RoleofHemichannelsinCNS Inflammationandthe InflammasomePathway
YuriKim*,JoanneO.Davidson†,KatherineC.Gunn† , AnthonyR.Phillips
{,ColinR.Green*,AlistairJ.Gunn†,1
*DepartmentofOphthalmologyandNewZealandNationalEyeCentre,Auckland,NewZealand †DepartmentofPhysiology,FacultyofMedicalandHealthSciences,Auckland,NewZealand {SchoolofBiologicalSciences,UniversityofAuckland,Auckland,NewZealand 1Correspondingauthor:e-mailaddress:aj.gunn@auckland.ac.nz
1. Introduction
2. RoleofConnexinHemichannelsintheInflammatoryCascadeoftheCNS4
3. ConnexinHemichannelsandTheirRolesinResponsetoInjuryandInflammation12
3.1 LinkingConnexinHemichannelstotheInflammasomePathway
3.2 AnimalModelsShowTherapeuticBenefitsofConnexinHemichannelBlock19
Neurodegenerative,cardiovascular,andmetabolicdisorders,oncetriggered,sharea numberofcommonfeatures,includingsustainedinflammatorycellactivationandvasculardisruption.Thesesharedpathwaysareinducedindependentlyofanygeneticpredispositiontothediseaseorthepreciseexternalstimulus.Glialcellsrespondtoinjury withaninnateimmuneresponsethatincludesreleaseofproinflammatorycytokines andchemokines.Vascularendothelialcellsmayalsobeaffected,leadingtoopening oftheblood–brainbarrierthatfacilitatesinvasionbycirculatinginflammatorycells. Inflammationcantriggeracuteneuralinjuryfollowedbychronicinflammationthat playsakeyroleinneurodegenerativeconditions.Gapjunctionchannelsnormallyallow directcell-to-cellcommunication.Theyareformedbythedockingoftwo hemichannels,onecontributedbyeachoftheneighboringcells.Whiletheopening probabilityofthesechannelsistightlycontrolledunderrestingconditions, hemichannelscanopeninresponsetoinjuryorinflammatoryfactors,formingalarge,
relativelynonselectivemembranepore.Inthisreview,weconsidertheCNSimmune systemfromtheperspectivethatmodulatingconnexinhemichannelopeningcanpreventtissuedamagearisingfromexcessiveanduncontrolledinflammation.Wediscuss connexinchannelrolesinmicroglia,astrocytes,andendothelialcellsinbothacuteand chronicinflammatoryconditions,andinparticulardescribetheroleofconnexin hemichannelsintheinflammasomepathwaywheretheycontributetobothitsactivationanditsspreadtoneighboringcells.Finally,wedescribethebenefitsofhemichannel blockinanimalmodelsofbraininjury.
1.INTRODUCTION
Neurodegenerative,cardiovascular,andmetabolicdisorderscantriggeranumberofsharedprocesses,includingsustainedinflammatorycellactivationandvasculardisruption(withaccompanyingtissueischemia),thatin effectresemblepersistent,nonhealing“wounds.”Thesesharedprocesses occurregardlessofgeneticpredispositiontothediseaseortheinitialexternal stimulus(suchascirculatinginflammatorycytokinesoranischemicevent) (Green&Nicholson,2008).
Thecentral nervous system(CNS),whichconsistsofthebrainandthe spinalcord,hasacontinuoussurveillancemechanismtodetectandeliminate mediatorsofinfectionand/orinjury(Boulayetal.,2015).Evenintheirquiescentstate, microglial cellscontinuouslymonitortheirenvironment,asthe primaryresidentimmunecellsoftheCNS(Nimmerjahn,Kirchhoff,& Helmchen,2005). Infection orsterileinjury(eg,stroke)stimulatesmicroglia totransformto anactivatedphenotypeandtorecruitneighboringimmune cellsinanattempttocleartheimmuneprivilegedsiteofinjuryanddebris (Davalosetal.,2005).Theresponseofthemicrogliatoinjuryand/orinfectionis usually veryrapidandprecedesthatofothercellsoftheCNS (Kreutzberg,1996).Activatedmicroglialcellsareprimedtorelease proinflammatorycytokines and chemokinestotriggertheinnateimmune response—thehost’sfirstlineofdefenseagainstinjuryand/orinfection ( Janzer&Raff,1987).Activatedmicrogliacommunicatewithsurrounding CNScells, including astrocytesandpericytesthatprojecttheirend-feet aroundvesselstoformahighlyspecializedneurovascularunit(Bertossi etal., 1993; Mulligan&MacVicar,2004).Theneurovascularunit,together withendothelialcells, maintainstheintegrityoftheblood–brainbarrier (BBB)thatprotectsthebrainfrominfiltrationofforeignmoleculesand immunecells( Janzer&Raff,1987).
