2. primarily recognize viral nucleic acids and
activate antiviral measures including the
type I interferons.
TLR expression is significantly up-
regulated in experimental models of sep-
sis and in patients with sepsis (11–14).
Trauma including thermal injury gener-
ates danger-associated molecular pat-
terns (i.e., high mobility group box-1,
heat shock proteins, S100 proteins, hya-
luran, etc.) that augment TLR expression
like PAMPs. It also primes the innate im-
mune system for enhanced TLR reactiv-
ity, resulting in excess lipopolysaccharide
(LPS)-induced mortality (15). Multiple
positive feedback loops between danger-
associated molecular patterns and
PAMPs, and their overlapping receptors
temporally and spatially drive these pro-
cesses and may represent the molecular
basis for the observation that infections,
as well as nonspecific stress factors, can
trigger flares in systemic inflammatory
response. The degree to which TLR reg-
ulation mediates the ultimate outcome in
sepsis in individual patients remains elu-
sive and is an active area of clinical in-
vestigation (13, 16).
Inflammasome and Signalosome
Pathways
TLRs induce pro-interleukin(IL)-1beta
production and prime NLR-containing
multiprotein complexes, termed “inflam-
masomes,” to respond to bacterial prod-
ucts and products of damaged cells (17,
19). This results in caspase-1 activation
and the subsequent processing of pro-
IL-1 to its active extracellular form
IL-1 (19). Caspases are a set of cysteine
proteases that alter the enzymatic activity
of target proteins at specific peptide se-
quences adjacent to aspartate moieties.
Caspases are important in process of ap-
optosis, cellular regulation, and inflam-
mation (Figs. 1 and 3). One of the many
targets of the caspase cascade is caspase
activated DNase (CAD). CAD activation
induces DNA fragmentation characteris-
tic of programmed cell death (apoptosis).
The posttranslational activation of
caspase-1 is tightly regulated by inflam-
masome, the known components of
which include caspase-1, ASC (apoptosis-
associated speck-like protein containing a
caspase activation and recruiting do-
main), NALP1 (NACHT, leucine rich re-
peat and pyrin domain containing 1), and
caspase-5 (20). Additionally, alternate in-
flammasome constructions have been
suggested to contain pyrin, NALP3, and
other members of the NOD-LRR family
(21–23). ASC facilitates inflammasome
assembly thus triggering caspase-1 acti-
vation and IL-1 processing. Interest-
ingly, ASC can also regulate the nuclear
factor (NF)-B pathway, thus linking the
inflammasome to the signalosome
(Fig. 1) (24).
The inflammasome pathways contrib-
ute to the inflammatory response in sep-
sis (25). Caspase-1 knock out mice are
protected from sepsis (26) while a natu-
rally occurring polymorphism for human
caspase-12, a putative regulator of
Figure 1. Specific host immune response to each pathogen is mediated by various sets of pathogen
associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) as detailed in Figure
1. PRRs are essential for initiating the host’s immune defenses against invading pathogens, yet they
can also contribute to persistent and deleterious systemic inflammation. PRRs also serve as receptors
for endogenous danger signals, hemodynamic changes in sepsis (tissue hypoperfusion and ischemia/
reperfusion phenomenon), thus same signaling systems that alerts the highly advantageous, host
defense mechanisms, also contributes to the disadvantageous, pathologic events of systemic inflam-
mation, coagulation, tissue damage in target organs in sepsis. Heat shock proteins, fibrinogen,
fibronectin, hyaluran, biglycans and high mobility group box-1 (HMGB-1) have been defined as danger
associated molecular patterns (DAMPs) which are likely relevant for sepsis. Toll-like receptors (TLRs),
especially TLR4, are involved in the recognition of these endogenous or harmful self-antigens ligands
which are released during noninfectious injury, such as trauma or ischemia/reperfusion suggesting
their function may not be restricted to the recognition of extrinsic pathogens. NOD-LRR, nucleotide-
oligomerization domain leucine-rich repeat; ASC, apoptosis-associated speck-like protein containing
caspase activation and recruiting domain; NF-, nuclear factor .
Figure 2. Binding of toll-like receptors (TLRs) activates intracellular signal-transduction pathways that
lead to the activation of transcriptional activators such as interferon regulator factors, phosphoino-
sitide 3-kinase (PI3K)/Akt, activator protein-1, and cytosolic nuclear factor-kappa  (NF-). Activated
NF- moves from the cytoplasm to the nucleus, binds to transcription sites and induces activation
of an array of genes for acute phase proteins, inducible nitric oxide synthase, coagulation factors,
proinflammatory cytokines, as well as enzymatic activation of cellular proteases. TLR9 DNA, TLR 3
dsRNA, and TLR7/8 ss RNA are endosomal. TLR 10 ligand is not defined and TLR1 forms heterodimers
with TLR2. LPS, lipopolysaccharide; IRF, interferon regulatory factor; JNK; c Jun N-terminal kinase.
292 Crit Care Med 2009 Vol. 37, No. 1
3. caspase-1, has been linked to sepsis (27,
28). Thus, caspase-1 activation appears to
be a prerequisite for a competent im-
mune response (29). Recently, the in-
flammasome components have been
shown to be significantly lower in septic
shock patients during the early stages of
systemic inflammatory response with el-
evated plasma cytokines levels (30). This
monocyte deactivation process may be
maladaptive in the later phases of sepsis
and predispose to secondary infection.
