Integrating Inflammasome Signaling in Sexually Transmitted Infections

Review

Integrating

In

flammasome

Signaling

in

Sexually

Transmitted

Infections

Christopher

Lupfer

and

Paras

K.

Anand

*

Inflammasomesarecytosolicmultiproteinplatformswithpivotalrolesin

infec-tiousdiseases. Activation of inflammasomesresultsin proinflammatory

cyto-kinesignalingandpyroptosis.Sexuallytransmittedinfections(STIs)areamajor

healthproblemworldwide,yetfew studieshaveprobed theimpactof

inflam-masome signaling during these infections. Due to the dearth of appropriate

infectionmodels,ourcurrentunderstandingofinflammasomesinSTIsismostly

drawnfromresultsobtainedinvitro,fromdistantinfectionsites,orfromrelated

microbialstrainsthatarenotsexuallytransmitted.Understandinghowin

flam-masomesinfluencetheoutcomeofSTIsmayleadtothedevelopmentofnovel

andeffective strategiestocontroldiseaseandpreventtransmission.Here we

discussandhighlighttherecent progressinthisfield.

Inflammasomes

Inflammasomesaremultiproteincomplexesactivatedinresponsetovarious pathogen-associ-atedmolecularpatterns(PAMPs),suchasbacteriallipopolysaccharide(LPS)andflagellin,orin responseto cellular damage-associated molecular patterns (DAMPs), includingextracellular ATPandfluctuationsincytoplasmicionconcentrations[1–3].DetectionofPAMPsorDAMPs canoccur viaoneofthecytoplasmicpatternrecognitionreceptors oftheNOD-likereceptor (NLR)orAIM2-likereceptor(ALR)(alsoknownasPyhinproteins)family.Theseinclude,butmay notbelimitedto,NLRP1,NLRP3,NLRC4,AIM2,andIFI16[4].Followingactivation,theNLRor ALRinteractswiththe adaptorproteinapoptosis-associatedspeck-likeproteincontaininga CARD (ASC), which then recruits caspase-1 into the complex. Under certain conditions, caspase-8isalsorecruitedintotheinflammasome complex[5,6].Activationofcaspase-1in theinflammasome results intheproteolytic cleavageand activationoftwo important proin-flammatory cytokines,IL-1b andIL-18. Furthermore,inflammasomeactivation results inthe cleavageandactivationofgasderminD,which inducesaproinflammatory formofcelldeath knownaspyroptosis[7,8].Caspase-11inmice(theorthologofhumancaspase-4/5) isalso involvedinNLRP3-mediatedinflammasome activationinresponsetocertain Gram-negative bacterial infections[5,9–11].NLRP3 inflammasome activation dependent oncaspase-11 is termed the noncanonical NLRP3 inflammasome and is initiated by cytoplasmic LPS that activates caspase-11,which in turn activatesgasdermin D andfacilitates NLRP3-mediated activationofcaspase-1 [7–9].Importantly,caspase-11caninducepyroptosis,butnot IL-1b maturation,independentlyofcaspase-1.[12].Someinflammasomes,suchasNLRP1b and NAIP/NLRC4,aredirectly activated bybacterialPAMPs[13–15].Otherinflammasomes,like NLRP3and,undercertainconditions,AIM2require aprimingstepwheretheirexpressionis increasedthroughactivationofothersignalingpathwayssuchasToll-likereceptor(TLR)ortypeI interferon(IFN-//b)signaling.ThisinturnactivatestheNF-kB,IRF-1,orSTAT1/2transcription factorstoincreasetheexpressionofNLRP3andAIM2aswellasguanylate-bindingproteins (GBPs),caspase-11,pro-IL-1b,andpro-IL-18[9,16–18].

Trends

Distinctinflammasomes can be acti-vatedduringsexuallytransmitted infec-tions (STIs), often in discrete cell compartments.Thecrosstalkbetween multipleinflammasomesensorsis cen-traltomaintainingtissuehomeostasis. Contrarytopopularbelief,in flamma-some activation can generate detri-mental inflammatory responses duringSTIsinvivo. Theoutcomeof inflammasomeactivityisgovernedby thecomplexhost–pathogeninteraction intheinfectedtissue.

Inflammasomeeffectormoleculescan yield disparate functional conse-quencesindifferenttissues, suggest-ing the need toemploy appropriate infectionmodels.

Ligandinternalizationisnota neces-saryprerequisiteforcytosolicNLRP3 activation. Engagement of certain plasmamembrane-localizedreceptors bysexuallytransmittedpathogensor theirligands elicitsboth primingand activation signals for the NLRP3 inflammasome.

