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REVIEWCell deathmediated cytokine release and itstherapeutic implicationsDavid E.Place and Thirumala-Devi KannegantiTargeting apoptosis to treat diseases has seen tremendous success over the past decades.More recently,alternative forms ofregulated cell death,including pyroptosis and necroptosis,have been described.Understanding the molecular cascadesregulating both pyroptosis and necroptosis will yield even more targets to treat diseases.These lytic forms of cell death aredistinct from apoptosis due to their characteristic lysis and release of cellular components that promote disease or direct abeneficial immune response.In this review,we focus on how pyroptosis and necroptosis,which release potent immunecytokines such as IL-1 and IL-18,contribute to various diseases.We also consider the important role that the executioners ofthese cell death pathways,GSDMD and MLKL,play in the progression of inflammatory diseases.Crosstalk between thedifferent cell death pathways likely plays a major role physiologically.New therapeutic strategies targeting these specificmolecules hold enormous potential for managing inflammatory diseases.IntroductionRegulated cell death has been understood as a concept for dec-ades,with apoptosis being the first well-defined process inwhich cells dismantle themselves in a process that is generallyimmunologically quiet(Kerr et al.,1972;Elmore,2007).Apo-ptosis is induced homeostatically and upon exposure to a widevariety of insults,leading to the activation of initiator caspases(caspase-8,-9,-10)and effector caspases(caspase-3,-6,-7),re-sulting in a nonlytic cell death characterized by membraneblebbing,cellshrinkage,andchromosomalcondensation(Elmore,2007).While apoptosis facilitates the controlled deg-radation of intracellular proteins and organelles,pyroptosis andnecroptosis lead to cell lysis and the release of a wide range ofintracellular components and inflammatory cytokines.We focusin this review on the lytic forms of cell death(pyroptosis andnecroptosis)and the consequences of their cytokine release,with aneye toward new waysof treating inflammatory diseases.Unlike apoptotic cell death,in which plasma membrane in-tegrity is maintained and intracellular components are seques-tered,pyroptosis is a lytic form of cell death that,through atightly regulated pathway,activates and releases the potentcytokines IL-1 and IL-18(Fig.1;Dinarello et al.,1974;Dinarello,1998;Cookson and Brennan,2001;Martinon et al.,2002).Py-roptosis plays a major role in the control of intracellularpathogens by destroying their replicative niche and drivingimmune responses through the release of matured cytokines(Man et al.,2017).Like apoptosis,pyroptosis is regulated bycaspases,in particular caspase-1 and caspase-11(in mice)orcaspase-1/4/5(in humans).Caspase-4/5/11 binds intracellularLPS directly,leading to cleavage of the substrate gasdermin D(GSDMD),the recently discovered executioner of pyroptosis(Heet al.,2015;Kayagaki et al.,2015;Shi et al.,2015).Upon cleavage,the autoinhibitory C-terminal domain of GSDMD is releasedfrom the pore-forming N-terminal domain,leading to insertionof the GSDMD N-terminal domain into the plasma membraneand oligomerization with other GSDMD fragments,therebygenerating a large pore 1020 nm in diameter(Ding et al.,2016).Similar to caspase-4/5/11,caspase-1 activation by upstreamsensor proteins leads to caspase-1mediated cleavage of GSDMD.The upstream regulators of caspase-1 activation differ in theirspecificity to different ligands and cellular states and include theproteins NLRP3,NLRC4,AIM2,NLRP1,and pyrin,with otherrelated proteins being suggested as additional sensors(Place andKanneganti,2018).A subset of these