Caspase-1 and its proinflammatory cy-
tokine products are likely to contribute to
the pathogenesis of sepsis in overwhelm-
ing inflammation. However, like many
other essential elements of innate immu-
nity, caspase-1 also has a positive impact
on host defense against several infections
up-regulating microbial killing mecha-
nisms such as the production of reactive
oxygen and nitrogen species (ROS and
RNS) (31, 32). Negative regulation of
TLRs and TLR-induced programmed cell
death has been defined (7). Apoptosis in-
duction is used by Pseudomonas aerugi-
nosa to inhibit the secretion of immune
and proinflammatory mediators by target
cells (33).
MyD88 protein and IL-1 receptor-
associated kinase pathways activate
NF-B during the innate immune re-
sponse (34). NF-B is a transcription fac-
tor that is constituted by homo- or het-
erodimers of the Rel protein family with a
pivotal role in inflammation, cell sur-
vival, and proliferation. In unstimulated
cells, NF-B is maintained in a latent
form in the cytoplasm by means of se-
questration by inhibitory B (IB) pro-
teins. NF-B activating stimuli, such as
cytokines, viruses, and lipopolysaccharide
(LPS), induce the degradation of inhibi-
tory Bs by the proteasome, unmasking
the nuclear localization signal of NF-B,
resulting in its nuclear translocation,
binding to NF-B motifs, and gene tran-
scription. Dynamic redox control of NF-
B through glutaredoxin-regulated S-
glutathionylation of inhibitory Bs has
been demonstrated (35). NF-B, subject
to regulation by redox changes, has been
shown to be involved in the transcrip-
tional regulation of more than 150 genes
with a significant portion demonstrating
proinflammatory properties (36). NF-B
is readily activated upon intraperitoneally
LPS challenge within 4 hrs in lung, liver,
and spleen (37). A variant IL-1 receptor-
associated kinase Ϫ1 haplotype has re-
cently been demonstrated to affect the
magnitude of NF-B activation and di-
rectly correlates with an increased inci-
dence of septic shock and significantly
reduced survival rates (38). The impact of
NF-B signaling is tissue-specific as defi-
cient NF-B activation in intestinal epi-
thelium is associated with increased in-
flammation in vivo (39, 40). Both studies
demonstrate that defects of NF-B signal-
ing cause immunosuppression which
triggers and maintains inflammation.
Thus, it can be suggested that inhibition
of massive NF-B activation in vivo leads
to reduced inflammatory responses, at
least during certain phases and certain
tissues (i.e., parenchymal tissues) in sep-
sis. On the other hand, this activation is
followed by negative NF-B regulation
favoring apoptosis in immune cells which
Figure 3. Pathogenic mechanisms during sepsis or in response to tissue injury can lead to organ
dysfunction. PRRs, pattern recognition receptors; TLRs, toll-like receptors; NOD-LRR, nucleotide-
oligomerization domain leucine-rich repeat protein receptors; RLHs; retinoic-acid-inducible gene I
(RIG-I)-like helicases; TNF-␣, tumor necrosis factor alpha; IL-1, interleukin 1; HMGB-1, high mobility
group box-1; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PGN, peptidoglycan; VSMCs, vascular
smooth muscle cells; RBC, red blood cell; MPO, myeloperoxidase; XOR, xanthine oxidoreductase;
iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase; RAGE, receptor for advanced glycation
end products; ROCK; RhoA/Rho kinase; PARP-1, poly(ADP ribose) polymerase-1; PAR, protease
activated receptor; ROS/RNS, reactive oxygen and nitrogen species; NF, nuclear factor; NADPH,
nicotimanide adenosine dinucleotide phosphate; PAMPs, pathogen-associated molecular patterns;
DAMPs, danger-associated molecular patterns.
293Crit Care Med 2009 Vol. 37, No. 1
4. may lead to immunosuppression and fatal
outcome in severe sepsis. Recently, using
specific gene-targeted deletions, it has
been shown that deletion of MyD88
caused a worsened survival in a model of
severe peritonitis, despite the marked de-
crease in sepsis-induced T and B lympho-
cyte apoptosis suggesting MyD88, like
NF-B, is also critical host survival in
sepsis (41).
Toll-Like Receptor Signaling
The Role of Phosphoinositide 3-Kinase.
Phosphoinositide 3-kinase (PI3K), a sig-
nal transduction enzyme, and the down-
stream serine/threonine kinase Akt (also
known as protein kinase B) have been
reported in cellular activation, inflamma-
tory responses, chemotaxis, and apoptosis
(Figs. 2 and 3) (42). PI3K can function
either as a positive or negative regulator
of TLR signaling. PI3Ks (three types) act
at several steps downstream of TLRs, de-
pending on the cell type and/or the en-
gagement of a specific TLR regulating
downstream signaling to NF-KB transac-
tivation or to mitogen-activated phos-
phokinase activation (43, 44). As a posi-
tive mediator of TLR signaling, PI3K
together with p38 and extracellular reg-
ulated kinase (ERK)1/2 mitogen-acti-
vated phosphokinases, lead to production
of proinflammatory cytokines IL-1,
IL-6, and IL-8 upon microbial challenge
(44, 45). In detail, PI3K activation ap-
peared to have a significantly promoting
function for these mediators in mono-
cytes, whereas activation appeared to
limit the LPS response for generation of
these cytokines in neutrophils (46). How-
ever, PI3K inhibition resulted in im-
paired oxidative burst and phagocytosis
activity in both neutrophils and mono-
cytes. By limiting C5a-mediated effects
on neutrophil cytokine generation, and
promoting oxidative burst and phagocy-
tosis, PI3K activation seems to be a ther-
apeutic approach for limiting inflamma-
tion in sepsis.
On the other hand, PI3K/Akt signaling
pathway acts as an endogenous negative
feedback mechanism that serves to limit
proinflammatory and apoptotic events as
seen in monocytes in response to endo-
toxin (47). The suppressive role in in-
flammation and coagulation was con-
firmed in mouse model of endotoxemia
(48). It can also promote the generation
of anti-inflammatory cytokine IL-10 (49).
