The inflammasome regulatory pathway and-2010

REVIEWThe inflammasome regulatory pathway and infections:Role in pathophysiology and clinical implicationsRyosuke Osawa a ,c ,Kristi L.Williams b ,Nina Singh a ,*a Department of Medicine,VA Pittsburgh Healthcare System and University of Pittsburgh,Pittsburgh,PA,USA bDepartment of Medicine,Duke University Medical Center,Durham,NC,USAAccepted 5October 2010Available online 13October 2010KEYWORDSInnate immunity;Inflammasome;Infection;IL-1bSummary The innate immune system serves to generate immediate host defenses against pathogens.Advance in the mechanism of innate immunity has provided new insights into host-pathogen microbial interactions.The cytosolic multi-protein complex called the inflam-masome,which regulates the caspase-1dependent processing of inflammatory cytokines IL-1b and IL-18,is critical for the innate defense against pathogens.We summarize the cur-rent knowledge regarding the regulatory functions of the inflammasome in the pathogenesis of infections by various microbes (e.g.,bacteria,fungi,viruses,and protozoa),and discuss its potential application in a clinical setting.Understanding of the unique role of the inflam-masome signaling pathway in initiating and regulating inflammation is pivotal for the devel-opment of innovative approaches to optimize management of these infections.ª2010The British Infection Association.Published by Elsevier Ltd.All rights reserved.Abbreviations:AIM2,absent in melanoma 2;ASC,apoptosis-associated speck-like protein containing a CARD;CARD,caspase-recruitment domain;DAMP,danger-associated molecular patterns;ICE,IL-1b converting enzyme;MDA-5,melanoma differentiation-associated gene 5;NAIP,neuronal apoptosis inhibitor protein;NLR,nucleotide-binding domain and leucine-rich repeat;NLRC,NLR family,CARD domain con-taining;NLRP,NLR family,pyrin domain containing;PAMP,pathogen-associated molecular patterns;RIG-I,retinoic acid inducible gene I;TLR,toll-like receptor.*Corresponding author.Infectious Diseases Section,VA Pittsburgh Healthcare System,University Drive C,Pittsburgh,PA 15240,USA.Tel.:þ14123601688;fax:þ14123606950.E-mail address:nis5@ (N.Singh).cPresent address:Department of Medicine,State University of New York at Buffalo,NY;Buffalo General Hospital,NY;Roswell Park Cancer Institute,NY,USA.0163-4453/$36ª2010The British Infection Association.Published by Elsevier Ltd.All rights reserved.doi:10.1016/j.jinf.2010.10.002/journals/jinfJournal of Infection (2011)62,119e 129IntroductionThe innate immune system is thefirst barrier to protect human hosts from pathogens.The host recognizes various pathogens via pattern recognition receptors including toll-like receptors(TLRs)and specific NLRs.1TLRs reside on the membrane of host cells and sense pathogen-associated mo-lecular patterns(PAMP)and danger-associated molecular patterns(DAMP).For example,lipopolysaccharide,a major component of gram-negative bacteria is recognized by TLR4.Engagement of TLRs leads to activation of NF-k B sig-naling pathway with subsequent production of precursors of proinflammatory cytokines(e.g.,pro-IL-1b).NLRs are cyto-plasmic counterparts of TLRs,which sense intracellular mi-crobial molecules or danger signals.NLRs are also involved in the assembly of a novel cytosolic multi-protein complex termed the“inflammasome”,the components of which can include an NLR serving as a sensing protein(i.e.,NLRP1, NLRP3,NLRC4),an adapter protein i.e.,apoptosis-associ-ated speck-like protein containing a CARD(ASC),and the effector protein pro-caspase-1(Fig.1).2e6AIM2,a HIN200 family protein,and RIG-I also form an inflammasome to ac-tivate caspase-1through their interactions with ASC.7,8The inflammasome functions as a molecular scaffold for the maturation of caspase-1into its active form and thus serves as a critical regulator of inflammatory responses.Indeed, the recent discovery of the inflammasome has been touted as the“Rosetta stone”of inflammatory responses with the potential to yield new insights into the biologic basis of in-flammatory disorders.9A key function of caspase-1is the cleavage of inflammatory cytokines IL-1b and IL-18from their inactive precursor mole-cules(pro-IL-1b and pro-IL-18)into mature biologically active forms(IL-1b and IL-18)that can be the released from cells.10IL-1b is one of the most potent proinflammatory cytokines that functions in the generation of systemic and localized responses to infection,injury,and inflammatory challenges.11Excessive or inappropriate IL-1b production can lead to unfettered in-flammation that is a hallmark of several autoimmune and in-flammatory disorders.12Gout is one of the most acute inflammatory arthritides,and