sensors(NLRP3,AIM2,andpyrin)require the adaptor protein ASC to activate caspase-1 af-ter ligand sensing.A single specific trigger for NLRP3 activation is not known,but many cellular insults induce NLRP3 inflammasome activa-tion and cleavage of caspase-1,including loss of cellular ionhomeostasis and plasma membrane disruption(Kannegantiet al.,2006;He et al.,2016;Jo et al.,2016).NLRC4 is activatedby a set of sensor proteins including human NAIP/mouse NAIP1,NAIP2,and NAIP5/6,which recognize the bacterial type 3 se-cretion system(T3SS)needle,T3SS rod,or flagellin proteins,.Department of Immunology,St.Jude Childrens Research Hospital,Memphis,TN.Correspondence to Thirumala-Devi Kanneganti:thirumala-devi.kannegantistjude.org.2019 Place and Kanneganti.This article is distributed under the terms of an AttributionNoncommercialShare AlikeNo Mirror Sites license for the first six monthsafterthe publication date(see http:/www.rupress.org/terms/).After six months itis available under a CreativeCommons License(AttributionNoncommercialShare Alike4.0 International license,as described at https:/creativecommons.org/licenses/by-nc-sa/4.0/).Rockefeller University Presshttps:/doi.org/10.1084/jem.201818921474J.Exp.Med.2019 Vol.216 No.714741486respectively(Sharma and Kanneganti,2016).The upstreamNAIP proteins are also transcriptionally regulated by the tran-scription factor IRF8(Karki et al.,2018).The AIM2 in-flammasome is activated by cytosolic double-stranded DNAbinding with AIM2,which is detected upon viral entry into thecytosol or downstream of IRF1-mediated expression of IFN-stimulated genes that lyse intracellular bacteria(Fernandes-Alnemri et al.,2009;Hornung et al.,2009;Man et al.,2015;Meunier et al.,2015).The mouse NLRP1b inflammasome is ac-tivated following proteolysis after exposure to Bacillus anthracislethal toxin and DPP8/9 inhibitors(Chavarr a-Smith and Vance,2013;Okondo et al.,2017).Newer studies have shown that en-dogenous human DPP9 binds and negatively regulates NLRP1,and a mutation in this binding domain is associated with NLRP1-associated autoinflammation with arthritis and dyskeratosis(NAIAD;Zhong et al.,2018).DPP8/9 inhibitors activate murineNLRP1b by inducing proteasomal degradation of the auto-inhibitory N-terminal fragment,while anthrax lethal toxinfunctions by induction of N-end rulemediated proteolysis ofthe N-terminal fragment of NLRP1(Okondo et al.,2018;Chuiet al.,2019;de Vasconcelos et al.,2019b;Sandstrom et al.,2019).Pyrin inflammasome activation is triggered by RhoA-GTPasemodifying toxins(Xu et al.,2014).Concurrent withGSDMD cleavage,caspase-1 activation cleaves inactive pro-IL-1 and pro-IL-18 into their active forms.The formation ofGSDMD pores in the plasma membrane facilitates the release ofintracellular contents and mature IL-1 and IL-18,leading toinflammatory signaling in responding cells,which can have bothbeneficial and detrimental effects.Following pore formation byGSDMD,other inflammatory cytokines and alarmins that do notFigure1.Overviewofpyroptosis.PyroptosisismediatedbytheinflammasomesensorproteinsNLRC4,NLRP1,AIM2,Pyrin,andNLRP3,leadingtoactivationof caspase-1.The NLRP1 and NLRC4 sensor proteins do not require the adaptor protein ASC,while the remaining sensors require ASC to oligomerize withcaspase-1.Caspase-1 directly cleaves GSDMD to release the autoinhibitory C-terminal domain from its pore-forming N-terminal domain and also cleaves pro-IL-1 and pro-IL-18 into their active forms,which are released through the GSDMD pore.Caspase-11 binding to intracellular LPS activates caspase-11 to cleaveGSDMD,which drives pore formation that leads to cell lysis and downstream NLRP3 inflammasome activation through the loss of ion homeostasis.dsDNA,double-stranded DNA.Place and KannegantiJournal