PI3K/Akt has an effect in balancing Th1
vs. Th2 responses (50). Overexpression of
Akt in lymphocytes decreases lymphocyte
apoptosis, a Th1 cytokine propensity, and
improve outcome in cecal ligation and
puncture-induced sepsis (51). Peptide-
mediated activation of Akt and extracel-
lular regulated kinase signaling protects
lymphocytes from numerous apoptotic
stimuli both in vitro and in vivo (52).
This suggests again the survival advan-
tage of PI3K/Akt pathway activation for
the later stages of sepsis.
The Role of Rho GTPases. The Rho
family of small GTPases is one of the
master regulators of cell motility, as they
control actin cytoskeleton remodeling.
RhoA, Rac1, and CDc42 are the well
known family members which act molec-
ular switches regulating responses of in-
nate immune cells related to pathogen
sensing, intracellular uptake, and de-
struction (53). These GTPases play both
unique and overlapping roles in phago-
cyte functions including migration, che-
motaxis, and optimal bacterial killing
(54). It has been shown that PAMPs may
utilize GTPases through TLRs (i.e.,
TLR-2, -4, -3, and -9) in which Rac1 ac-
tivation is required for PI3K activation
upon TLR2 stimulation (55). RhoA regu-
lates not only cytoskeletal events, which
mediate neutrophil migration, but also
contributes to NF--dependent proin-
flammatory gene transcription (Fig. 3)
(56). It has been suggested that ROCK
inhibition could attenuate cytoskeletal
rearrangement of endothelial cells, lead-
ing to decreased neutrophil emigration
into the lung parenchyma in LPS-
induced lung injury (57). An important
role for rho kinase in leukocyte recruit-
ment is also supported in an endotoxemic
liver injury model (58). The recently
emerged connections between TLR sig-
naling and small Rho-GTPases seem to
provide new therapeutic avenue of re-
search in sepsis.
Toll-Like Receptor Signaling
and Regulatory T Cells (Tregs)
To balance self-tolerance and immu-
nity against pathogens, the immune sys-
tem depends on both up-regulatory and
down-regulatory mechanisms. Recent
studies have suggested that several lym-
phocyte subpopulations (i.e., CD4ϩCD25ϩ
Foxp3ϩ T regulatory-cell ͓Tregs͔) may
have the capacity to actively suppress an
adaptive immune response and may po-
tentially be involved in septic immune
dysfunction. Naturally occurring Tregs
express the transcription factor forkhead
box protein (FoxP3), which is induced by
the anti-inflammatory cytokine trans-
forming growth factor- (59, 60). TLR
triggering induces dendritic cell matura-
tion also, which is essential for the induc-
tion of adaptive immune responses (61).
Studies have recently highlightened the
importance of TLRs on Tregs (62). In this
regard, the dominant role of TLR2 signal-
ing on the Treg-mediated immune sup-
pression has been demonstrated (63, 64).
Tregs control inflammatory reactions
to commensal bacteria and opportunist
pathogens and play a major role in sup-
pressing immune reactivity, ranging
from autoimmunity to infectious disease
(65) and to injury (66). Monneret et al
(67) observed that sepsis increases
CD4ϩCD25ϩ T cells in the peripheral
blood of septic patients. This was subse-
quently found to be a relative increase in
Tregs due to a decrease in the CD4ϩCD25–
T effector cell populations (68). Further-
more, Treg-mediated induction of “alter-
natively activated” macrophages has been
demonstrated suggesting as one of the
causes of immune dysfunction in sys-
temic inflammatory response syndrome
and sepsis (69, 70). Targeting apoptosis of
Tregs may be a new therapeutic approach
in preventing the continuum of sepsis to
severe sepsis. However, it has been re-
ported that depletion of CD25ϩ cells be-
fore inducing sepsis did not alter septic
mortality pointing the need of more stud-
ies to clarify the significance of this cell
population’s expansion in sepsis morbid-
ity (71, 72).
Adoptive transfer of Tregs before or
following the initiation of polymicrobial
sepsis improved survival by enhancing
tumor necrosis factor (TNF)-␣ produc-
tion and bacterial clearance (73). Tregs
inhibit LPS-induced monocyte survival
through the Fas/FasL dependent pro-
apoptotic mechanism, which might play
a role in the resolution of exaggerated
inflammation (74). The positive and neg-
ative effects of TLR on Tregs are curious
in sepsis, and the dynamics of TLR ex-
pression on immune-suppressive Tregs
upon inflammation or in relation to type
of pathogen are needed to illuminate.
Neutrophils and
Monocyte/Macrophages in
Inflammation
Myeloid cells including neutrophils
and elements of monocyte/macrophage
lineage are heavily armed with large
stores of proteolytic enzymes and with
294 Crit Care Med 2009 Vol. 37, No. 1
5. the capacity to rapidly generate ROS and
RNS to degrade internalized pathogens.
The highly proapoptotic nature of neu-
trophils is designed to maintain a balance
between antimicrobial effectiveness and
the potential for neutrophil-associated
damage to the host in septic challenge or
in other injurious processes, such as
trauma or ischemia/reperfusion (75).
Host tissue damage in severe sepsis may
arise via a variety of mechanisms includ-
ing premature neutrophil activation dur-
ing migration, extracellular release of cy-
totoxic molecules and toxins during
microbial killing, removal of infected or
damaged host cells or debris during host
tissue remodeling, and failure to termi-
nate acute inflammatory responses (76).