inflammasomecomplex(reviewedin Takeuchi&Akira,2010).Iftheinnate inflammatoryresponseis unsuccessfulinclearingthestimulus,theacquired immuneresponsepromotesinfiltrationofTandBcellsfromtheperipheral nervoussystem(Metcalf,Baxter,Nilaratanakul,&Griffin,2013),leadingto vascularleak and afterasufficientlyseverestimulus,neuronalcelldeath. Adaptiveimmunitycanrestoreandrepairtheinjuredtissueorleadto chronicinflammation(reviewedin Hagbergetal.,2015).
2.2MicrogliainAcuteInflammation
Microglialglialcellscandynamicallyswitchbetweensurveying(“resting”) andactivatedstates(reviewedin Kreutzberg,1996).Inits“resting”phenotype,the microglial cellhasaramifiedmorphologywithalowlevelofCx43 (Garg,MdSyed,&Kielian,2005).However,cuessuchasbraininjuryor infectionreadily activate microgliaandareassociatedwithupregulation ofCx43expression(Eugeninetal.,2001).Activatedmicrogliaalsoproliferateand migrate tothesiteofinjurywithinthefirst24hfromtheonset (Eugeninetal.,2001).Forexample, Staphylococcusaureus-derived PGN induces Cx43upregulationandgapjunctioncouplinginadditionto upregulationoftumornecrosisfactor(TNF)-α andinterleukin(IL)-1β (Gargetal.,2005).Microglialaccumulationatthesiteofinjurypeaksafter approximately2–4 d butinsomesettingscanpersistformanyweeks(Mcrae, Gilland,Bona,& Hagberg,1995).
2.3AstrocytesinAcuteInflammation
AstrocytesabundantlyexpressCx43underbasalconditions(Giaume& McCarthy,1996).However, astrocytesshowalteredCx43expressionin responsetoproinflammatory cytokines.Inmousecorticalastrocytecultures, treatmentwithacombinationofIL-1β andTNF-α for24h invitro reduced totalCx43expressiononthecellmembranescomparedtountreatedcontrols(Retamaletal.,2007).Thiscombinationofproinflammatorycytokines isrequired to reduceCx43expressionandcell–cellcoupling,asTNF-α or IL-1β aloneat24hhadnosignificanteffectcomparedtocontrol(Meme etal., 2006).
Lipopolysaccharide (LPS), amajorcomponentofthecellwallsofgramnegativebacteria(Bolon,Peng,Kidder,&Tyml,2008;Frost,Nystrom,& Lang,2002; Smolinski &Pestka,2003),canalsoreduceCx43expression, throughinductionof proinflammatorycytokines(TNF-α andIL-6). Short-termexposureofratcorticalastrocytestoLPS(1–6h)didnotaffect
Cx43expression,butalongerexposurefor10–72hwasassociatedwitha significantreductioninCx43viatheubiquitin–proteasomepathway (Liao,Jeng,Wang,Wang,&Wu,2013;Liao,Wang,Chen,Wang,& Wu,2010). Spinal cordastrocytesalsodisplayedatime-dependentreduction inCx43mRNA andproteininresponseto24and48hexposuretoTNF-α andIFN-γ (Zhang,Morioka,Nakashima-Hisaoka,&Nakata,2013).Strikingly,the combination ofTNF-α,andIFN-γ wasassociatedwithreduced Cx43mRNAexpressionasearlyas3-hpostincubation(Zhang,Morioka, etal., 2013). Similarlytobrainastrocytes,achangeinCx43expressionin thespinalcord wasattributedtoenhanceddegradationviatheubiquitin–proteasomepathway(Zhang,Morioka,Kitamura,Hisaoka-Nakashima,& Nakata,2015). It isimportanttoappreciate,however,thatthereisa distinctionbetweenreduced cell–cellcouplingandincreasedhemichannel openingundersuchconditions,aswillbecomeapparent.