1

DepartmentofBiology,Missouri StateUniversity,Springfield,MO 65809,USA

2

InfectiousDiseasesandImmunity, ImperialCollegeLondon,LondonW12 0NN,UK

*Correspondence:

paras.anand@imperial.ac.uk

STIsareamajorhealthproblemworldwide.Despiteextensiveeffortsandrecentawareness,only limitedsuccess has beenachieved indefying STIs. The World Health Organization(WHO) estimatesthatmorethan1millionnewSTIsoccureverydayglobally.IntheUSAalone,thereare 110milliontotalSTIsand20millionnewinfectionsareacquiredeachyear,withyoungpeople beingparticularlyatriskandcostingtensofbillionsofdollarsintreatment[19,20].Althoughexact statisticsareunavailable,itisbelievedthatthereisasignificantlyhigherburdeninless-developed countrieswhere thesocialstigma associatedwith STIsresults inunderreporting. In flamma-someshaveemergedascriticalhubsofinnateimmunityininfectiousdiseases,yetonlyalimited number of studies have investigated the impact of inflammasome signaling in STIs. It is becoming increasingly clear that innate immunity and inflammasomes mediate important functionsinthegenitaltract(Box1).Table1liststheinflammasomereceptorsandtheirknown activationsignalsduringSTIs.However,lackofappropriatemodelsthatemployvaginalorother relevantinvivoinfectionrouteslimitsourunderstandingoftheimportanceofinflammasomes duringSTIs.Furthermore, severalpathogensthatcause STIs arerestricted to humansand thedevelopmentofvalidatedsurrogatesornewinfectionmodelsisneeded.Belowwereview thecurrentstateofunderstandingofinflammasomesignalingduringSTIswithemphasisonthe involvementoftheinflammasomeinanimalmodelsorclinicalsamples.

MechanismsofInflammasomeActivationinSTIs

Understanding the mechanism by which pathogens activate the inflammasome not only enhances ourbasic understanding of the mechanisms ofdisease butalso provides useful insightintopotentialtherapeuticstrategies.Candidaalbicansisthemajorfungalspeciesknown tocauseSTI.C.albicansisgenerallyacommensalorganismbutmaycausesuperficialmucosal diseases,oropharyngealcandidiasis(thrush),orvulvovaginalcandidiasis(VVC)in immunocom-promisedindividuals. Studiesestimatethat 75%ofhealthy women arealsoatrisk ofVVC.

Box1.InnateImmunityintheGenitalTract

Thereproductivesystem,ofbothmalesandfemales,isintegraltotheinnermucosalliningofthehumanbody.Similarto themucosaltissuesoftheintestineandlungs,thereproductivetractisabletomountcomprehensiveimmuneresponses topathogens.Inadditiontoinvadingmicrobes,thefemalelowergenitaltractalsocontainsauniquemicroflora.Thus,the cervicalandvaginalepithelialcellsareinfrequentcontactwithmicroorganismswhilealsooccasionallyfacingantigenic andinflammatorystimuliduringintercourse[100].Therefore,thegenitaltractisfortifiedbybothinnateandadaptive immuneresponses.However,inkeepingwithitsparamountroleinreproduction,distinctadaptationshaveevolved. Thefemalegenitalmucosacomprisesasinglelayeroftightlypackedcolumnarepithelialcellsintheupperreproductive tract.Bycontrast,thelowerreproductivetractismadeofmultiplelayersofstratifiedsquamousepithelium.Theouter epitheliallayerisfurthercoatedontheluminalsidebyathickmucuslayercomprisingglycosylatedproteinsknownas mucins.Together,epithelialcellsandthemucuslayerformbothphysicalandimmunologicalbarrierstopreventinfection andtransmissionofSTIs;nonetheless,invasiondoesoccur.Oncethesebarriersarebreachedbypathogens,epithelial cellsareinfectedfollowedbytheactivationofmacrophagesandDCs.Thesetissue-residentmacrophagesandDCs endeavortoeliminatetheinfectiousagentbyphagocytosiswhileaugmentingcytokineandchemokineproductionto expandcellularrecruitment.Moreover,activatedDCsservetobridgeinnateandadaptiveimmunitybymigratinginto draininglymphnodeswheretheyprimeantigen-specificTcellsandinitiateBcellresponses[100–103].

Inadditiontotheexpressionofinflammasomesensors(whicharediscussedindetailbelow),severalPRRsareexpressed intheepithelialcellsofthereproductivetract.TheNLRsNOD1andNOD2andtheiradaptorproteinRIP2areexpressedin thehumanendometrium[104,105].Inaddition,TLRsincludingTLR1,2,3,and6arefoundinthegenitalmucosal epithelium[101].SomeoftheTLRshaveauniquesite-specificexpressionpatterninthereproductivetracttissue[105– 110].Forexample,TLR4ismainlyexpressedintheuppergenitaltractandtheexpressiongraduallydecreasesinthe lowergenitaltract[105,108,110].IthasbeensuggestedthatthisgradientofTLRdistributionislikelytoreflectthe immunologictoleranceofthelowergenitaltracttocommensalorganismswhile maintainingafirmintolerance to commensalsandpathogensintheuppergenitaltract,whichisimportantforreproduction.Thus,commensalflora, butalsoreproductivehormonesandlocallyavailablecues,shapetheimmuneresponsesanddifferentiationofcellsinthe upperandlowergenitaltract.Theroleofthesefactorsaswellasnon-inflammasomePRRsinthereproductivesystemare discussedindepthelsewhere[101,103,111,112].However,manynon-inflammasomePRRshaveessentialrolesin priminginflammasomes,asdiscussedaboveandthusa discussionoftheirimportancerelativetoinflammasome activationduringSTIsisincluded.