Therefore, to maximize host defense ca-
pabilities while minimizing damage to
host tissues, neutrophil microbial re-
sponses are tightly regulated. On the
other hand, quorum sensing (the ability
of bacteria to assess their population den-
sity) has been found to have a crucial role
in regulating tissue invasion by bacterial
pathogens, and inhibitors of quorum
sensing system provide new avenues for
intervention against invasive pathogens
(1). Evidence now exists that quorum
sensing system can even open up bidirec-
tional lines of communication between
bacteria and the human host.
Leukocyte-endothelial interactions,
which may also contribute to inflamma-
tion-mediated injury, involve two sets of
adhesion molecules, selectins, and inte-
grins (77, 78). P-selectin is expressed on
platelets; E- and P-selectins are expressed
by endothelial cells, whereas L-selectin is
expressed on leukocytes. Selectins mediate
neutrophil rolling along activated endothe-
lial surfaces as circulating neutrophils de-
celerate to engage endothelial receptors.
The beta-2 intergrins (CD11/CD18 com-
plexes) mediate tight adhesion to endothe-
lial membranes, allowing subsequent
egress of neutrophils to extravascular sites
of inflammation (78). Activated neutrophils
stimulate transendothelial albumin trans-
port through intracellular adhesion mole-
cule-1 mediated, Scr-dependent caveolin
phosphorylation. The role of caveolin in
inflammatory response in sepsis has been
recently defined (Fig. 3) (79–82).
Neutrophils contribute to blood coagu-
lation in localized inflammation and in
generalized sepsis (1, 3, 75). During sys-
temic inflammation, homeostatic mecha-
nisms are compromised in the microcircu-
lation including endothelial hyperactivity,
fibrin deposition, microvascular occlusion,
and cellular exudates that further impede
adequate tissue oxygenation. Neutrophils
participate in these rheologic changes
through their augmented binding to blood
vessel walls and through the formation of
platelet-leukocyte aggregates (83). Neutro-
phil elastase, other proteases, glycases and
inflammatory cytokines degrade endoge-
nous anticoagulant activity, and impair fi-
brinolysis on endothelial surfaces favoring
a procoagulant state (1).
The Role of Apoptosis. Sepsis-induced
neutrophil-mediated tissue injury has
been demonstrated in a variety of organs
including the lungs (84–86), diaphragm
(87), kidneys (86), intestine (88, 89), and
liver (86). Apoptosis is a counter-regula-
tor of the initial inflammatory response
in sepsis (90). Neutrophils are constitu-
tively proapoptotic and apoptosis is fun-
damental for the resolution of inflamma-
tion and cell turnover. Neutrophils can
undergo apoptosis via intrinsic and ex-
trinsic pathways; the latter also requires
mitochondrial amplification (Fig. 4) (91).
The role played by mitochondria in the
regulation of neutrophil life span is more
crucial than in other cell types in the
body (92). As neutrophils kill pathogens
using ROS and RNS and a mixture of lytic
enzymes, delayed clearance of neutro-
phils in sepsis can potentially contribute
Figure 4. Summary of apoptotic signaling pathways as seen through activation of death receptor
(extrinsic) or mitochondrial (intrinsic) pathway. Extrinsic signals bind to their receptors and trigger
intracellular signaling, leading to caspase-8 activation. Activation of caspase-8 by extrinsic stimuli
(such as tumor necrosis factor-␣ [TNF-␣], Fas ligand) involves mitochondria-dependent signaling in
type II cells. In type I cells, on the other hand, execution of apoptosis occurs without significant
participation of mitochondria. MAPK, mitogen-activated phosphokinase; PI3K/Akt, phosphoinositide
3-kinase/Akt; APAF-1, apoptosis protease activating factor 1; ER, endoplasmic reticulum; IL, interleukin.
Figure 5. The recognition of apoptotic cells by macrophages is largely dependent on the cell surface
appearance of phosphatidylserine (PS). S-nitrosylation of critical cysteine residues inhibits aminophospho-
lipid translocase (APLT), leading to PS externalization. It generates an “eat me” signal during apoptosis.
iNOS, inducible nitric oxide synthase; .
NO, nitric oxide; O2, superoxide; ONOO؊
, peroxynitrite.
295Crit Care Med 2009 Vol. 37, No. 1
6. to cell/organ injury. Importantly, the
phagocytosis of bacteria and fungi accel-
erates neutrophil apoptosis. Apoptotic
cell clearance induces anti-inflammatory
effects in tissues. It has been shown that
intratracheal administration of killed E.
coli attenuated lung injury and improved
survival in an intestinal ischemia/reper-
fusion injury associated with marked pul-
monary neutrophil infiltration (93).
Cytokine-induced prolonged neutro-
phil survival is accompanied by evidence
of increased neutrophil activation, in-
cluding augmented respiratory burst ac-
tivity (90). Neutrophils from patients
with sepsis manifest markedly prolonged
survival in vitro in association with evi-
dence of cellular activation (94). Phago-
cytosis of apoptotic neutrophils by
macrophages inhibits the release of
proinflammatory cytokines and promotes
the secretion of anti-inflammatory cyto-
kines (95). In contrast, inefficient apopto-
tic cell clearance is proinflammatory and
immunogenic (96). The recognition of
apoptotic cells by macrophages is largely
dependent on the cell surface appearance
of an anionic phospholipid, phosphatidyl-
serine (PS), which is normally confined
to the inner leaflet of the plasma mem-
brane (97). Asymmetric distribution of
PS across the plasma membrane is
mainly because of the activity of a spe-
cialized enzymatic mechanism, amin-
ophospholipid translocase. S-nitrosyla-
tion of critical cysteine residues inhibits
aminophospholipid translocase, leading
to PS externalization. PS expression dur-
ing apoptosis generates an “eat-me” sig-
nal (Fig. 5), which in turn triggers clear-
ance of apoptotic cells and suppresses the
inflammatory response (98). It has been
demonstrated that S-nitrosylation of crit-
ical cysteine residues in aminophospho-
lipid translocase using a cell-permeable
transnitrosylating agent, S-nitroso-acetyl-
cysteine, resulted in egression of PS to
the outer surface of the plasma mem-
brane, rendering these cells recognizable
by macrophages (99). The therapeutic po-
tential of regulating neutrophil life-span
in sepsis remains to be determined. Care
must be exercised in regulating of this
pathway, as sepsis enhances the capacity
of macrophages to clear expanded apo-
ptotic populations, a mechanism contrib-
uting to septic immune suppression
(100).