Studiesexaminingtheoveralleffectofinjury invivo reportsignificant upregulationofCx43.Forexample,upregulationofCx43immunoreactivitywasobservedat1–2,4,8,and24hafterischemia–reperfusionretinal injury,andcolocalizedwithastrocytes,Mullercellsandvascularendothelial cells(Danesh-Meyeretal.,2012).Similarly,spinalcordinjurywasassociated with upregulation of astrocyticCx43expressionintheperitraumaticregions (Cronin,Anderson,Cook,Green,&Becker,2008;Theriault,Frankenstein, Hertzberg,& Nagy, 1997).Inlight-damagedretinaintherat,upregulation ofCx43in thechoroidhasbeenreportedtocolocalizewithnitrotyrosine,a markerofoxidativestress,andwithinflammatorycells(Guo,Tran,Green, Danesh-Meyer,& Acosta, 2014).
Undernormalconditions, in vitro arapidyetbalancedtranslationand internalizationofcellsurfaceCx43ismaintained,withahalf-lifeofapproximately1–2h(Laird,Puranam,&Revel,1991).Turnoverisdynamicand amenableto external cues.Theapparentdiscrepanciesintheexpressionof Cx43couldbeattributedtothereactivestatusoftheinflammatorycascade. Themajorproinflammatorycytokines,TNF-α andIL-1,aredetectedas earlyas1hafterinjury,beforeneuronaldeath(Liuetal.,1994;Wang etal., 1994).Clinicalstudieshavecorrelatedtheseverityofinjurywith increasedcytokinelevels inpostmortembraintissue(Griffinetal.,1994; Krupinski,Kumar,Kumar, &Kaluza,1996).AstrocytesareparticularlysensitivetoTNF-α, whichdirectlyelevatesintracellularCa2+ concentration andinducescelldepolarization(Koller,Thiem,&Siebler,1996;Koller, Trimborn,Von Giesen, Schroeter,&Arendt,2001).Additionally,IL-1β promotestherelease ofvascularendothelialgrowthfactorfromactivated
astrocytesthatcanenhancethepermeabilityoftheBBBandleakageof plasmaprotein-richfluid(Argawetal.,2012).Proinflammatorycytokines uncouplethe astrocytic gapjunctionnetworkbutenhancehemichannel activity(Memeetal.,2006;Retamaletal.,2007).InthebrainTNF-α andIL-1β have beenidentifiedasthemostpotentinhibitorsofastrocyte–astrocytecommunication(Memeetal.,2006).
Whileit is clearthatgapjunctionshelptomaintainneuronalhomeostasis undernormalconditions(Wallraffetal.,2006),aroleforgapjunctions ininflammation has beendebated,particularlyastowhetheruncoupled gapjunctionsaffecttheoverallviabilityoftheCNS.“Bystanderdeath” effectwasinitiallytermedby Freemanetal.(1993) todescribehow Ganciclovir-sensitivetumor cells couldalsoinduceapoptosisinacoculture ofGanciclovir-insensitivetumorcells(Freemanetal.,1993).However, Cx43gene knockout modelstudiesintheCNSprovidedanargument againstthe“bystander”deathinCNSinjurybydemonstratingthatlossof Cx43didnotconferprotectionandindeedexacerbatedthesizeoftheinjury (Nakase,Sohl,Theis,Willecke,&Naus,2004;Siushansian,Bechberger, Cechetto,Hachinski, & Naus,2001).Insupport,inhibitinggapjunction channelsfollowinginjury compromisesastrocyticfunctionleadingtoglutamatecytotoxicityandincreasedneuronalinjury(Ozog,Siushansian,& Naus,2002). Hence, thealternatehypothesissuggeststhatgapjunctionspreventtheaccumulation oftoxicmetabolitesandsynchronizetissuesfollowinginjurythrough“spatial-buffering.”Itispossiblethatbothprodeathand prosurvivalrolesofgapjunctionsinastrocytesaresignificant,butarehighly dependentonthetimefromtheonsetofinjuryorinfection.Whilegap junctionsmayinitiallypropagatetheinjurysignalstoneighboringcells, recentevidenceintheliteratureassociateslong-terminhibitionofgapjunctioncommunicationwithenhancedsecondarylesionvolume.