the Nlrp3inflammasome is es-sential for urate crystal induced inflammation.13A group of autoinflammatory disorders known as cryopyrin-associated pe-riodic syndromes(CAPS)are characterized by mutations in NLRP3and an enhanced propensity to induce caspase-1activa-tion.14In fact,inflammasome-associated aberrant proinflam-matory responses are thought to contribute to the pathogenesis of virtually all inflammatory disorders which in-volve IL-1b and IL-18.Inhibition of these novel regulators of in-flammation has been proposed as a promising therapeutic strategy for autoinflammatory disorders.14In clinical practice, agents that block IL-1b production have proven effective in the management of these disorders.15Activation of caspase-1and IL-1b is also crucial for the induction of immune responses to pathogens.6Modulation and active suppression of the inflammasome pathway compromise host immunity and are significant components of the virulence and pathogenicity of the microorganisms.Animal and human studies have highlighted the importance of the inflammasome pathway in host responses to bacteria,fungi,andviruses. Figure1Activation of the Nlrp3inflammasome.Hosts recognize pathogen-associated molecular patterns(PAMP)and danger-as-sociated molecular patterns(DAMP)via pattern recognition receptors such as toll-like receptors(TLRs).Engagement of TLRs leads to activation of NF-k B signaling pathway with subsequent production of precursors of proinflammatory cytokines(e.g.,pro-IL-1b and pro-IL-18)and activation of Nlrp3.Activated Nlrp3form oligomers and interacts with ASC,bridging the association with pro-caspase-1.The formation of this multi-protein complex,Nlrp3inflammasome,leads to activation of caspase-1and subse-quently production of IL-1b and IL-18.120R.Osawa et al.While,inflammasome-mediated host responses are well char-acterized and reviewed in autoimmune inflammatory disor-ders,15,16little is known about the regulatory functions of inflammasome in the pathogenesis of infection.In this report,we aim to summarize the existing state of knowledge regarding the role of inflammasome in the host-pathogen microbial interactions and as a regulatory signal-ing pathway modulating disease expression in infections. BacteriaInnate immune system recognizes common structures of bacteria such as peptidoglycan and lipopolysaccharide as well as toxins via pattern recognition receptors.These receptors trigger signaling pathways that can lead to production of precursors of proinflammatory cytokines.A second stimulus typically results in activation of caspase-1 through the inflammasome,of which Nlrp3and Nlrc4 inflammasomes have been best studied in the pathogenesis of bacterial infections(Table1).6Bacillus anthracisVirulence of anthrax depends on the production of an exotoxin called lethal toxin,consisting of protective antigen and lethal factor.Protective antigen interacts with the host’s cell surface receptors,allowing endocytosis of lethal toxin.Nlrp1in mice is highly polymorphic and only a subset of the alleles was associated with susceptibility to lethal toxin.17Nlrp1senses lethal factor present in the host’s cyto-sol,which leads to the activation of caspase-1and subse-quent IL-1b production.17,18Spores of Bacillus anthracis also induce caspase-1mediated IL-1b expression but via sig-naling pathways distinct from those for lethal toxin.Whereas lethal toxin induced caspase-1activation leads to IL-1b secretion that was cleaved from pro-IL-1b released as a result of cell death,B.anthracis spores-induced caspase-1acts on the newly synthesized pro-IL-1b and did not induce cell death.19This study suggests that the spores-induced cas-pase-1activation is critical to host defense.19 Staphylococcus aureusInnate immune responses to Staphylococcus aureus require ASC and Nlrp3for caspase-1activation.20Alpha-hemolysin is a major virulence factor for S.aureus and is believed to be responsible for serious sequelae such as necrotizing staphylococcal pneumonia.21Nlrp3induced caspase-1acti-vation is the primary mediator of alpha-hemolysin associ-ated severe inflammation and necrosis.22It has been also suggested that degradation of S.aureus peptidoglycan by lysozyme in the phagosome is essential for the activation of Nlrp3inflammasome.23IL-1b production mediated by the inflammasome component ASC promoted neutrophil re-cruitment and was key in facilitating bacterial clearance in a mouse model of cutaneous S.aureus infection.24 Listeria monocytogenesListeria monocytogenes is a gram-positive intracellular microbe with the ability to cause severe infection in im-munocompromised hosts.It produces a major virulence factor,listeriolysin O(LLO)which