sepsis. Future studies should also explore
the therapeutic potentials of iNKT cell glycolipid agonists when
used in combination with antibiotics.
There is a paucity of information on the role of vNKT cells in
sepsis. This is due, at least largely, to a lack of powerful or stable
experimental tools to study these cells. CD1d−/− mice are devoid
of both vNKT and iNKT cells (50). Once exclusively iNKT celldeficient mice become available, they can be used in parallel with
CD1d−/− animals to address the relative contribution of iNKT
and vNKT cells to the septic response. Sulfatide-loaded CD1d
tetramer reagents invented by Vipin Kumar’s laboratory provide
a very useful tool for detection of vNKT cells (46) but are not very
stable. Treatment with native sulfatide has been used as a means
of vNKT cell activation in vivo. However, it is likely that sulfatide
exerts other effects and engages other cell types in the body.
Sulfatide-reactive, CD1d-restricted γδ T cells have been described
in human (242, 243), but their presence in mice is not completely
clear. Future studies will need to test the effect of other vNKT
cell ligands on sepsis. One such ligand is lysophosphatidylcholine
whose levels are in fact altered during inflammatory processes
(244). Finally, vNKT and iNKT cells are known to cross-regulate
each other in tumor models (245). Whether a similar cross-talk
exists during sepsis remains an open question.
Exploration of MAIT cell roles in sepsis is still in its infancy.
Mouse and human MAIT cells have distinct tissue distribution
Outstanding Questions and Concluding
Remarks
Despite decades of active research and numerous clinical trials, sepsis continues to take its Toll on human lives and cause
significant morbidity, thus imposing a heavy burden on human
populations and healthcare systems worldwide. Standardized
management protocols and better ICU facilities have improved
sepsis outcomes. However, there is currently no “cure” for this
devastating syndrome. Targeting conventional T cells, APCs or
individual inflammatory cytokines has not met with success. We
propose that NKT and MAIT cells provide attractive targets for
immunotherapy of sepsis. This is because: (i) they are abundant
in certain anatomic locations where microbial pathogens are first
encountered. For instance, iNKT cells are enriched in the human
omentum (16), for which the term “policeman of the abdomen”
was coined by a British surgeon, Rutherford Morison, in 1906
(240). Human MAIT cells may serve as “gate-keepers” in mucosal
layers and within the liver where they are highly abundant (66);
(ii) NKT and MAIT cells can be activated early in the course of
sepsis; (iii) they produce large quantities of immunomodulatory
cytokines that control the function of downstream innate and
adaptive effector cells, thus setting the tone for subsequent host
responses; (iv) NKT and MAIT cells are restricted by monomorphic Ag-presenting molecules (i.e., CD1d and MR1, respectively)
as opposed to distinct HLA allomorphs. Therefore, they can be
stimulated and potentially manipulated by universal ligands in
many, if not most, septic patients. This saves time and allows for
therapeutic interventions to be implemented speedily; (v) using
Th1- or Th2-polarizing ligands, typically in the case of iNKT cells,
provides flexibility in tailoring therapies to sepsis stages in which
hyper- or hypoinflammatory responses predominate.
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and frequencies. However, inducing sepsis in MAIT cell-deficient MR1−/− mice may still provide useful clues toward understanding the role of these cells in sepsis. In addition, mouse and
human MR1 tetramer reagents (68), once more widely available,
will undoubtedly advance the field of MAIT cell immunology.
They will enable mechanistic and functional studies on MAIT
cells and elucidate their effector and/or regulatory functions
during sepsis.
CD1d- and MR1-restricted T cells have become a focus of
intense investigation in recent years. The advent of novel and reliable tools, techniques and models by which to study these cells will
better our understanding of their basic biology and therapeutic
potentials in various disorders including sepsis. We remain
optimistic that the remarkable, quick-acting and wide-ranging
immunomodulatory functions of these cells can be harnessed to
invent efficacious treatments for different stages of sepsis.
Acknowledgments
This work was supported by a Canadian Institutes of Health
Research (CIHR) operating grant (MOP-130465) to S.M. Mansour
Haeryfar. We thank members of the Haeryfar laboratory for
helpful discussions. We apologize to investigators whose relevant
work was not cited due to space limitations.
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