Candidasp.arerecognizedbymultiplepatternrecognitionreceptors(PRRs),buttheDectin-1/ Sykpathwayinparticular has emergedas animportantcomponentof thehostarsenalfor Candidarecognitionandmodulatesvariousimmunefunctions[6,21–25].DeficiencyinDectin-1 islinkedtothedevelopmentofmucocutaneousinfectionsinhumansandhighlightstheessential natureofthispathway[26].NLRP3istheprimaryinflammasomeactivatedbyC.albicansinvitro andinvivo.EngagementofDectin-1andthedownstreamadaptorSykprovidesthenecessary primingandactivationsignalsforthecanonicalNLRP3inflammasome[24].Bycontrast,certain C.albicansstrainstriggerthenoncanonicalcaspase-8inflammasomeindendriticcells(DCs) throughtheassemblyofaCARD9–Bcl-10–MALT1complex[6].Intriguingly,thisnoncanonical pathwayofinflammasomeactivationistriggeredextracellularlybyDectin-1ligationandCandida internalizationisnotnecessary[6].Overall,thesestudiessuggestredundancyinpathwaysfor inflammasomeassembly through thesame upstream sensor.In addition, Candidadisplays remarkablemorphologicalplasticitybyswitchingfromtheyeasttoafilamentoushyphalform,a keyfeatureenablingadherenceandtissueinvasionatmucosalsurfaces.Thisalterationresultsin NLRP3activationbyexposingtheDectin-1ligand,b-glucan,whichisnormallyshieldedfrom recognitionbymannoproteins.Accordingly,boththeyeastformandmutantstrainslackingEgf1, aprincipalregulatoroffilamentation,areincompetentinNLRP3activation[24,27,28].Highly polarizedhyphaemayalsoinflictphysicaldamageonthehostcellviaruptureofphagosomaland cellmembranes,therebygeneratingDAMPsforNLRP3activation.However,thesefindingshave beenchallengedbythediscoveryofmutantstrainsthatfailtoactivateNLRP3yetassemble normalhyphalfilaments[29,30],therebysignifyingtheinvolvementofadditional,unrecognized microbialfactorsinNLRP3activation.

AnotherimportantdeterminantofCandidapathogenesis,andinflammasomeactivation,isthe extracellularproteolyticactivityproducedbyafamilyoftensecretedaspartylproteinases(Saps). Besidestheir plausibleroleintheevasionofhostimmunity,Sapsalso enablethefungusto adhereandinvadehosttissues.FamilymemberSap2activatestheNLRP3inflammasomein vitro andin vivo,where vaginal inoculation of the purified full-length Sap2 resulted in local neutrophil influx and IL-1b accumulation in the vaginal fluid [31,32]. These measurements decreasedontreatmentwith anti-Sap2antibodyorinfectionwith mutantCandidaDSap1–3 Table1.LigandsofIn_flammasomeSensorsinSTIs.

PRR STIPAMP/DAMP Pathogen Refs

NLRP3 Secretedaspartylproteinases, ROS,K+efflux,andothercellular damagesignals HPV [87] HSV [58,64] HIV [73,80,93–96] Chlamydiatrachomatis, Chlamydiamuridarum [46,47,81] Neisseriagonorrhoeae [37] Treponemapallidum [39,41] Candidaalbicans [6,24,27,28,31,32,36,68] NLRC4 Flagellin,T3SScomponents, orunknownfungalligands

C.albicans [36,68]

AIM2 CytoplasmicdsDNA HPV [92]

HSV [65]

Chlamydia [97]

IFI16 NucleardsDNA HSV [64,98,99]

[32].WhetherSap2'senzymaticactivityisalsorequiredtotriggertheinflammasomeisunclear, ascontradictoryresultsexistinvitroandinvivo.IthasbeensuggestedthatSap2activityserves onlytohydrolyzethemucosallayerforefficientCandidainvasioninvivo[31].Anydirectrolefor Sap2enzymaticactivityininflammasomeactivationremainstobedetermined.Inaddition,these studiesneedfurthervalidation, asthe yeastform ofCandidaproduces Sap2 butisnot an effectiveinflammasomeactivator[33–36].Hypha-associatedmembersSap4,5,and6arealso expressedrobustlyduringVVC[33–36].Incontrasttothepreviousstudy,intravaginalchallenge withDSap5mutantbutnotthetripleknockoutDSap1–3resultedinreducedIL-1bsecretionand polymorphonuclearleukocyte(PMN)flux[36].Thesedifferencesinresultscouldbeduetothe differentgeneticbackgroundofanimalsusedand/ordifferencesinthepreparationanddoseof Candidainfection.Althoughconflicting,thesestudiesproposeessentialfunctionsforSapsin inflammasome-mediatedimmunopathogenesisofVVC.However,moreevidenceisneededto identify the important Saps, which should enable improved understanding of the complex interplaybetweenhostandpathogenatthevaginalinterface.