The Role of ROS and RNS. ROS and
RNS exert several beneficial physiologic
functions, such as intracellular signaling
for several cytokines and growth factors,
second messengers for hormones and re-
dox regulation. Despite their importance
as a defense mechanism against invading
pathogens, an overwhelming production
of ROS and RNS or a deficit in oxidant
scavenger and antioxidant defenses result
in oxidative/nitrosative stress, a key ele-
ment in the deleterious processes in sep-
sis (Fig. 6) (101, 102).
Stimulated neutrophils produce ROS
and RNS through the nicotinamide ade-
nine dinucleotide phosphate oxidase
complex, myeloperoxidase and xanthine
oxidoreductase and represent a defense
mechanism against invading microor-
ganisms (103). Lipopolysaccharide and
other proinflammatory mediators acti-
vate nicotinamide adenine dinucleotide
phosphate oxidase to produce superoxide
radical (O2
Ϫ
). In aqueous environments,
superoxide radical is rapidly catalyzed by
superoxide dismutase hydrogen peroxide
(H2O2) and hydroxyl radicals. Myeloper-
oxidase from neutrophil azurophilic
granules produces hypochlorous acid
from hydrogen peroxide (H2O2) and chlo-
ride anion (Cl؊
) during respiratory burst.
These radicals are highly cytotoxic, and
neutrophils used them to kill bacteria
and other pathogens. Expression of hu-
man xanthine oxidoreductase is markedly
up-regulated by hypoxia, ischemia/reper-
fusion, LPS, and TNF-␣. Increased activ-
ity of xanthine oxide, one the important
contributors of ROS production, has been
reported in adult and pediatric patients
with sepsis (104).
O2
؊
in the presence of nitric oxide,
generates peroxynitrite (ONOOϪ
), a key
player in the pathogenesis of sepsis-
induced organ dysfunction. ONOOϪ
can
cause DNA strand breakage, which trig-
gers the activation of DNA repair en-
zymes such as poly (adenine dinucleotide
phosphate-ribose) polymerase (Fig. 3).
Poly (adenine dinucleotide phosphate-
ribose) polymerase inhibitors protect
against oxidative and nitrosative stress-
induced organ dysfunction in endotox-
emia (87–89). Recently, the potential role
of poly (adenine dinucleotide phosphate-
ribose) polymerase activation has been
implicated in the pathogenesis of myo-
cardial contractile dysfunction associated
with human septic shock (105).
Novel Cytokines in Inflammation
High Mobility Group Box-1 and Re-
ceptor for Advanced Glycation End-
Products. High mobility group box-1
(HMGB-1) is a nonhistone, nuclear DNA-
binding protein involved in nucleosome
stabilization and gene transcription. How-
ever, when HMGB-1 is released in large
quantities into the extracellular environ-
ment, it becomes a lethal mediator of sys-
temic inflammation (Fig. 3) (106). Indeed,
Figure 6. Proinflammatory/anti-inflammatory cytokines and reactive oxygen and nitrogen species have
important effects within the microcirculatory unit: the arteriole, endothelial cell, capillary bed, and the
venule. The arteriole is where the characteristic intractable vasodilation of sepsis occurs. The capillary bed
is where the effects of endothelial cell activation/dysfunction are most pronounced and microvascular
thromboses are formed. The postcapillary venule is where leukocyte trafficking is most disordered. All of
these causes impair flow through the microcirculation leading to microcirculatory dysfunction. TLR,
toll-like receptor; NOD-LRR, nucleotide-oligomerization domain leucine-rich repeat; RLH, (RIG-I)-like
helicase; VSM, vascular smooth muscle; IL, interleukin; TGF, transforming growth factor; MIF, migration
inhibitory factor; TNF, tumor necrosis factor; HMGB, high mobility group box; PAF, protease activating
factor; sTNFR, soluble tumor necrosis factor receptor.
296 Crit Care Med 2009 Vol. 37, No. 1
7. it has been recently shown that it has a
weak proinflammatory activity by itself and
binding to bacterial substances including
lipid molecules such as phophatidylserine
strengthens its effects (107). Interestingly,
phosphatidylserine has been implicated in
the regulation of inflammation and
HMGB-1 might thus regulate its anti-
inflammatory activities (108).
HMGB-1 is released into the extracel-
lular space through acetylation or phos-
phorylation (109, 110). HMGB-1 is either
“passively released” from necrotic cells,
and a mechanism that represents a pro-
cess adopted by the innate immune sys-
tem to recognize damaged and necrotic
cells, or “actively secreted” by immune
cells including macrophages and neutro-
phils to trigger inflammation (106). Re-
cently, HMGB-1 release from macro-
phages has been shown during the course
of apoptosis as well as necrosis and de-
fined as a downstream event of cell apo-
ptosis during severe sepsis (111, 112).
