Induction of regulatory Tr1 cells and inhibition of T(H)17 cells by IL-27

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Reference

Induction of regulatory Tr1 cells and inhibition of T(H)17 cells by IL-27

POT, Caroline, et al.

Abstract

Accumulating evidence indicates that IL-27, a member of the IL-12 family of cytokines, alleviates the severity of autoimmune diseases in both mice and men. The IL-27-induced activation of signal transducer and activator of transcription (Stat)1 and Stat3 promotes the generation of IL-10- producing type 1 regulatory T (Tr1) cells that inhibit effector T cells. In addition, IL-27 also suppresses the development of pathogenic IL-17-producing CD4(+) T cells (T(H)17) cells suggesting that pharmacological manipulations of IL-27 signaling pathway could be exploited therapeutically in regulating tissue inflammation. Here, we review how IL-27 controls inflammation through the regulation of Tr1 and T(H)17 responses.

POT, Caroline, et al . Induction of regulatory Tr1 cells and inhibition of T(H)17 cells by IL-27.

Seminars in Immunology , 2011, vol. 23, no. 6, p. 438-45

DOI : 10.1016/j.smim.2011.08.003 PMID : 21893418

Available at:

http://archive-ouverte.unige.ch/unige:32745

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ContentslistsavailableatScienceDirect

Seminars in Immunology

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / y s m i m

Review

Induction of regulatory Tr1 cells and inhibition of T H 17 cells by IL-27

Caroline Pot

, Lionel Apetoh

, Amit Awasthi

, Vijay K. Kuchroo

aDepartmentofPathologyandImmunology,UniversityofGeneva,Switzerland

bDivisionofNeurology,Geneva,UniversityHospital,Switzerland

cCentreGeorgesFranc¸oisLeclerc,Dijon,France

dINSERM,U866,Dijon,France

eUniversitédeBourgogne,Dijon,France

fCenterforNeurologicDiseases,BrighamandWomen’s,Hospital,HarvardMedicalSchool,Boston,MA,UnitedStates

a r t i c l e i n f o

Keywords:

IL-27 TH17cells Tr1cells

Foxp3⁺regulatoryTcells Stat

Maf

a b s t r a c t

AccumulatingevidenceindicatesthatIL-27,amemberoftheIL-12familyofcytokines,alleviatesthe severityofautoimmunediseasesinbothmiceandmen.TheIL-27-inducedactivationofsignaltrans- ducerandactivatoroftranscription(Stat)1andStat3promotesthegenerationofIL-10-producingtype1 regulatoryT(Tr1)cellsthatinhibiteffectorTcells.Inaddition,IL-27alsosuppressesthedevelopmentof pathogenicIL-17-producingCD4⁺Tcells(TH17)cellssuggestingthatpharmacologicalmanipulationsof IL-27signalingpathwaycouldbeexploitedtherapeuticallyinregulatingtissueinflammation.Here,we reviewhowIL-27controlsinflammationthroughtheregulationofTr1andTH17responses.

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

SincetheoriginalclassificationbyMosmannand Coffmanof CD4⁺helperT(T_H)lymphocytesintoT_H1andT_H2subsets[1],the repertoireofT_Hsubsetshasexpandedtoincludeadditionaleffec- torandregulatoryTcellsubsetssuchasTH17cellsandregulatory T cells (Foxp3⁺Tregs and Tr1 cells). TH1 cells, which predomi- nantlyproduceinterferon(IFN)-␥andlymphotoxin,areessential for eliminating intracellularpathogens, but werealso regarded asthemajoreffectorT cellsin inducingtissue inflammationin organ-specificautoimmunity.However,micelackingthecompo- nentofT_H1-IFN-␥pathway(Il12^−/−,Ifng^−/−,Ifngr1^−/−,Il12rb2^−/−) werenotprotectedbutoverlysusceptibletoautoimmunediseases includingExperimentalAutoimmuneEncephalomyelitis(EAE)[2], ExperimentalAutoimmuneUveitis(EAU)[3]andcollagen-induced arthritis (CIA)[4].Subsequent studiesrevealed that TH17 cells, insteadofT_H1cells, inducetissueinflammationinautoimmune diseases.AlthoughT_H17cellsareessentialforeliminatingextra- cellular pathogens [5,6], exaggerated TH17 response promotes autoimmunity.ElevatedamountsofIL-17AandIL-17Faredetected inseveralautoimmunediseasesincludingmultiplesclerosis(MS) [7],rheumatoidarthritis (RA)[8]andpsoriasis[9].Theinvolve-

Abbreviations:Tr1cells,type1regulatoryTcells;TH17,Thelper17;Stat,signal transducerandactivatoroftranscription;Maf,transcriptionfactorMaf;Ahr,Aryl hydrocarbonreceptor.

