Emerging Activators and Regulators of Inflamma.pdf

ReviewEmerging Activators and Regulatorsof Inflammasomes and PyroptosisYansong Xue,1,2Daniel Enosi Tuipulotu,1,2Wei Hong Tan,1Callum Kay,1and Si Ming Man1,*The inflammasomeisacytosolicimmune signaling complex that induces inflammation andpyrop-tosis.Inflammasome complexes respond to a variety of pathogens,as well as danger or homeo-stasis-altering signals;they can play critical roles in the development of autoinflammatory condi-tions and cancer.Studies have now provided additional insights into the activation mechanismsand regulation of established inflammasome complexes,including NLRP1b,NLRP3,NOD-like re-ceptor family apoptosis inhibitory protein(NAIP)NLRC4,absent in melanoma(AIM)2,caspase-11,and pyrin.New activators and regulators of emerging NLRP6 and NLRP9b inflammasomecomplexes have also been described.We highlight the latest progress in our understanding ofthe molecular mechanisms governing inflammasome activation and pyroptosis,including the dis-covery of the pore-forming protein gasdermin D(GSDMD).We also discuss the importance ofinflammasome activators and regulators in health and disease.An Overview of Mammalian Inflammasome ActivationThe inflammasome a cytosolic multimeric innate immune signaling complex has emerged as acentral pillar of mammalian innate immunity since its discovery in 2002 1.Formation of a functionalinflammasome is initiated by germline-encoded pattern recognition receptors(PRRs;see Glos-sary),which are capable of sensing pathogen-associated molecular patterns(PAMPs),danger-associated molecular patterns(DAMPs),and homeostasis-altering molecular processes(HAMPs)2,3.Inflammasomes have mostly been studied in the context of macrophages,but have beendescribed in other myeloid cells,notably dendritic cells and neutrophils 4,5.Upon sensing a spe-cific PAMP,DAMP,or HAMP,certain PRRs recruit the adaptor protein apoptosis-associated speck-like protein containing a CARD(ASC).ASC contains two domains:a pyrin domain(PYD,not to beconfused with the inflammasome sensor pyrin)and a caspase activation and recruitment domain(CARD),which,through homotypic CARDCARD interactions,allow ASC to couple the upstreamPRR to the effector cysteine protease caspase-1 6(Figure 1,Key Figure).Active caspase-1 canthen proteolytically cleave the proinflammatory cytokines pro-interleukin(IL)-1b and pro-IL-18into their mature forms 6.Alternatively,PRRs containing a CARD may directly bind to the CARDof caspase-1 and form an inflammasome complex without ASC(Figure 1A)6.Major progress inthe field has been the discovery that inflammasome-associated caspases(human caspase-1,-4,and-5,and murine caspase-1 and-11)have the capacity to mediate cleavage and activation ofthe pore-forming protein gasdermin D(GSDMD),liberating an N-terminal domain that oligomer-izes in the host plasma membrane to drive pyroptosis in macrophages and other immune cell sub-sets such as neutrophils 4,7,8.Oligomerization of the N-terminal domains leads to pore formation,cell swelling,and release of cytoplasmic contents,including biologically active IL-1b and IL-18,which generate an inflammasome-associated inflammatory response in the host 913(Figure 1).SeveralfamiliesofPRRshavebeen described to forminflammasomes,namelythenucleotide-bindingdomain and leucine-rich repeat receptors(NLRs),the absent in melanoma 2-like receptors(ALRs)and pyrin.In particular,we focus on the latest advances elucidating the activation mechanisms ofNLRP1,NLRP3,NOD-like receptor family apoptosis inhibitory protein(NAIP)NLRC4,AIM2,cas-pase-11,pyrin,and GSDMD.We also discuss two sensor proteins that have emerging functions in in-flammasome assembly and activation,NLRP6 and NLRP9b(Figure 1).While other sensors,such as