After treatment with LPS or various cy-
tokines such as TNF-␣, IL-1, or IFN-␥,
HMGB-1 is released from activated mac-
rophages within 4 hrs and reaches a pla-
teau around 18–24 hrs. It binds to several
transmembrane receptors such as recep-
tor for advanced glycation end products
(RAGE), TLR-2, and -4, activating NF-B
and extracellular regulated kinase 1/2
(113, 114).
Although HMGB-1 was originally de-
scribed as a late mediator of endotoxin-
induced lethality (115), recent studies in-
dicate a role for HMGB-1 in angiogenesis,
tissue repair, and regeneration (116).
HMGB-1 appears to be a novel myocardial
depressant factor upon release by resi-
dent myocardial cells following tissue in-
jury. HMGB-1 might decrease energy uti-
lization in ischemic tissue, thereby
preventing injured myocytes from wors-
ening ATP depletion that eventuate in
necrosis (117). On other hand, excessive
release of HMGB-1 in sepsis might con-
tribute to sustained inflammation and to
profound myocardial depression.
The cholinergic anti-inflammatory
pathway is a neural mechanism that in-
hibits the expression of HMGB-1 and
other cytokines (118–121). Signals trans-
mitted via the vagus nerve, the principal
nerve of the parasympathetic nervous
system, significantly attenuate the re-
lease of HMGB-1 and other cytokines in
inflammation in animal and human stud-
ies (122).
The remarkable binding characteristics
of HMGB-1 suggest another important role
for this protein in the extracellular fluid.
HMGB-1 might serve as a shuttle platform
for LPS and other microbial mediators for
docking to CD14 and recognition by the
TLRs (108, 123) or through binding to
host-derived proinflammatory mediators,
such as IL-1beta (124).
Elevated levels of HMGB-1 are measur-
able in the majority of patients up to 1 wk
after the diagnosis of sepsis or septic shock
and are correlated with the degree of organ
dysfunction (125, 126). However, serum
HMGB-1 levels do not consistently identify
nonsurvivors from survivors as a predictor
of hospital mortality (127). As HMGB-1 is
late inflammatory cytokine of sepsis, it pro-
vides a wide therapeutic time window for
clinical intervention and remains an attrac-
tive target for sepsis treatment.
RAGE, a member of the immunoglobu-
lin superfamily, is a pattern-recognition re-
ceptor that binds diverse classes of endog-
enous molecules including HMGB-1. It has
been defined as part of a newly appreciated
component of the innate immune system
referred to as the danger associated molec-
ular pattern system (Fig. 3) (128). Mem-
brane bound and soluble forms of RAGE
(sRAGE) have been detected in plasma. Sol-
uble RAGE, anti-RAGE antibody (Fab 2
fragment) or data from RAGEϪ/Ϫ animals
have been shown to decrease inflammation,
reduce neutrophil extravasation, and re-
duce migration (129). Recently, the dem-
onstration of survival benefit after delayed
administration of anti-RAGE antibody in a
murine model of polymicrobial sepsis, even
when delayed up to 24 hrs, provides a ther-
apeutic rationale for the use of anti-RAGE
mAb as a salvage therapy for established
severe sepsis (130).
Macrophage Migration Inhibitory
Factor. Migration inhibitory factor (MIF)
acts as a stress response mediator and
proinflammatory cytokine upon induc-
tion by glucocorticoids (131). This pro-
tein is readily measurable in patients with
sepsis, and MIF probably contributes to
the pathogenesis of sepsis. Inhibition of
MIF or its targeted deletion attenuates
TNF-␣ and IL-1 expression and protects
mice from lethality is experimental sepsis
(132, 133). Systemic challenge of animals
with MIF increases LPS-related lethality
(133). MIF promotes the expression of
TLR4 on macrophages, and thereby sen-
sitizes these immune effector cells to LPS
(134).
The immunoregulatory effects of MIF
might be crucial to the control and resolu-
tion of the inflammatory response as a con-
sequence of its ability to regulate activa-
tion-induced apoptosis (135). High MIF
levels delay the removal of activated mono-
cytes/macrophages by apoptosis. This pro-
longs monocyte/macrophage survival, in-
creases cytokine production, and sustains
an ongoing proinflammatory response.
Mitochondrial Dysfunction in
Inflammation
Although microvascular flow abnormal-
ities occur, findings of decreased oxygen
consumption (136) and elevated tissue ox-
ygen tension (137), yet minimal cell death
despite functional and biochemical de-
rangements (138), suggest that the prob-
lem lies more in cellular oxygen utilization
rather than a problem with oxygen delivery
(139). It is postulated that prolonged and
systemic inflammatory insult is accompa-
nied by a basic tissue survival response me-
diated by switching off its energy-consum-
ing biophysiological processes. Recent
evidence suggests that sepsis and septic
shock severely impair the mitochondria
(140, 141), and the severity and outcome of
organ dysfunction could be related to mi-
tochondrial dysfunction (142). Depleted
levels of reduced glutathione, an important
intramitochondrial antioxidant, in combi-
nation with excess generation of ROS and
RNS severely inhibit oxidative phosphory-
lation and ATP generation (142). This ac-
quired intrinsic derangement in cellular
energy metabolism which has also been
termed “cytopathic hypoxia,” contributes to
reduced activities of mitochondrial electron
transport chain enzyme complexes and im-
paired ATP biosynthesis, potentially organ
dysfunction in sepsis (141, 143, 144).
Sepsis-related derangements in mito-
chondrial function can activate the ubiq-
uitin proteolytic pathway in skeletal mus-
cle of septic patients (145). Mitochondrial
permeability transition pore seems to be
involved in sepsis-induced mitochondrial
damage, since its inhibition significantly
improved organ function and reduced
mortality in rodents (146). Whether sep-
sis-related reduction in energy supply
could result in a state of cellular shut-
down analogous to myocardial “stun-
ning” (or hibernation) following coronary
occlusion, allowing for eventual restora-
tion of organ function and survival, has
not yet been determined.