Neisseriagonorrhoeae,thecausativeagentofgonorrhea,resultsinacuteurethritisandcervicitis inmalesandfemales,respectively.Neisserialipooligosaccharide(LOS),amodifiedformofLPS, hasbeensuggestedtoactivatetheNLRP3inflammasomeandIL-1bsecretionina cathepsin-B-dependentmanner[37](Figure1).However,invasionofthehostcytosolbyN.gonorrhoeae,ora roleforadditionalgonococcalantigensaspossibleNLRP3activators,cannotbecompletely excluded.SimilartogonorrheaLOS,TreponemapallidumTpF1,abacterioferritinandamajor virulencefactorofthisspirochete,alsoactivatestheNLRP3inflammasome.T.pallidumcauses syphilis,asexuallytransmittedchronicinflammatorydisorderthatischaracterizedby mucocu-taneousrashwithenhancedvascularinflammationandangiogenesis[38].TpF1elicitspro-IL-1b productionbymonocytes,thusprimingtheinflammasome,andtriggersthesecretionofATP,a knownactivatorofNLRP3[39].Thus,TpF1deliversbothofthesignalsrequiredforin flamma-someactivation(Figure1).Arelatedtreponeme,Treponemadenticola,implicatedinhuman periodontaldisease,alsoactivatestheNLRP3inflammasome.Althoughrare,T.denticolamay cause vaginitis andaffect preterm delivery[40].Interaction ofTd92, aT. denticola surface protein,withmonocytemembraneintegrin/5b1promptedATPreleaseandK+_{effluxpreceding}

NLRP3 activation[41].Contrary to the requisite cytosolicpresence ofmicrobial ligands for NLRP3trigger,activationbyTd92isindependentofitsinternalization,anddirectbindingof recombinantTd92totheglycosylatedb1subunitofintegrinismandatory forinflammasome activation(Figure1).

PositiveandNegativeConsequencesofNLRP3InflammasomeActivation Duringinfection,micelackingNlrp3displayenhancedsusceptibilitytovariousinfectiousagents. Bycontrast,gain-of-functionmutationsintheNlrp3geneleadtoinflammatorydiseasestogether known as cryopyrin-associated periodic syndromes (CAPSs). Thus, the role ofthe NLRP3 inflammasomeishighlycontextdependent.Inthecaseofsexuallytransmitteddiseases,the importanceofinflammasome activationinvivoissimilarly contextdependentandtheuse of appropriatemodelsforthestudyofSTIsisneeded.Chlamydiatrachomatisinfectionresultsin scarringoftheovariesandFallopiantubesandisconsideredtheleadingcauseoftubalinfertility. Evenwhenprocreationisachieved,infectionmayresultinectopicpregnancy,pretermbirth,and verticaltransmissiontothedevelopingfetus[42,43].Muchofourunderstandingofthe patho-genesisofandimmuneresponsestoC.trachomatishasdevelopedthroughequivalentmouse modelsofChlamydia muridarum,wherethegenitaltractpathology iscomparableto thatin humans[44,45].Chlamydiainfection activatestheNLRP3inflammasome (Figure1). Surpris-ingly,caspase-1deficiencyresultedinsimilarC.muridarumgrowthintheintravaginallyinfected mousemodel,andthelevelsofshedliveorganismswerecomparableatdays17and21 post-infection[46].Intermsofgenitaltractpathology,abolitionofcaspase-1orIL-1receptor(IL-1R) signalingreducedinflammatorydamageintheoviducts[46,47].Ofnote,thepathologydoesnot

appeartobeaffectedinNlrp3-deficientmice[47],suggestingthatotherinflammasomesmaybe involved.Onehypothesispostulatesthatinflammasome-mediatedpathologyisduetorapid IL-1b-mediatedPMNinflux.Accordingly,abrogationofIL-1Rsignalingdiminishedpathologyinthe genitaltissueandcorrelatedwithreducedPMNrecruitment[47].Itisintriguingtoconsiderthat theinfiltratedneutrophilscanalsosupplementactiveIL-1bthroughcleavageoftheprecursor formbyneutrophilproteasesandthusexacerbateoviductpathologyduringintravaginal chal-lenge.Overall,thesestudiessuggestthattheinflammasomepersedoesnotaffectintravaginal Chlamydiacolonizationbutaugmentsdetrimentalpathologyintheuppergenitaltractduringthe innatephaseofinfection.