Conversely,undernormalconditionsCx43hemichannelsarepredominantlyintheclosedstate(Contrerasetal.,2003),butaretriggeredtoopen after injury. Uncontrolled exchangebetweenthecytoplasmandtheextracellularspaceviaopenCx43hemichannelscontributestoastrocyticcell death(Contrerasetal.,2002).Cx43gapjunctionchannelsandhemichannelsare oppositely regulatedduringacuteinflammation.Theadverse outcomewithinflammationislikelytoreflectbothuncoupledgapjunctionsandopenhemichannels.Proinflammatorycytokinesincreaseastrocyte permeabilitytotheextracellularspacethroughCx43hemichannelswhile intercellularcommunicationisreduced(Retamaletal.,2007).Thishas directimplications for themetabolicstatusofastrocytesduringacute
Asinanyvesselwound,injurytotheBBBcompromisesvesselintegrity wherebytheendotheliumbecomespermeabletoplasmaproteinswithinthe first24hfromtheonsetofinjury(Barzo,Marmarou,Fatouros,Corwin,& Dunbar,1996; Noble &Wrathall,1989).Thevascularleakinessiscausedby impairedintracellularCa2+ transmissionintheendothelium(DeBocketal., 2012)astheriseinintracellularCa2+ concentration([Ca2+]i)causesadropin thetransendothelialelectricalresistancethatfacilitatescirculatingblood derivedmicrogliatoenteracrosstheendothelialbarrier(Luetal.,2008).
Connexinhemichannels are Ca2+-permeablechannelsthathavebimodal dependenceon[Ca2+]i (<500nM)foropening,whichunderpinsCa2+ oscillationsinprimarybrainendothelialcells(DeBocketal.,2012).
Calciumoscillationis triggeredbybradykinin,aninflammatorypeptide, andisrapidlyinhibitedbyGap26andGap27peptides(withinminutes), reflectingthefastnatureofendothelialcellhemichannelactivity (Romanovetal.,2007).Gap27alsoinhibitedbradykinin-mediatedpermeabilityin in vitro and in vivo experiments(DeBocketal.,2012).Bradykinininduced[Ca2+]i oscillationswereinterruptedwithCx37/43knockdown, furtherconfirmingaroleforconnexinchannelsinendothelialcells (DeBocketal.,2012).TheCa2+ oscillationwereassociatedwithpurinergic signalinginresponsetobradykinin.Hemichannel-independentCa2+ oscillationswerealsosuggestedwiththeuseofATP(DeBocketal.,2012).
Gapjunctionsin thebloodvesselwallregulateintercellularcommunicationacrossdifferentcelltypesatthebarrierjunction.Thecross-talk betweenastrogliaandtheendothelialcellsiscrucialformaintainingBBB integrity(Ezanetal.,2012).Undernormalconditions,theend-feetof theastrocytes are opposedtotheoutersurfaceoftheendothelium (Mathiisen,Lehre,Danbolt,&Ottersen,2010).However,anabsenceof astroglialconnexins compromises theintegrityofBBBbyincreasingthe hydrostaticvascularpressureandshearstress(Ezanetal.,2012).
2.5ChronicInflammation
Chronicconditionsarecharacterizedbypersistentinflammationthatcan resultfrominefficientclearingofdebrisandrecurrentinflammatorysignals (reviewedin Perry,Cunningham,&Holmes,2007).IntheCNS,itisalso characterizedby “glial scarring”intheinjuredarea(McKeon,Schreiber, Rudge,&Silver, 1991).Glialscarringischaracterizedbydenseaccumulationof astrocytes withextensiveproductionofabnormalextracellularmatrix (Gallo&Bertolotto,1990).Thisisoftenseenasabarriertorestorationof
eliminationofby-products(Dienel&Cruz,2003).Lossofthissyncytium can,forexample, contributetotheonsetofdemyelinatingdiseasessuch asmultiplesclerosisorneuromylitisoptica(Masaki,2015).