Impact of Inflammation on
Coagulation
The clotting system is almost invari-
ably activated by systemic microbial in-
297Crit Care Med 2009 Vol. 37, No. 1
8. vasion (147). Clotting is one most prom-
inent features of sepsis. Coagulation
contributes significantly to the outcome
in sepsis with concurrent down-regula-
tion of anticoagulant systems and fibri-
nolysis (Fig. 7). Inflammation-induced
coagulation in turn contributes to fur-
ther inflammation (148). Indeed, collab-
oration between clotting and inflamma-
tion accounts for the basic survival
strategy of walling off the damaged and
infected tissues from the rest of the host
(149). The key determinant of survival in
sepsis is to limit excess systemic inflam-
matory and coagulopathic damage while
retaining the benefits of controlled anti-
microbial clearance and localized clot
formation (147, 150).
The inflammatory reaction to tissue
injury activates the clotting system, and
coagulation promotes inflammation (3,
151, 152). The role of procoagulant apo-
ptotic microparticles have also been dem-
onstrated in sepsis (153–155). Linkage of
coagulation enzymes with their serine
protease activity with protease activated
receptors (PAR 1-4) on endothelial sur-
faces increases P-selectin, cytokine produc-
tion, and adhesion molecule expression,
leading to microcirculatory dysfunction in
severe sepsis (156, 157).
Activated Protein C. Activated protein
C (APC) is derived from its zymogen pro-
tein C in contact with thrombin: thrombo-
modulin complexes on endothelial sur-
faces. Protein C was originally thought to
be synthesized exclusively by the liver
(158). It has recently shown that it is
strongly expressed by the endothelium and
keratinocytes (159). The conversion to APC
is augmented by endothelial PC receptor
(EPCR) which is present on endothelial
cells, neutrophils, monocytes, and keratin-
ocytes (160, 161) whereas soluble EPCR
inhibits APC anticoagulant activity (162).
Soluble EPCR is released constitutively and
levels increase in patients with Gram-
negative sepsis (163). Levels of APC, protein
C, and its cofactor protein S are depleted in
sepsis (164, 165). Furthermore, peripheral
conversion of Protein C by the thrombin:
thrombomodulin complex is impaired in
sepsis, further contributing to microvascu-
lar thrombosis and vascular leakage (166).
APC is a central endogenous anticoag-
ulant protein with antithrombotic, anti-
inflammatory, antiapoptotic, and profi-
brinolytic activities (Fig. 8) (167, 168).
Although an anti-inflammatory role for
APC may be an indirect consequence of
its ability to reduce thrombin generation,
APC also has direct anti-inflammatory
Figure 7. The extrinsic pathway (tissue factor pathway) is the primary mechanism by which thrombin is
generated in sepsis. The intrinsic cascade (contact factor pathway) primarily serves an accessory role in
amplifying the prothrombotic events that are initiated in sepsis. Thrombin, factor Xa and the TF-factor VIIa
complex interact with the PAR (protease activated receptors) system and directly activate endothelial cells,
platelets and white blood cells, and induce a proinflammatory response. Platelet-derived microparticles
(MPs) express functional adhesion receptors including P-selectin on their surface, attach to the site of
injury on the vessel wall, and support the rolling of leukocytes in the presence of shear stress or severe
insult. When bearing appropriate counter-ligands, MPs can transfer their procoagulant potential to target
cells. Platelet-derived MPs can bind to soluble and immobilized fibrinogen, thus delivering procoagulant
entities to the thrombus via the formation of aggregates. In vitro, interaction between endothelial MPs and
monocytes promotes TF mRNA expression and TF-dependent procoagulant activity. Activated platelets and
platelet-derived MPs thus amplify leukocyte-mediated tissue injury in thrombotic and inflammatory
disorders. EPCR, endothelial protein C receptor; PMN, polymorphonuclear leukocyte; APC, activated
protein C; ADAMST13, a disintegrin and metalloproteinase with a thrombospondin type 1 motifs 13;
ULVWF, unusually large von Willebrand factor; ICAM, intracellular adhesion molecule; VCAM, vascular cell
adhesion molecule-1; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon.
Figure 8. The homeostatic balance between thrombin and activated protein C (APC) in coagulation and
inflammation is given. APC is a central endogenous anticoagulant protein with antithrombotic, anti-
inflammatory, anti-apoptotic and pro-fibrinolytic activities. The ligand occupancy of endothelial protein C
receptor (EPCR) switches the protease-activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin
from a permeability-enhancing to a barrier-protective response. Other pleiotropic effects are also explained. TAFI,
thrombin activatable fibrinolysis inhibitor; NF-, nuclear factor .
298 Crit Care Med 2009 Vol. 37, No. 1
9. properties (169). APC can cleave and ac-
tivate PAR1-dependent cellular pathways
(170). APC competes for PAR-1 binding
with thrombin, but in vitro studies sug-
gest that APC is 103
- to 104
-fold less po-
tent that thrombin in cleaving PAR-1.
The critical receptors required for both
PC activation and APC cellular signaling
(i.e., thrombomodulin, EPCR and PAR-1)
are co-localized in lipid rafts on endothe-
lial cells (171). EPCR is associated with
caveolin-1 on lipid rafts and EPCR bind-
ing to the gamma-carboxyglutamic acid
domain of protein C/APC leads to its dis-
sociation from caveolin-1 (Fig. 7). APC
then engages PAR-1 generating a protec-
tive signaling pathway through coupling
of PAR-1 to the pertussis toxin-sensitive
G(i)-protein. Thus, when EPCR is bound
by protein C, the PAR-1-dependent pro-
tective signaling responses in endothelial
cells can be mediated by either thrombin
or APC. These results explain how PAR-1
and EPCR participate in protective sig-
naling events in endothelial cells (172).