hu-man NLRP7,mouse NLRP12 and human interferon(IFN)g-inducible protein(IFI)16 have beensuggested to induce caspase-1 activation 1416,at present,their exact role remains unclear andwill not be discussed here.This review aims to provide a timely overview of the latest findings propel-ling forward our understanding of the activation mechanisms and regulation of inflammasome1Department of Immunology andInfectiousDisease,TheJohnCurtinSchoolof Medical Research,The AustralianNational University,Canberra,Australia2These authors contributed equally*Correspondence:siming.mananu.edu.auHighlightsThe inflammasome family is ex-panding and now includes the ca-nonicalAIM2,NAIP-NLRC4,NLRP1,NLRP3,NLRP6,NLRP9b,and pyrin inflammasomes and thenoncanonicalcaspase-11inflammasome.Inflammasomesensorsdetectagrowing constellation of pathogen-associatedmolecularpatterns(PAMPs),danger-associatedmo-lecular patterns(DAMPs),and ho-meostasis-altering molecular pro-cesses(HAMPs).Activation of human caspase-1,-4,and-5,or mouse caspase-1 and-11,induces cleavage of the pore-forming protein GSDMD,medi-ating pyroptosis.Assembly and signaling of the in-flammasome often requires tran-scriptional and translational regu-lation,and spatial localization ofinflammasome components at thesite of organelles,such as the Golgiand mitochondria.Targeting inflammasome compo-nentshaspotentialtherapeuticvalue in the treatment of certaininfections,cancers,and inflamma-tory conditions.Trends in Immunology,-,Vol.-,No.-https:/doi.org/10.1016/j.it.2019.09.005 2019 Elsevier Ltd.All rights reserved.1Please cite this article in press as:Xue et al.,Emerging Activators and Regulators of Inflammasomes and Pyroptosis,Trends in Immunology(2019),https:/doi.org/10.1016/j.it.2019.09.005Trends in Immunologycomplexes leading to inflammation.We also highlight unanswered questions and future avenues forresearch in inflammasome biology undoubtedly a focus in coming years.NLRP1 InflammasomeHuman NLRP1 was the first protein identified to assemble into an inflammasome complex 1.Unlikehuman NLRP1,mice carry three NLRP1 paralogs(NLRP1ac),all of which lack a PYD 17.Of these pa-ralogs,NLRP1b is the most well characterized and is activated by the anthrax lethal toxin secreted byBacillus anthracis 18,19(Figure 2).Other activators of human NLRP1 and murine NLRP1b have beenidentified,includingtheeukaryotictoxoplasmosis-causingparasiteToxoplasmagondii20,theubiq-uitin ligase secreted effector IpaH7.8 from the bacterium Shigella flexneri 21,and the compoundVal-boro-Pro,an inhibitor of cytosolic serine proteases DPP8 and DPP9 22.A unifying mechanism of NLRP1b inflammasome activation has been described by means of func-tional degradation 21,23(Figure 2).In this scenario,autoproteolytic processing within the FIINDdomain splits NLRP1b into the N-terminal and C-terminal fragments bound by a noncovalent inter-action 21,24.Subsequent cleavage of murine NLRP1b by lethal factor exposes a destabilizingneo-N terminus 21,23,initiating the ubiquitination and proteasomal degradation of the N-terminalfragment 21,23 and liberating the C-terminal fragment to initiate inflammasome assembly 21.Furthermore,ubiquitination of NLRP1b driven by the E3 ligase UBR2,the ubiquitinating proteinUBR4,and the ubiquitin-activating enzyme UBA6 of the host 22,23,or the E3 ligase IpaH7.8 ofS.flexneri 21,can lead to N-terminal degradation and NLRP1b inflammasome activation.Previousstudies have also shown that NLRP1b activation can be blocked by proteasome inhibition in murinemacrophages,indicating that proteasomal activity is essential for murine NLRP1b activation 25(Fig-ure 2).Thus,N-terminal degradation is potentially a unifying mechanism of NLRP1b inflammasomeactivation induced by a diverse group of activators.How