InflammasomeactivationalsoappearsdetrimentalduringVVC.Experimentsinwild-type(WT) miceimplicatedNLRP3activityasthesourceofincreasedPMNrecruitment,increased pro-ductionofalarmins,andelevatedlevelsofIL-1binvaginallavagefluidduringVVC.Consequently, infectioninNlrp3-deficientmiceortreatmentofWTmicewith theNLRP3 inhibitorglyburide reducedC.albicansvaginitiswithoutaffectingmicrobialcolonization [36].

IL-1β transcripon NOD1 RIP2 NF-κB Chlamydia _Neisseria Treponema IL-1β Neutrophil flux NLRP3 inflammasome AIM2 Cathepsin B Lipooligosaccharide (LOS) TpF1 Td92 Integrin β1 subunit (glycosylated) TLRs RBs associated with inclusion membrane Pyroptosis K+ efflux IL-1β

MyD88

Figure1.InflammasomeActivationMechanismsduringBacterialSexuallyTransmittedInfections(STIs).ActivationoftheNLRP3inflammasomeoccurs throughtheligationofvariouspatternrecognitionreceptors(PRRs).Chlamydiaistakenupinavacuoleknownasaninclusion,whereactivityoftheChlamydiatypeIII secretionsystemtriggerstheNLRP3inflammasome.Neisseriareleasesmembranelipooligosaccharide,whichhasbeensuggestedtoactivatetheNLRP3inflammasome throughacathepsin-B-dependentpathway.TreponemasurfaceproteinsTpF1andTd92activatetheNLRP3inflammasomedependentonK+_efflux.

Sinceour understandingofinflammasomesandtheiractivationmechanismsisincompleteinbacterialSTIs,othermechanismsmayalsobeinvolved.Itisalsounclearwhetherthe samemechanismsareimportantinvivo.

Contrary to results described during genital infection, inflammasome activation during C. pneumoniaelunginfectioniscriticalforbotheliminationofthepathogenandprotectionfrom lung fibrosis [48,49]. Independent reports suggest that C. pneumoniae may also activate cytosolicsensors distinctfrom thoseactivated by C.muridarum orC.trachomatis [50,51]. Similarly,micelackingcomponentsoftheNLRP3inflammasomeandupstreamfungal recogni-tionreceptorsaresusceptibletodisseminatedcandidiasis[24,28,52].Inaddition,deficiencyin IL-1b,orlossofIL-1Rsignaling,promotessusceptibilityduetotheimpactofthispathwayon granulocyteinfluxandsuperoxideproduction[53].Finally,administrationofrecombinantIL-18 protectsagainstinfection,andthisoccursthroughtherestorationoftype1immunity[54–56]. Overall,theresultsobtainedfromvaginalinfectionwitheitherChlamydiaorCandidasuggestthat systemicinfectionorinfectionatdistalsiteswithsimilarpathogenscannotbeusedtoinferthe roles of the inflammasome in the genital tract. Instead, appropriate pathogen strains and infectionroutesareessentialtoelucidateaclearpictureofthefunctionofinflammasomesin STIs.

VaginalinfectioninamousemodelofHSV-2demonstratedthatIl18 / _{micediedsoonerthan}

WTmiceandviraltiterswerehigher inIl18 / _{miceonday3 afterinfection [57].However,}

followingsecondarychallengewithHSV-2inamemoryrecallexperiment,Il18 / micewerefully protected,suggestingthatIL-18isnot requiredforthedevelopmentofappropriateimmune memory [57]. Unfortunately, little else has been reported regarding the importance of the inflammasome for HSV-2. Increased inflammasome activation was also associated with increased protectionfrom HSV-1 infection but inan ocular infection model [58]. A second reportshowedthatNlrp3 / micearemoresusceptibletoHSV-1infectioninanocularinfection model, butthis was independent of inflammasome activation [59]. Indeed,the latter study reportedthatIL-1blevelswerehigherinNlrp3 / micefollowingHSV-1infectionandnotedthat NLRP3waslocalizedtothenucleus.TheauthorshypothesizedthatNLRP3hasanin flamma-some-independentfunctioninimmuneregulationthathelpstosuppressdeleteriousin flamma-tion in the ocular model of HSV-1 infection [59]. NLRP3 reportedly has inflammasome-independent roles [60–63]butmoreresearch is neededto fully understand these potential functions. Furthermore, inflammasome activation may be important in ocular models, but whetherthisactivitywill berecapitulatedduringsexual transmissionisunclear asactivation of the inflammasome in human keratinocytes did not affect HSV-1 replication [64,65] and inflammasomeactivationinvaginalmodelsofHSV-1havenotbeenreported.