There isincreasingevidencethatconnexinhemichannelsarethepathologicalporesassociatedwiththespreadofCNSinjuryafterischemicstroke orhypoxicinjury(Davidson,Drury,etal.,2014;Davidson,Green, Nicholson, Bennet,&Gunn,2013;Davidsonetal.,2012;Orellanaetal., 2010),spinalcordtrauma,andretinalstroke(Danesh-Meyeretal.,2012; O’Carroll,Alkadhi, Nicholson, &Green,2008;O’Carroll,Gorrie, Velamoor,Green,&Nicholson,2013),theonsetofdemyelinatingdiseases referredto in thepreviousparagraph(Masaki,2015)ordenervationleading tomuscle degeneration (Ceaetal.,2013).Pannexinchannels,similarinstructuretoconnexin hemichannels,mayalsocontributetodamagespreadafter CNSinjury(Thompsonetal.,2008)althoughtherelationshipbetweenconnexin hemichannelsandpannexinchannels,ortherelativecontributionofeach channeltype,remainstobedetermined.Thefactthatthesumoftheparts appearstoexceedthewhole,withblockadeofeithershowingamarkedreductioninlesionspread,maysuggestafunctionalrelationshipbetweenthesetwo channelstypes.
Gapjunctionchannelsareformedbythedockingoftwohemichannels (orconnexons)atareasofcloseplasmamembraneappositionbetween cells.Becausehemichannelsarecontinuouslyrecruitedfromsurrounding unopposedplasmamembraneareas,migratingwithintheplasmamembrane tositesofclosemembraneappositionadjacenttoexistinggapjunction plaques(Goodenough&Paul,2009;Laird,2006;Theveninetal.,2013), they potentiallyoffer asignalingpathwaybetweentheintra-andextracellularenvironment(Evans,DeVuyst,&Leybaert,2006;Wangetal., 2013).However,whileconnexinhemichannelsaresaidtobeactiveunder “normal”physiological conditions, therearefewstudiesinwhichcells undercompletely“normal”conditionsexhibitspontaneoushemichannel opening.PurinergicsignalingbyregulatedreleaseofATPfromhemichannels,forexample,requiresatriggeringeventsuchasmechanical stress,quinine-inducedhemichannelopening,loweredextracellularCa2+, ortheapplicationoftransmembranevoltages(Hofer&Dermietzel,1998; Kangetal., 2008;Lietal.,1996;Stoutetal.,2002).Althoughincreased intracellularCa2+ uptoabout500nMopenshemichannels,theiropening isinhibitedathigherintracellularcalciumlevels(Wangetal.,2012). Nonetheless,lowered extracellular calciumalsoinducesnicotinamide adeninedinucleotide(NAD+)perfusionthroughCx43hemichannels
Cx43levels,asassessedbybothimmunohistochemistryandWesternblotting (Kandasamy,Escue,Manna,Adebiyi,&Parthasarathi,2015).InabrightlightdamagedalbinoratmodelofretinaldegenerationCx43expressioninthechoroid,assessedbyWesternblot,increasedbygreaterthantriplethecontrolat 6hpostlightexposure(Guoetal.,2014).InotherCNSmodels,Cx43protein expressionismorethandoubledwithin2hafterratspinalcordinjury (O’Carroll,Gorrie,etal.,2013),Cx43mRNAlevelsquadruplewithin4h ofbrainischemiaintheterm-equivalentfetalsheep(Davidsonetal., 2012),andCx43increasesaftertraumaticbraininjury,againassessedbyboth immunohistochemistryandWesternblotting(Li,Zhang,Zhang,Zhou,& Meng, 2015).Increasedconnexinexpressionmeanstherewillbegreater numbersofunopposedhemichannelsintheplasmamembrane,atleastuntil thenewlyexpressedconnexonsdockwiththoseofaneighboringcell.Furthermore,atthesametimethatthesehemichannelsmaybeshowingincreased openingprobabilityunderpathologicalconditions,whereascell-to-cellcoupling(gapjunctioncommunication)canbereduced.Asnotedearlier,factors thatoppositelyinfluencechannelopeninginthismannerincludekinaseactivatingstimuli(LPSandbasicFGForFGF-1)(DeVuystetal.,2007; Schalperetal.,2012),proinflammatorycytokines(TNF-α and IL-1β) (Retamaletal.,2007),intercellularCa2+ concentrationchanges(DeVuyst etal.,2006),ormetabolicinhibition(Contrerasetal.,2002).Interestingly, quininehasbeenevokedasaconnexinhemichannelactivator,triggering ATPreleaseandcalciumsignalinginastrocytesandinC6cellsexpressing Cx43(Stoutetal.,2002),butisalsousedtoblockgapjunctionchannels (Medina-Ceja,Pardo-pena,&Ventura-Mejia,2014;Rubinos,Sanchez, Verselis,&Srinivas,2012;Ventura-Mejia&Medina-Ceja,2014).