human NLRP1 is regulated has remained unclear.However,point mutations within PYD(A54T,A66V,and M77T)or deletions of the PYD or LRR of human NLRP1 have been reported to increase IL-1b production in immortalized keratinocytes relative to wild-type(WT)controls 26.Furthermore,hu-man germline gain-of-function mutations in the PYD and LRR of the NLRP1 disrupt the autoinhibitorystate leading to self-oligomerization and hyperactivation of the NLRP1 inflammasome 26.Suchconstitutive inflammasome activation has been shown to predispose individuals to skin disease,including multiple self-healing palmoplantar carcinoma,and familiar keratosis lichenoides chronica26.The same study also reported that overexpression of human NLRP1 fails to induce ASC speckformation;however,NLRP1 mutants lacking PYD readily form specks 26.This is different fromNLRP3 and AIM2,in which PYD enhances ASC oligomer assembly 26 and provides evidence thatPYD has diverse roles for different inflammasome sensors.NLRP3 InflammasomeNLRP3 responds to a diverse range of stimuli,including pathogens,microbial toxins,nucleic acids,ATP,and crystalline substances 27,28.These stimuli exert cellular perturbations often elicited byplasma membrane pore formation and ion flux,lysosomal rupture,and mitochondrial dysfunction28.An important advance in the field has been the identification of the kinase NIMA-related kinase(NEK)7 as a new component of the murine NLRP3 inflammasome 2931.NEK7 binds directly toNLRP3 and is required for inflammasome activation to all known NLRP3 activators 2931.The struc-ture of human NEK7 and inactive NLRP3 has also been described,with NEK7 binding to the LRR andnucleotide-binding domains of NLRP3 32.In the active conformation of the NEK7NLRP3 complex,NEK7 interactswith aneighboring NLRP3 to facilitate oligomerization of NLRP3proteins 32.Bindingbetween NEK7 and NLRP3 is not sufficient to drive NLRP3 inflammasome activation 32,therefore,additional components may trigger this process.Transcriptional RegulationA two-step mechanism,defined by a priming step and an activation step,is generally required for fullactivation of NLRP3(Figure 3A)33.The initial priming step induces upregulation of the NLRP3GlossaryAbsent in melanoma 2-like re-ceptors(ALRs):family of re-ceptors involved in the recogni-tion of DNA.Cryopyrin-associated periodicsyndrome(CAPS):auto-inflammatory syndrome charac-terized by NLRP3 mutations.Cyclic GMPAMP synthase(cGAS):cytosolic DNA sensortriggering the production of type IIFNs.Danger-associated molecularpatterns(DAMPs):host or envi-ronmental molecules that caninitiate an inflammatory response.Endoplasmic reticulum(ER)stress:disturbances in the normalphysiology of the ER.Endosomal sorting complexesrequired for transport(ESCRT):multiprotein machinery that me-diates receptor sorting,mem-brane remodeling and membranescission.Extrinsic apoptosis:cell deaththat is initiated through the tumornecrosis factor receptor(TNFR)superfamily.Familial cold autoinflammatorysyndrome(FCAS):an inheritedinflammatory disorder triggeredby exposure to coldtemperatures.Familial Mediterranean fever(FMF):autoinflammatory diseasecharacterized by MEFV mutations.High-mobility group box 1(HMGB1):nuclear and cytosolicprotein that is released into circu-lation during endotoxemia andsepsis.Homeostasis-altering molecularprocesses(HAMPs):perturba-tions in cellular homeostasis.Macrophage activation syndrome(MAS):acquired form of hemo-phagocytic lymphohistiocytosis.Neutrophil extracellular traps:chromatinprotein structuresreleased into the extracellularspace during neutrophil cell deathto capture microbes,promotecoagulation and activate myeloidcells.NLRP3 speckles:NLRP3 aggre-gates that form cytoplasmicpuncta.NOD-like receptor familyapoptosis inhibitory protein(NAIPs):acts as a sensor compo-nent of the NLRC4 inflammasome.Nucleotide-binding domain andleucine-rich repeat receptors2Trends in Immunology,-,Vol.