InterplaybetweenDistinctInflammasomes

Infectionwithapathogencanconcurrentlyengagemultipleinflammasomesensors[66,67].A recentfindingdemonstratedthecomplexinterplayofNLRP3andNLRC4duringC.albicans infection in the vaginal tissue [68].The expression ofboth of the inflammasome-activating sensorswasaugmentedduringVVC;however,NLRP3expressionpeakedearlierinthevaginal tissuethantheactivephosphorylatedformofNLRC4(pNLRC4),whichincreasedevenfurther underNlrp3-deficientconditions[68].FurthermechanisticstudiesassociatedNLRC4activation, throughanIL-22-andPKCd-mediatedpathway, withdampening exaggeratedinflammation throughtheproductionofIL-1receptorantagonist(IL-Ra)(Figure2,KeyFigure).Intriguingly, PKCdiscriticaldownstreamofseveralSyk-coupledCLRswith rolesinantifungalimmunity, includingDectin-1,Dectin-2,andMincle[69].Accordingly,IL-22administrationinvivo damp-enedcytotoxicdamageinthevaginasofinfectedmice.Conversely,treatmentwithaninhibitorof PKCddecreasedpNLRC4expressionandenhancedNLRP3-associatedvaginitis[68].These studiessuggestthatNLRC4negativelyregulatesNLRP3activity(Figure2).Additionally,they suggestthatsustainedproductionofIL-1RabyNLRC4dampensNLRP3-mediatedin flamma-tionduringVVC.AlthoughVVCandoropharyngealcandidiasisinvolvesimilarinflammasomes, theiractivationseemstoproduceoppositeresults.Duringoropharyngealcandidiasis,akinto VVC,bothNLRP3andNLRC4inflammasomesregulateIL-1bproduction.However,epithelial

Nlrc4 deficiency, more than Nlrp3, resulted in significantly enhanced Candida buccal load throughout the 21-day infection period and increased inflammatory cell recruitment inthe tongueepitheliumdespitethepresenceoferosivelesionsandhyphae[70](Figure2).Notably, Nlrp3deficiencyresultedinonlyslightlyelevatedoralcolonizationandgrossclinicalscore[70]. Thus,althoughbothoralandvaginalinfectionsareclinicalmanifestationsofmucosalinfection, inflammasomeactivationandenhancedPMNinfiltrationleadstocontrastingresultsatthetwo sites.

Inflammasome-InducedPyroptosisinSTIs

HIVisoneofthemostconcerningworldwidepandemics,withapproximately37millionpeople infectedwith avirus thatcauseslifelongmorbidity andeventualmortality [71].HIV infection resultsintheactivationofboththeNLRP3andIFI16inflammasomes(Figure3).Inmonocytes, theNLRP3inflammasomeisactivatedinresponsetoHIVinfectionasaresultofTLR8-mediated

KeyFigure

NLRP3

and

NLRC4

Inflammasomes

Mediate

Distinct

Host

Immune

Responses

during

Candida

albicans

Vaginal

and

Oral

Infection

NLRP3

NLRC4

IL-Ra

Vulvovaginal

NLRC4

NLRP3

Oropharyngeal

Fungal colonizaon Neutrophil flux

Fungal colonizaon

(A) (B)

Figure2.(A)Duringvulvovaginalcandidiasis,NLRP3activationleadstoIL-1bandIL-18production.NLRP3activityrestrainsfungalcolonizationwhileincreasing polymorphonuclearleukocyte(PMN)fluxtothevaginaltissue,throughIL-1bproduction,leadingtovaginitis.IL-18,bycontrast,increasestheamountofavailableIL-22by decreasingthelevelsofIL-22-bindingprotein(IL-22bp).Furthermore,IL-22leadstophosphorylation,andthusactivation,ofepithelialNLRC4andresultsinincreased productionofIL-1receptorantagonist(IL-1Ra)therebylimitingNLRP3activity.(B)Duringoropharyngealcandidiasis(thrush),epithelialNLRC4,morethanNLRP3, regulatesfungalcolonizationandPMNfluxinthebuccalcavity.NLRP3activity,inbothepithelialandmyeloidcells,servescriticalrolesinprotectionagainstdisseminated candidiasis.

primingandreactiveoxygenspecies(ROS)production[72–74].However,IFI16appearstobe thepredominantinflammasomeactivatedinCD4+TcellsandmayleadtoAIDSprogression[75]. Activationofinflammasomesresultsinaprogrammedcelldeathtermedpyroptosis.Pyroptosis oftheinfectedcellsresultsindestructionofthepathogenreplicativeniche.However,becauseof theinherentlyinflammatorynatureofthisformofcelldeath,itmaypromotetissuedamage.IFI16 activationandpyroptosisinresponsetoHIVinfectionresultsinthedepletionofrestingCD4+T cells,whichfurtherexacerbatesimmunodeficiency[75–78].DirectinfectionofCD4+_Tcellsdoes

notappeartoresultinpyroptosis.Instead,cell-to-celltransmissionthoughthevirussynapse resultsinabortiveinfectionofrestingCD4+_{Tcellsandtheaccumulationofreverse-transcribed}