of Experimental Medicine1475Inflammatory cell death and diseasehttps:/doi.org/10.1084/jem.20181892need processing by caspase-1,such as IL-1,IL-33,HMGB1,ATP,and ASC complexes,are released,further contributing to theinflammatory response(Vande Walle et al.,2011;Basiorka et al.,2018;Frank and Vince,2019).Together,the majority of theseinflammasome complexes function to expel intracellular patho-gensfrom the cellor haltviral replicationinaddition toreleasinginflammatory cytokines,serving a critical role in host defense.Excessive or dysregulated activation of these complexes can alsobe detrimental to the host and is associated with inflammatorydiseases,as discussed later in this review.Similar to pyroptosis,necroptosis is a lytic form of cell deaththat is activated by a different pathway.In cells where caspase-8is inhibited or missing,inflammatory signaling via TNF super-family receptors,IFNs,TLR3,or TLR4 can lead to the phospho-rylation of RIPK1,RIPK3,and ultimately mixed-lineage kinasedomain-like pseudokinase(MLKL;Fig.2;Holleretal.,2000;Choet al.,2009;He et al.,2009;Linkermann and Green,2014;Wanget al.,2014;Mocarski et al.,2015).Following phosphorylation,MLKL assembles into oligomers that create a channel in theplasma membrane,disrupting the ion homeostasis of the cell,similar to GSDMD-mediated lysis(Holler et al.,2000;Sun et al.,2012;Zhaoetal.,2012;LinkermannandGreen,2014;Wangetal.,2014).The conditions for necroptosis are fairly strict in com-parison to pyroptosis,but recent research has identified sce-narios in which this pathway is engaged.Physiologically,it seems that necroptosis plays a backup role for cell deathwhen the apoptotic pathway becomes inhibited by pathogens,mutations,changes in the regulators of apoptosis,or cancer(Pasparakis and Vandenabeele,2015).Due to the complicatedrole of Fas-associated protein with death domain(FADD),caspase-8,RIPK1,RIPK3,and MLKL in regulating this form ofcell death and also in regulating cytokine production,it is dif-ficult to definitively attribute necroptosis to the progression ofmany diseases.New mouse models,chemical inhibitors,andassays that probe for phosphorylation of MLKL are helping toadvance our understanding of the role of necroptosis in drivingdiseases(Pasparakis and Vandenabeele,2015;Weinlich et al.,2017).Crosstalk between apoptosis,necroptosis,and pyroptosismakes it difficult to determine how each pathway contributes tocomplex inflammatory diseases(Fig.2).For example,apoptosissignaling through FADD and caspase-8 has been shown tolink signaling between TLRs and priming of the NLRP3 in-flammasome,and manyof the proteins ofthe apoptosis pathwayFigure 2.Cell death crosstalk overview.Activation of ZBP1 recruits RIPK3,FADD,and caspase-8 to drive parallel cell death pathways of apoptosis andpyroptosis.NLRP3 activation leads to ASC and caspase-1 inflammasome assembly and cleavage of the pyroptosis executioner GSDMD.TNF-mediated celldeath induces apoptosis via TRADD,RIPK1,RIPK3,caspase-8,caspase-3,and caspase-7,but,upon inhibition of caspase-8(by zVAD),TNF drives RIPK1,RIPK3,and MLKL phosphorylation to drive necroptotic cell death.The nucleic acid binding protein ZBP1 can also be directly activated by viral infection(influenza Avirus IAV,HSV,mouse cytomegalovirus MCMV,and vaccinia virus VV)to induce cell death through parallel pathways of necroptosis,apoptosis,andpyroptosis.Place and KannegantiJournal of Experimental Medicine1476Inflammatory cell death and diseasehttps:/doi.org/10.1084/jem.20181892also regulate necroptosis(Elmore,2007;Gurung et al.,2014).Inaddition to caspase-8mediated priming of the inflammasome,others have observed that caspase-8 can localize to caspase-1and ASC-containing specks,facilitating inflammasome activa-tion and downstream