mediates escape from the phagosomes into the cytosol and promotes replication within the macrophage.25This phagosomal disruption ap-pears to trigger the activation of Nlrp3,leading to a strong production of IL-1b and IL-18via ASC-dependent caspase-1 activation.26e28Indeed,an LLO-deficient mutant incapa-ble of escaping from the phagosomes lacked the ability to promote secretions of IL-1b and IL-18.26,28It has beenThe inflammasome regulatory pathway and infections121suggested that Nlrc4also contributes to caspase-1activa-tion in listeriosis.29Finally,the role of Aim2as a cytosolic sensor of Listeria-derived DNA has been proposed.30e32 Francisella tularensisFrancisella tularensis is an intracellular pathogen,responsi-ble for the zoonotic disease tularemia.It has been specu-lated that sensing of cytosolic F.tularensis leads to the induction of type1interferons as well as activation of cas-pase-1and death of the infected macrophages.33e35How-ever,it has not been until recently that Aim2deficient macrophages were shown to be defective in caspase-1acti-vation,IL-1b secretion and cell death in response to infection with F.tularensis,36indicating that the Aim2inflammasome is critical in the innate immune system against F.tularensis.A unique characteristic of F.tularensis is that genes essential for its pathogenesis,FTT0584and FTT0748suppress innate immune defense pathway by limiting IL-1b.37This is another example of how pathogens actively modulate and suppress the inflammasome pathway.Salmonella typhimurium and ShigellaflexneriSalmonella typhimurium and Shigellaflexneri are capable of inducing caspase-1activation,leading to host cell death via IL-1b production.Translocation of bacterial effectors to host’s cytoplasm via a type III secretion system is required for caspase-1activation.38Nlrc4also plays a major role in activating caspase-1by sensingflagellin.39,40Macrophages with S.typhimurium lackingflagellin are incapable of cas-pase-1activation.39,40On the other hand,S.flexneri is not flagellated and caspase-1activation occurs independently offlagellin.41Caspase-1activation in response to S.typhi-murium is also mediated by Nlrp3and ASC independently of Nlrc4,42,43and Nlrp3-dependent activation is associated with proinflammatory response to a greater extent than cell death.44Finally,it was recently shown that macro-phages were able to directly detect a component of type III secretion systems in microbes such as S.typhimurium, S.flexneri and activate caspase-1through Nlrc4.38 Legionella pneumophilaLegionella pneumophila is another intracellular pathogen that can activate caspase-1.Legionella inject effector pro-teins into the host’s cytosol via a Dot/Icm type IV secretion system.45It multiplies in an intracellular vacuole of macro-phages while avoiding fusion with lysosomes for survival.In mice models,Nlrc4that sensesflagellin of L.pneumophila in conjunction with Naip5results in caspase-1activation,46e49 which leads to restriction of intracellular replication of L.pneumophila by allowing delivery of Legionella-containing phagosome to lysosomes and its degradation.50 Pseudomonas aeruginosaLike other microbes described above,Pseudomonas aerugi-nosa induces caspase-1activation in an Nlrc4-dependent manner.51,52P.aeruginosa utilizes a type III secretion system to translocate effector proteins into the host’s cytoplasm that contribute to its virulence.Exoenzyme U(ExoU)is one of the four known effector proteins of P.aeruginosa and its secretion is associated with increased virulence in a mouse model of acute pneumonia.53In patients with ventilator-as-sociated pneumonia due to P.aeruginosa,ExoU secreting iso-lates were also associated with worse outcomes than nonsecreting isolates.54Notably,ExoU is capable of inhibit-ing caspase-1activation and IL-1b production.55 Mycobacterium tuberculosisMycobacterium tuberculosis resides in host macrophages and possesses multiple traits that allow evasion of the innate and adaptive immune systems of the host.56IL-1receptor-medi-ated signaling is crucial for the development of innate re-sponse that controls acute M.tuberculosis infection.57,58 The recognition of ESAT-6,a potent T-cell antigen for TB,by Nlrp3inflammasome is required for caspase-1activation and subsequent IL-1b responses.59A peculiar attribute of M.tu-berculosis that facilitates its pathogenicity is its ability to block inflammasome activation.M.tuberculosis actively sup-presses IL-1b production via a metalloprotease encoded by a gene known as zmp1.In a mouse model of tuberculosis,mu-tation in zmp1gene was associated with increased IL-1b pro-duction,decreased intracellular survival of mycobacteria and inability to maintain