HIV genomes in the cell (Figure 3). These DNA molecules are then sensed by the IFI16 inflammasomeresultinginpyroptosis[79].Interestingly, cell-to-cellspreadoccursmostef fi-cientlyinthelymph nodeenvironmentandnot inblood-circulating CD4+Tcells[76].Inthe kidneys, HIV-associated nephropathy results from the lossof podocytes. Recent research suggests that NLRP3 inflammasome activation in the kidneys during HIV infection causes pyroptoticcelldeathofpodocytesandcontributestokidneydamage(Figure3)[80]. Further-more,inhibitionofROSortheNLRP3inflammasomeresultedinimprovedpodocytesurvivalin theTg26transgenicmousemodelofHIVinfection[80].Overall,inflammasomeactivationbyHIV appearstodomoreharmthan good,andit willbeofinteresttodeterminethe therapeutic potentialofinflammasomeinhibition.

InductionofpyroptosisduringChlamydia infectioncauses injurytotheupper genitaltract resultingindegenerationofoviductepithelia,swollenoviducts,andwidespreadnecrosisofthe endometrium[81].Inflammasomeassemblywasdemonstratedtoinducepyroptosisin anti-gen-presenting DCs in an IL-10-dependent manner [81]. Consequently, IL-10 abolition reduced inflammasome activation and limited necrosis in the endometrium. Additionally, Chlamydia-infectedIl10-deficient micehad100%fertilitybut Chlamydia-infected WTmice suffered significant fertility impairment.However, mechanistic pathways coupling IL-10 to NLRP3inDCsremainunclear.Furthermore,theseresultsappearcontradictorytothe emerg-ingroleofIL-10asanegativeregulatorofinflammasomesignaling[82,83].Nonetheless,the inflammasome-dependent pathology encountered by the host seems to be restricted to

CD4+ _{T cells} Parally reverse transcribed DNA ROS, vRNA, K+ _efflux, gp120 NLRP3 Inflammasome Pyroptosis Pyroptosis IFI16 Inflammasome

Pro-IL-1β IL-1β Pro-IL-1β

IL-18

IL-1β

IL-18

Pro-IL-18 Pro-IL-18

Macrophages and kidney podocytes

Figure3.PyroptosisduringHIVInfectionContributestoDisease.DuringHIVinfection,differentinflammasomesare activatedindifferentcelltypesandinresponsetodifferentstimuli.However,inallcelltypesinflammasomeactivationcauses maturationof IL-1bandtheinflammatory formofcell death,pyroptosis.Pyroptosisin CD4+

Tcells contributesto immunodeficiencybyfurtherdepletingCD4+

Tcellnumbers,whereaspyroptosisofpodocytesinthekidneyresultsin HIV-associatednephropathy.

primaryinfection,as pathologyencountered during recurrentinfections is propagated by adaptiveimmunity[46].

PolymorphismorExpressionChangesinInflammasome-CodingGenes The role of inflammasomes during VVC is also corroborated by studies inhumans where polymorphisminthegeneencodingNlrp3isassociatedwithincreasedincidenceofrecurrent VVC(RVVC),whichischaracterizedbyatleastthreeepisodesofinfectionperyear[84,85].One studymeasured inflammasome-dependent cytokine productionatthemucosal surfaceand observedenhancedIL-1blevelsinthevaginalfluidofRVVCpatientscomparedwithhealthy controls.Intriguingly,RVVCpatientsbearingtheriskalleledemonstratedevenhigherlevelsof IL-1bproduction[84].Inagreement,IL-1RalevelswerelowerinrecurrentVVCpatients. Addition-ally,IL-18levelswereunalteredinthevaginalfluidofpatientsbearingtheriskallele[84].These studiesthusarguethatgeneticvariationsintheNlrp3genemayinfluencetheprogressionofVVC andidentifyIL-1basatherapeutictargetinthemanagementofRVVC.

Severaltargetedgenetic associationstudies have foundthat certain allelesof IL-1b, IL-18, NLRP3,andNLRP1 areassociatedwith resistanceorsusceptibility tomoresevere human papillomavirus(HPV)outcomessuchascervicalcancer[86,87].Twootherstudiesreported downregulationoftheexpressionofIL-1bandotherinflammasome-relatedgenesinpatients whoareHPVinfectedorhavedevelopedcervicalcancer[88,89].Furthermore,elevatedIFI16 andAIM2expressionisassociatedwithHPVinfectionandHPV-associatedcancerdevelopment [88,90,91].AIM2mayrespondtoHPVinfectionofhumankeratinocytesbydetectingviralDNAin thecytoplasm.However,thisfindingwasnotbasedonanaturalinfection.Instead,viralgenomic DNAwastransfectedintokeratinocytes;thus,theroleofAIM2duringnaturalHPVinfectionis unknown[92].Finally,inflammasomeactivationduringHIVinfectionresultsinnegative immu-nopathologiceffectsasdescribedabove.Itisthusinterestingtonotethatpolymorphismsin NLRP3 and IL-1b are foundmore commonly inHIV-positive individualsthan in uninfected individuals [93,94]. Although the functional consequences of these polymorphisms are unknown, it willbe ofinterest to determinewhether they enhanceor inhibitinflammasome activation,potentiallyfacilitating pyroptoticcell death andleadingto diseaseprogressionor resultinginimpairedimmunitywithincreaseddiseasesusceptibility.