cytokine maturation(Gringhuiset al.,2012;Manetal.,2013;Karkietal.,2015).Treatmentofcolorectalcancercells with the platinum-based,DNA-damaging chemotherapydrug lobaplatin resulted in caspase-3dependent cleavage ofGSDME,a second gasdermin family pore-forming protein(Yuet al.,2019).Infection with Yersinia spp.has also revealed a rolefor caspase-8mediated cleavage of GSDMD,suggesting this playsa previously understudied role in regulating crosstalk betweenapoptosis and pyroptosis(Orning et al.,2018;Sarhan et al.,2018a).While recent studies have shown that crosstalk betweenapoptotic signaling pathways with necroptosis and pyroptosisoccurs,we focus here on the lytic forms of cell death as the finaloutcome that drives inflammatory pathology.Inhuman cell lines,others have found that MLKL ion channel formation can leadto NLRP3 inflammasome activation in a cell-intrinsic manner,suggesting that this crosstalk between necroptosis and pyroptosisplays a larger role than previously appreciated(Conos et al.,2017;Gutierrez et al.,2017).Other recent studies have described keynew players that regulate crosstalk between cell death pathways.InhibitionorlossofTAK1leadstoautocrineTNF-mediatedRIPK1-dependent cell death through combined apoptosis,pyroptosis,and necroptosis pathways(Malireddi et al.,2018).In some viralinfections,it also appears that the protein ZBP1 can act as amaster regulator of all three cell death pathways,which requiresfurther study in other contexts(Kuriakose et al.,2016).Impor-tantly,these lytic forms of cell death also regulate the release ofinflammatory cytokines that have been targeted successfully inmany disease treatments.New studies examining the execu-tioners of pyroptosis and necroptosis and their crosstalk in dif-ferent contexts will shed more light on their physiologicalrelevance.Monogenic inflammatory diseasesDue to the potent inflammatory signaling role of IL-1 and IL-18,the dysregulated activation and release of these cytokines con-tribute to many diseases(Table 1).Well-characterized gain-of-function mutations in inflammasome sensors lead to excessiveproduction of these cytokines and cell death,underlying thepathogenesis of a number of monogenic diseases.Gain-of-function mutations in pyrin are associated with the auto-inflammatory disease familial Mediterranean fever(FMF)anddrive the excessive release of IL-1 via GSDMD-mediated py-roptosis in a mouse model of FMF(Sharma et al.,2017;Kanneganti et al.,2018).While commonly treated with colchi-cine,recent work has shown that inhibition of IL-1 withanakinra(recombinant IL-1R antagonist),canakinumab(antiIL-1 monoclonal antibody),or rilonacept(decoy IL-1R/IL1-RA/IgG1 fusion protein)can improve outcomes in colchicine-resistant FMF and that GSDMD inhibitors may also play afuture role(Varan et al.,2019).In addition to the role ofpyroptosis-mediatedIL-1release,arecentstudyshowedthatTNFsignalingpromotedtheexpressionofpyrinina positive feedbackloop(Sharma et al.,2019).Patients with FMF also exhibitelevated serum IL-18,but whether this promotes disease and is apromising target for inhibition is unclear(Wada et al.,2018).Autoinflammatory diseases associated with NLRP3 muta-tions,collectively described as cryopyrin-associated periodicsyndromes(CAPS),include familial cold autoinflammatorysyndrome,Muckle-Wells syndrome,and neonatal-onset multi-system inflammatory disorder/chronic infantile neurological,cutaneous,and articular syndrome(Manthiram et al.,2017).Together,these diseases are associated with systemic inflam-mation,neutrophilia,fever,rashes,and joint pain and are suc-cessfully treated with anakinra,rilonacept,and canakinumaband may be treated with NLRP3 or GSDMD inhibitors in thefuture(Lachmann et al.,2009;Gillespie et al.,2010;Coll et al.,2015;X