phagosome maturation arrest.60 These data demonstrate how inflammasome activation can be manipulated by microbial pathogens to promote their propagation.Indeed,zmp1hasbeensuggested asa potentially useful target for abrogating M.tuberculosis infection.60 SepsisProgression to septic shock and systemic inflammatory response syndrome is a major cause of death in patients with sepsis.61Given that IL-1b is one of the important proin-flammatory cytokines in sepsis,the association between sepsis and inflammasomes is not surprising.In animal models,IL-1b-deficient mice are susceptible to endotoxin-induced shock.62However,caspase-1-deficient mice are protected when exposed to lethal doses of endotoxin63 and to lethal peritoneal Escherichia coli infection.64The fact that caspase-1-deficient mice cannot produce IL-1b suggests that a different role of caspase-1may be crucial in the pathogenesis of sepsis.In fact,although caspase-1 is not classified as an apoptotic caspase,it is known to cause apoptosis in macrophages.35Although the initial phase of sepsis is proinflammatory, it eventually progresses to a hypoinflammatory phase in which apoptosis of cells of the adaptive immune system, such as B cells and CD4T cells,occurs.This transition ensures the inhibition of excessive proinflammatory cyto-kine production however,it also leads to immune sup-pression that could lead to further worsening of infections.61Indeed,in the late phase of sepsis,anti-in-flammatory cytokines(e.g.,IL-10,TGF-b)that mediate immunosuppression via leukocyte deactivation are domi-nant.For example,in a murine model of P.aeruginosa pneumonia,concomitant intraabdominal sepsis markedly impaired pulmonary bacterial clearance and the early de-velopment of P.aeruginosa bacteremia,which was122R.Osawa et al.reversed by the administration of IL-10.65Prevention of apoptosis by either caspase inhibitors or antiapoptotic protein Bcl-2is also an effective therapy in mice model of sepsis.66,67To our knowledge,only one study has evaluated inflam-masome activity in clinical setting.This study,which assessed inflammasome expression in patients with early septic shock, showed that critically ill patients with septic shock compared to those without had significantly lower levels of inflamma-some components,including caspase-1,NLRP1,and NLRP12 (called Pypaf-7in this study).68Low expression of NLRP1also correlated with a higher severity of illness and poor outcome in patients with sepsis.These data suggest either a down reg-ulation or massive activation and subsequent exhaustion of the sensors and regulators of inflammation during systemic in-flammatory response syndrome.FungiIL-1b is also important for a protective antifungal immune response.69Existing,albeit limited data show that the in-teractions of pathogenic fungi with the inflammasome reg-ulate IL-1b production.Candida speciesIL-1a and IL-1b are essential for protective host response against invasive candidiasis.69IL-1b-deficient mice had decreased granulocyte recruitment upon intraperito-neal challenge with Candida albicans and had a higher mortality and an increased risk of disseminated disease compared with wild-type mice.69As in bacterial patho-gens,IL-1b-associated inflammatory response in C.albi-cans is mediated by caspase-1.Although activation of caspase-1can occur constitutively without the need of Nlrp3,70a growing body of work suggests that Nlrp3inflam-masome is required for caspase-1mediated IL-1b produc-tion in response to C.albicans.Nlrp3was essential in the transduction of fungal recognition signal to the inflam-masome adapter ASC for caspase-1activation.71This study also showed that Nlrp3-deficient mice infected with C.al-bicans had a higher mortality with higher fungal load in tissues.71In a murine model of oropharyngeal candidiasis,either ASC-or Nlrp3-deficient mice had an increased rate of dissemination and higher fungal burden.72It has long been recognized that nonfilamentous forms of C.albicans are avirulent and mutants defective in hyphal growth are noninvasive.73The ability of C. albicans to transform from a yeast into afilamentous is essen-tial for activation of the Nlrp3inflammasome and Nlrp3-defi-cient mice had increased susceptibility to C.albicans infection.74Although there are no human studies on the inflammasome and fungal infections,polymorphism in the gene CIAS1that codes for NLRP3correlated with an increased risk of vulvar vestibulitis syndrome-an entity characterized by a localized chronic inflammatory state and for which vaginal infection with C.albicans serves as a trigger.Women with vulvar vesti-bulitis syndrome had heightened localized Nlrp3expression and CIAS1gene polymorphism