Hemichannelopeningunder pathologicalconditionscontributestotissueedemaandcellswellingascellscannolongerosmoregulate.Thiscan leaddirectlytocellrupture,orbymediatingreleaseofregulatorysubstances orinflammatorycytokines,actastriggerforcelldeathprocessesinthe affectedcellsbutalsoinneighboringtissue. Quist,Rhee,Lin,andLal (2000) showedthatextracellularCa2+ concentrationchangeshaveasignificanteffectoncellvolume.Inconnexinnullcellsvolumechangedidnot occur,butafterCx43transfectioncellswellingoccurredinthepresence ofaloweredextracellularCa2+ ionconcentration.Swellingwasprevented bygapjunctionalchannelblockerssuchasoleamideandbeta-glycyrrhetinic acidandwasreversible;thecellvolumereturnedtonormalwhenextracellularcalciumlevelswereraisedagain.Cellswellingandrupturehas alsobeenassociatedwithhemichannelopeningaftercardiacischemia
Asaspecificexample,CeaandcolleaguesshowedthatalackofCx43/45 hemichannelsattenuatesinflammasomeactivationinskeletalmuscle(reducingatrophy)(Ceaetal.,2013).Membranechannel-mediatedpermeability wasslightly reduced bypannexinchannelblockade,butcompletely inhibitedbyconnexinhemichannelblock.Pannexinchannelshavebeen proposedtoplayaroleintheinflammasomepathwayalthoughmore recentlyithasbeensuggestedthatPannexin1isrequiredforATPrelease duringapoptosis,butnotforinflammasomeactivation(Quetal.,2011), whichwould leave connexinhemichannelsastheprimetherapeutictarget. Inperinatalinfection/inflammationasseeninpretermfetalsheepexposedto LPS,whichisassociatedwithreleaseofproinflammatorycytokines,particularlyTNF-α,ledtoreducedmaturationofbrainactivityandwhitematter injuryTNF-α isknowntoincreasehemichannelactivity(Mallardetal., 2014)withATPandcytokinerelease,andcalciumwaves,triggeringfurther injury(inflammasome activation) inneighboringcells.Theserolesforconnexinhemichannelsinboththeinitiationandpropagationofthe inflammasomepathwayareillustratedin Fig.1.Connexinhemichannel blockersmay have aroleinthelimitingtheinitiationandpropagationof theinflammasomeprocess.
3.2AnimalModelsShowTherapeuticBenefitsofConnexin HemichannelBlock
Interventionispossibleatseverallevels.Inthefirstinstance,bothacuteand chronicwoundscanleadtoendothelialdiebackandvascularleakassociated withhemichannelopening.Compromisedblood–brainorblood–retina structuralbarriercausesvesselleak,whichinducesinfiltrationofboth tissue-derived(activatedmicroglia)andblood-derivedinflammatorycells (neutrophils/leukocytes).Interventioninthesecasesaimsfirsttoreducevesselleak,andsecondarilytoreduceinflammationandtheinflammasome pathway.Insomescenarios,however,thereisnovesselleakcomponent andinterventiontargetstheinflammatoryresponseinaffectedtissues. Finally,itispossibletodirectlytargetneuronalconnexinhemichannels andstillshowbenefit.
Followingspinalcordinjuryinratmodels,lesionspreadoverthefirst 24hpostinjuryisattributedtothegapjunctionmediatedbystandereffect andtheopeningofconnexinhemichannels(Croninetal.,2008; O’Carroll et al., 2008;O’Carroll,Gorrie,etal.,2013).Hemichannelopeningcausestissue swelling,inflammation(withblood–borneinflammatory cellsenteringthelesion,astrocytosis,andmicrogliaactivation).Vesselleak,