-,No.-Please cite this article in press as:Xue et al.,Emerging Activators and Regulators of Inflammasomes and Pyroptosis,Trends in Immunology(2019),https:/doi.org/10.1016/j.it.2019.09.005Trends in Immunologyprotein.In both human and murine macrophages,this step requires recognition of PAMPs and/orDAMPsbyPRRs,includingToll-likereceptors(TLRs)andNLRsNOD1andNOD2,orrequirescytokinestimulation via IL-1 receptor(IL-1R)and tumor necrosis factor(TNF)receptors TNFR1 and TNFR2 33.Several mediators facilitating signal transduction of these receptors have been described,includingthe adaptor protein myeloid differentiation primary response 88(MyD88)33,the kinases interleukin1receptorassociatedkinase(IRAK)1andIRAK434,35,apoptosissignal-regulatingkinase(ASK)1andASK2 36,Fas-associated protein with death domain(FADD)37,caspase-8 37,ubiquitin-bindingprotein SHARPIN 38,and TRAF-interacting protein with forkhead-associated domain(TIFA)39(Figure 3A).These signaling components ultimately drive the transcription of human and murineNLRP3,and the proinflammatory gene IL1B,ensuring that the cell is ready or primed to respond toNLRP3 activators 33.Post-translational ModificationsNLRP3 activation is tightly regulated by multiple post-translational modifications;these pathwayshave been reviewed elsewhere 28.E3 ubiquitin ligases tripartite motif-containing protein(TRIM)31 40 and membrane-associated ring-CH-type finger(MARCH)7 in mice can negatively regulateNLRP3 by ubiquitination 41,whereas the E3 ubiquitin ligase Pellino2 promotes activation ofNLRP3 in both human and murine macrophages 42.Phosphorylation is another critical modificationinvolved in NLRP3 regulation.Protein kinase A suppresses NLRP3 by phosphorylating the nucleotide-binding domain 43,44,whereas phosphorylation of NLRP3 by protein kinase D at the same residuepromotes its activation in response to accumulated diacylglycerol at the Golgi in human monocyteTHP-1 cells and murine bone-marrow-derived macrophages(BMDMs)45.Additional studies havesuggested that human and murine NLRP3 can be negatively regulated by sumoylation 46.Thesepost-translational modifications highlight the complex regulation that governs the activation and in-hibition of NLRP3 28.Ion FluxesIon fluxes are linked to NLRP3 activation through several mechanisms:K+efflux is a well-establishedmechanism leading to NLRP3 activation in response to NLRP3 activators,such as ATP,crystalline sub-stances and bacterial toxins,which all trigger K+efflux in murine macrophages 47,48.Further,the K+efflux channels P2X purinoceptor 7(P2X7)and TWIK2 cooperate to induce activation of NLRP3 49.Although studies have highlighted the importance of K+efflux,K+has not been shown to directlyinteract with NLRP3 and K+efflux is not universally required in the activation of NLRP3 for a small sub-set of activators,such as the TLR7 and/or TLR8 agonists imiquimod and CL097 50,51.NLRP3 can be activated by increased intracellular Ca2+,sensed by the calcium-sensing receptor inmurine BMDMs 52.Elevation of intracellular Ca2+is also found in peripheral blood mononuclearcells(PBMCs)from patients with cryopyrin-associated periodic syndrome(CAPS)52.Another studyproposed that a disturbance in mitochondrial Ca2+homeostasis contributed to NLRP3 activation dur-ing Pseudomonas aeruginosa infection of primary human airway epithelial cells 53.By contrast,Ca2+influx is dispensable for NLRP3 activation in murine macrophages and dendritic cells(DCs);however,Ca2+influx does occur as a consequence of caspase-1 activation 54.Therefore,additional studiesare required to robustly elucidate the exact