was associated with reduced C.albicans induced IL-1b production.75CryptococcusFollowing pulmonary infection with Cryptococcus,adaptive immunity characterized by Th1response confers resistance to progressive infection.76Accumulating data however, show that innate immune system is a critical initial step that precedes and subsequently facilitates the generation of the adaptive T-cell immunity.77TLR induced activation of innate immune responses with the production of inflam-matory mediators including cytokines such as TNF-a,and IFN-g,chemokines such as MIP-1a/CCL3,and MCP-1/CCL2 and its receptor CCR2play a key role in anti-cryptococcal im-munity.78IL-1b is a key component of this inflammatory re-sponse.Cryptococcal capsular polysaccharide induces IL-1b release by neutrophils.79IL-1b and IL-Ra were differentially upregulated in a genetically resistant mouse model of Cryp-tococcus and mice with heightened proinflammatory re-sponses developed differential polarization towards protective Th1responses.80These data show that TLR path-way lies at the interface between innate and adaptive immu-nity in Cryptococcus neoformans infection.NLRP12regulates inflammatory responses that occur via TLR activation.81 NLRP12protein levels in human THP-1cell lines which ex-press NLRP12is reduced by TLR4agonists.81Taken together, these in vitro results suggest that NLRP12is a negative regu-lator of inflammatory gene expression in macrophages in re-sponse to infection.A robust innate immune response in experimental pulmonary cryptococcosis is associated with resistance to progressive pulmonary and extrapulmonary infection.80VirusesThe recognition of viral invasion relies on sensors including Toll-like receptors and RNA helicases retinoic acid inducible gene I(RIG-I)and melanoma differentiation-associated gene 5(MDA-5).82These sensors detect virus-associated PAMPs, namely genomic DNA and RNA,single-stranded RNA or dou-ble-stranded RNA,leading to activation of transcription fac-tors such as NF-k B and IRF3/IRF7.Signaling through these pathways produces type I interferons which suppress viral replication.83In addition to type I interferons,IL-1b and IL-18also play a critical role through the activation of caspase-1in the pathogenesis of viral infections.The pathogenic basis by which host cells sense viruses to induce caspase-1has only recently been elucidated.InfluenzaResearch on influenza virus in relation to innate immune system has garnered significant interest given severe morbid-ity and mortality in patients with highly pathogenic H5N1 avian influenza infection.Activation of the virus M2protein, a proton selective ion channel,and subsequent imbalances in the concentration of hydrogen ion are required for formation of the Nlrp3inflammasome complex.84Several studies have confirmed that Nlrp3inflammasomes are an essential compo-nent in the immune response against influenza virus A.85e88 For example,Nlrp3,ASC,or caspase-1-deficient mice had dra-matically increased mortality with attenuated airway inflam-mation after exposure to the influenza virus.85Secretions ofThe inflammasome regulatory pathway and infections123Nlrp3-dependent cytokines including IL-1b and IL-18were re-duced in caspase-1and Nlrp3-deficient mice infected with in-fluenza virus A.86Severe epithelial necrosis,extensive collagen depositing in the alveoli,and respiratory failure with hypoxia were observed in Nlrp3-deficient mice compared to wild-type mice,86suggesting a role of the inflammasome in tissue repair after infectious injury.Finally,in addition to TLR pathway,activation of ASC/caspase-1inflammasome ap-peared to be crucial in the development of adaptive antiviral immunity.87Other virusesNlrp3and Aim2inflammasomes also played a critical role in the innate immune response against other viruses such as adenovirus,89human herpesvirus-1,89Sendai virus,88vaccina virus,30,90mouse cytomegalovirus,30and modified vaccina virus Ankara.91Other viruses,including some RNA viruses such as reovirus and vesicular stomatitis virus89were not able to activate inflammasome with cellular challenge.It is notable that poxviruses down regulate inflammasomes to impair the host’s immune responses.Myxoma virus is a rab-bit-specific lethal poxvirus,which produces M13L-PYD pro-tein.This PYD-protein interferes with ASC-1and inhibits caspase-1activation and subsequent IL-1b production,block-ing the host’s proinflammatory responses to the virus infec-tion.92A Shope Fibroma virus-encoded PYD-only protein1 also inhibited PYD-mediated activation of caspase-1.93Cow-pox virus possessed the gene encoding cytokine response mod-ifier A(crmA),a broad-spectrum caspase inhibitor,94,95which allows