ConcludingRemarks

ThecontributionsofinflammasomesduringSTIsareonlybeginningtobeunderstood.Recent studies have depicted the significance of inflammasomes in vitro in response to sexually transmittedpathogens.However,fewinvivostudieshavebeenconductedandthisremains challengingbecauseofthetopologyofinfectionsiteandlackofappropriateanimalmodelsthat faithfullyrecapitulatetheinfection.Nevertheless,afewwell-controlledstudiesemploying intra-vaginalchallengemodelsofCandidaandChlamydiahavedepicteddetrimentalrolesof inflam-masomesinthegenitaltract,incontrasttoresultsobservedinvitroandinsystemicmodelsof infection.Thesesignificantdifferenceshighlighttheimportanceofperformingdiscovery-based experimentsusingspecificmodelsinsteadofdrawingconclusionssolelyfromrelatedstudies. Thesestudiesalsoillustratedactivationofdistinctinflammasomesinhematopoieticandstromal compartments,therebyhighlightingtheneedtodeveloptissue-specificmodelsandconditional knockoutsthataccuratelymeasurethecontributionofeachinflammasometype.Nevertheless, whetherthedetrimentalroleofinflammasomesinthegenitaltractextendstootherSTIsremains tobeexamined.Regardless oftheinfection,improved modelsofSTIs areneededtobetter understandtheroleofinflammasomesinSTIs.Especially,thereisaneedforthedevelopmentof modelsthatrecapitulatetheinitialsexualtransmissionoftheinfectionandallowexaminationof theinitialimmuneresponsesthatareinvolvedinfacilitatingorpreventingdiseasetransmission. ThereislittledoubtthatinflammasomesareactivatedduringSTIs.Themajorquestioniswhich inflammasometypesareimportantintheskinandmucosaltissues?Also,whataretheprecise

OutstandingQuestions

Whichinflammasometypesarecritical inthegenitaltract?Whatarethe molec-ularpathways thatactivate inflamma-somes in the genital mucosa? We have increased understanding of inflammasomesignalingin hematopoi-eticcellsbutourknowledgeofimmune receptorsandinflammasomeactivation mechanismsinthemucosalepithelium isratherlimited.

Whataretherolesofthenoncanonical NLRP3-dependent and NLRP3-inde-pendent inflammasomes? Recent reportshavesuggestedkeyrolesfor inflammasomesotherthanNLRP3 dur-ingawidevarietyofinfections. How-ever, their roles in STIs remain ambiguous.Forexample,activationof theAIM2 inflammasomeby Candida sp.wasrecentlydescribedin macro-phages.DoesAIM2alsoinfluence pro-gressionofvulvovaginalcandidiasis?

Whatistheroleofautophagyduring STIs?Both autophagy ofpathogens (xenophagy)andautophagic degrada-tionofinflammasomesandprecursor IL-1b by macroautophagy are now considered important mechanisms contributing to infection outcome. However, these mechanisms have notbeencharacterizedinSTIs. Whichhostpathwaysfunctionas rheo-statsbetweenpathogeneliminationand exaggeratedinflammatoryresponses? Sinceinflammasomeshaveboth bene-ficialanddetrimentalroles,thereisa needtoidentifytargetsthatcanspeci fi-callyactivateordampeninflammasome activity.Knowledgeinthisareacanhelp us develop appropriate therapeutic interventions.

Whatarethefunctionalconsequences ofinflammasomegenepolymorphisms inthehumanpopulation?Numerous studiesreportthatspecificallelesof genesencodinginflammasome com-ponentsareassociatedwithincreased propensitytoinfectionorsevere dis-ease,butthefunctionalconsequences oftheseallelesandhowthey predis-posepatientstodiseaseareunknown.

pathwaysthatpaireachSTItoaspecificinflammasome?Theseandotherquestionsremain enigmatic(see OutstandingQuestions), butby understanding the nature ofprotection and damagemediatedbyinflammasomesthesestudieswillfurtheradvanceourknowledgeandare essentialforreproductivehealth.Finally,animprovedgraspoftheroleofinflammasomesinthe genitaltractmaytranslateintonewtherapeuticopportunitiestoreducemorbidityandmortality duetoSTIs.

Acknowledgments

Theauthorsapologizetonumerousinvestigatorswhoseworkcouldnotbecitedduetospacelimitations.C.L.issupported bytheDepartmentofBiology,MissouriStateUniversity.WorkinthelaboratoryofP.K.A.issupportedbyfundsfromthe WellcomeTrust(108248/Z/15/Z)andtheRoyalSociety(RG150535)andcorefundsfromImperialCollegeLondon.

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