Therapeutic administration of recom-
binant human APC (rhAPC) is currently
in use as a treatment strategy for severe
sepsis patients with a high risk of death
(173, 174). Genetically engineered vari-
ants of APC have been designed with
greater antiapoptotic activity and reduced
anticoagulant activity relative to wild-
type APC to increase the risk/benefit ratio
of rhAPC regarding the bleeding compli-
cation (175). A nonanticoagulant from of
APC reduces mortality in experimental
models of endotoxemia and sepsis (176).
von Willebrand Factor, A Disintegrin-Like
and Metalloproteinase with Thrombospondin
Type-1 Motifs 13 and Ashwell Receptor.
The discovery of a disintegrin-like and met-
alloproteinase with thrombospondin type-1
motifs 13 (ADAMTS-13) has provided new
insights in pathogenesis of thrombosis in
sepsis. ADAMTS-13, the principal physio-
logic modulator of von Willebrand factor
(VWF) is produced mainly stellate cells in
the liver (177). VWF is synthesized in vas-
cular endothelial cells and released into the
plasma as unusually large VWF multimers
which are rapidly degraded into smaller
VWF multimers by ADAMTS-13. Deficiency
of the ADAMTS-13, as observed in most
forms of thrombotic thrombocytopenic
purpura, increases the level of unusually
large VWF multimers in plasma and leads
to platelet aggregation and/or thrombus
formation, especially in small arterioles, re-
sulting in microvascular failure (178–180).
Recently, inflammation associated-
ADAMTS-13 deficiency has been de-
scribed in patients with systemic inflam-
matory response syndrome and severe
sepsis (Fig. 7) (181–185). Decreased lev-
els of ADAMTS-13 have been reported in
healthy volunteers following endotoxin
infusion (186). Furthermore, reduced
ADAMTS-13 levels are associated with dif-
ferences in morbidity, mortality, and
variables of inflammation and endothelial
dysregulation in severe sepsis patients
(182, 187).
The Ashwell receptor, which is the
major lectin of hepatocytes, modulates
VWF homeostasis. It has been recently
demonstrated that the marked thrombo-
cytopenia associated with S. pneumoniae
sepsis is the result of Ashwell receptor-
dependent clearance of platelets (188).
The ensuing reduction in platelet counts
at the onset of sepsis protects the host
against the development of disseminated
intravascular coagulation. Homeostatic
adaptation by this receptor moderates the
onset and severity of disseminated intra-
vascular coagulation during sepsis sug-
gesting the improvement in host survival
probability (189). At present, it remains
unclear whether blocking the Ashwell re-
ceptor may have a beneficial effect in
severe sepsis.
CONCLUSION
A unifying concept of innate immu-
nity is based upon pathogen-, or damage-
associated molecular pattern molecules
and downstream signaling pathways. This
has facilitated significant advances in our
understanding of the pathophysiology of
sepsis and led to a multitude of clinical
Table 1. Ongoing and upcoming clinical studies with molecular targets
Target Sponsor or Institution Phase Comments
CytoFab (AZD9773) Fabs of IgG that bind to TNF-␣ AstraZeneca II Recruiting
Recombinant human lactoferrin LPS-neutralization Agennix II Recruiting
Lipid emulsion (GR-270773) TLR4 Glaxo Smith Kline II Suspended
E5564 TLR4 Eiasi III Ongoing
TAK-242 TLR4 Takeda III Recruiting
Recombinant human soluble thrombomodulin Coagulation Artisan Pharma II Recruiting
Recombinant antithrombin Coagulation Leo Pharma II Recruiting
Recombinant tissue factor pathway inhibitor Coagulation Novartis III Completed
Recombinant activated Protein Cϩ Erythropoietin Coagulation and inflammation LHRI III Recruiting
Recombinant activated Protein C Coagulation Lilly III Recruiting
Recombinant plasma gelsolin Replacement of circulating
actin-binding protein
CBC II Recruiting
L-Citrulline supplementation NO pathway MU II Not yet open for
participant
recruitment
Inhaled nitric oxide NO pathway NIMS III Not yet open for
participant
recruitment
Simvastatin Pleitrophic effects MUV, Austria and
Chicago, USA
IV Recruiting
Rosuvastatin Pleitrophic effects UASLP, Mexico and Beth
Israel
II Ongoing
Atorvastatin Pleitrophic effects HCPA, Brazil II Recruiting
TNF, tumor necrosis factor; LPS, lipopolysaccharide; NO, nitric oxide; TLR, toll-like receptor, LHRI, Lawson Human Resources Institute; CBC, Critical
Biologics Corporation; MU, Maastricht University; NIMS; National Institute of Medical Sciences; MUV, Medical University of Vienna; UASLP, Universidad
Autonoma de San Luis Potosi; HCPA, Hospital de Clinical de Porto Alegre.
299Crit Care Med 2009 Vol. 37, No. 1
10. trials (Table 1). However, further identi-
fication of the critical elements in severe
sepsis that drives the transition from lo-
calized inflammation to deleterious host
response remains incompletely illumi-
nated. The limitations in our current
knowledge of the molecular mechanisms
in sepsis have made the design of inter-
vention trials in clinical sepsis challeng-
ing (190). New findings as to biomarkers
and measures to detect genetic signa-
tures of sepsis are now making their way
into clinical trial designs. It is anticipated
that such innovations will improve the
outlook for successful development of
new sepsis treatments tailored to individ-
ual patient needs.
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