the virus to disable the host’s inflammatory responses by blocking the IL-1b production.ProtozoaMalariaMalaria is by far amongst the major threats in sub-Saharan Africa,resulting in at least one million deaths annually.96The pathogenesis of malaria involves activation of coagulation cascade,sequestration of red blood cells in the capillaries due to parasites’cytoadherence molecules,and excessive production of proinflammatory cytokines.Hemozoin,a breakdown product of hemoglobin,is re-leased systemically after erythrocytes are hemolyzed due to malaria infection.It has been recognized that hemozoin can activate macrophages and dendritic cells to produce proinflammatory cytokines,97but its exact mechanism has not been defined.It is noteworthy that hemozoin is an inor-ganic heme crystal similar to monosodium urate which has the capacity to promote inflammatory mediators such as IL-1b via Nlrp3,ASC,and caspase-1activation.13,98e100In-deed,murine experimental models of cerebral malaria have shown that IL-1b and Nlrp3-deficient mice had limited inflammatory characteristic pathological changes i.e.,leu-kocyte infiltrate and damaged endothelial cells,and had improved survival.98Uric acid may play a critical role in the inflammasome activation not only in patients with gout,but also in those with malaria.Plasmodium-infected erythrocytes accumulate hy-poxanthine,a precursor of uric acid,that upon erythrocyte rupture is released into the bloodstream and tissues.101Ma-ture infected erythrocytes induce inflammatory cytokines IL-6and IL-1b production by the peripheral blood mononu-clear cells in a dose-dependent manner.101Furthermore,allo-purinol,a xanthine oxidoreductase inhibitor that prevents conversion from hypoxanthine to uric acid,and uricase, an enzyme that degrades uric acid inhibited the inflammatory cytokine response.99The effect of allopurinol may be ex-plained by the ability of xanthine oxidoreductase to generate reactive oxygen species,102which is one of the known triggers for inflammasome activation.Finally,in a randomized controlled study,adjunctive allopurinol therapy to quinine in patients with acute Plasmo-dium falciparum infection shortened the duration of parasite-mia and fever.103Taken together these data show that hemozoin induced inflammasome activation is a significant contributor to inflammatory host response and the pathoge-nicity of Plasmodium spp.Implications for clinical researchMolecules targeting the inflammasomes or their signaling pathway have been successfully employed in the manage-ment of various inflammatory disorders.Amongst these agents,IL-1b blocking agents have been approved for use in clinical setting.Cryopyrin-associated periodic syndromes are autoinflam-matory disorders,comprising three clinically distinct disor-ders:familial cold autoinflammatory syndrome,Muckle-Wells syndrome,and neonatal onset multisystem inflammatory disease.These conditions are characterized by mutations in a single gene,which encodes NLRP3(NLRP3was previously called cryopyrin),104,105resulting in local and systemic inflam-mation via excessive caspase-1activation and IL-1b release.It has been shown that therapeutic inhibition of IL-1b by a re-combinant IL-1receptor antagonist(anakinra),106an IL-1re-ceptor immunoglobulin fusion protein(rilonacept,IL-1 Trap),107,108and a human anti-IL-1b monoclonal antibody (canakinumab)109rapidly led to remission of symptoms.Fa-milial mediterranean fever is another inflammatory disorder characterized by mutations in the MEFV gene that encodes pyrin.In patients who were unresponsive to its standard ther-apy such as colchicine,anakinra has been used anec-dotally.110e112Blockage of IL-1b has also been effective in ameliorating the symptoms of gout.113,114Sepsis,as a systemic response to infections,attributes to unfettered inflammation triggered by proinflammatory cy-tokines such as TNF-a and IL-1b.In animal models,adminis-tration of IL-1receptor antagonist reduced the mortality of septic shock.115,116However,in two large phase3random-ized controlled trials in patients with sepsis,the use of IL-1 receptor antagonist did not significantly improve the clinical outcomes.117,118While thesefindings could simply reflect its lack of efficacy,the precise effects of immunomodulatory agents in sepsis are inherently difficult to interpret given het-erogeneity of the patient population and the complexity of pathophysiology such as multiple interactions between cyto-kines and the immune system.Other agents have also been tested in animal studies or in early phase human trials.119Caspase inhibitors were initially used as research tools119and were categorized into whether124R.Osawa et al.。