∗Correspondingauthor.Tel.:+16175255537;fax:+16175255566.

E-mailaddress:vkuchroo@rics.bwh.harvard.edu(V.K.Kuchroo).

1 Authorsequallycontributedtothework.

mentofT_H17cellsintissueinflammationwasconfirmedinmouse modelssuchasEAEwhereIL-17-neutralizingantibodiesameliorate clinicalscores[10]orCIAwhereIL-17-deficientanimalsdevelop attenuateddisease[11].Thedifferentiationfactorsforbothmouse andhumanT_H17cellswerefoundtobeacombinationofTGF-␤1 andIL-6orTGF-␤1andIL-21[12].Theactivationofsignaltrans- ducerandactivatoroftranscription(Stat)3byIL-6orIL-21iscritical for inducing the expression of the T_H17 cell master transcrip- tionfactorsretinoid-relatedorphanreceptor(ROR)␥t,encodedby the gene Rorc, and ROR␣ (Rora) [13–15]. Rorc^−/− and Rora^−/−

miceshowdefectiveT_H17cellgeneration[15].Inaddition,Chip- SequencinganalysisrevealedStat3bindingsitesinthepromoters regionsofil17a andil17fgenes[12].FurthermoreROR␥tdrives theexpressionofGM-CSFthatisessentialforinducingpathogenic T_H17cells,andmicedeficientinmakingGM-CSFareresistantto developEAE[16].TheseobservationsindicatethatROR␥tisessen- tialforthedevelopmentofT_H17cells.IndeedT_H17cellgeneration canbeinhibitedbydirectlytargetingROR␥tusingsmallchemi- calcompoundssuchasdigoxinandSR1001[17].While IL-23is notrequiredfortheinductionofTH17celldifferentiation,IL-23 hasaprominentroleinexpansionandstabilizationofpathogenic TH17cells[18–20].BothIL-12p19^−/−andIL-23R^−/−miceareresis- tanttoEAE,andfewTH17cellsarefoundinthecentralnervous system(CNS)ofthosemice[21–23].TheIL-23-T_H17pathwayhas beenshowntobecriticalinmanyautoimmunediseases,which isconsistentwiththefactthatIL-23Rpolymorphismshavebeen geneticallyassociatedwithanumberofhumanautoimmunedis- easesincludingpsoriasis,inflammatoryboweldiseases(IBD)and ankylosing spondylitis[24]. More recentstudies suggestedthat T_H17cellscouldalsobeinducedwiththecombinationofIL-1␤,IL-6 1044-5323/$–seefrontmatter© 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.smim.2011.08.003

andIL-23intheabsenceofTGF-␤1,suggestingthatTH17cellsmight actuallyrepresentaheterogeneouspopulationofproinflammatory cellsthatarehighlypathogenicandcanbeinducedbymultiple differentways.

Exaggeratedinflammatory responsesare prevented byregu- latoryTcellsubsets thatsuppressactivationof effectorT cells.

CD4⁺regulatoryTcellscompriseFoxp3⁺regulatoryT-cells(Tregs) andIL-10-producingregulatorytypeI(Tr1)cells[25].Foxp3⁺Tregs are important to maintain self-tolerance as illustrated by the severeautoimmuneinflammationobservedin micedeficient in Foxp3[26]orinpatientswithdysfunctionalFOXP3protein[27].

AlthoughFoxp3⁺TregsinhibiteffectorTcellresponses,theylose theirsuppressivefunctionsininflammatoryconditions[28].There- fore,IL-10-producingTr1cellsmightbecrucialincontrollingtissue inflammation.Inhumans,Tr1cellswerefirstdescribedinsevere combinedimmunodeficient(SCID) patientswho had developed long-termtolerancetostemcellallografts, supportingtheexis- tenceofthesecellsinhumansandsuggestingthattheymayplay aroleinmediatingTcelltolerance[29].Tr1cellsmediateimmune suppressionbysecreting thesuppressivecytokine IL-10and by killingeffectorcellsviaGranzyme-BandPerforin[30,31].While IL-10wasinitiallydescribedtobethedifferentiationfactorforTr1 cells,theseTcellscouldnotexpandinthepresenceofIL-10.There- foretherewasanemphasisonidentifyinggrowth/differentiation factors for Tr1 cells. Recent identification of IL-27 as a differ- entiation/growthfactor forTr1 cells hasrevivedtheinterest in examiningtheirroleintissueinflammation[32–34].

2. IL-27dampensautoimmuneinflammation

IL-27,anheterodimericcytokinecomposedbythesubunitp28 (IL-27p28)and theEpstein–Barrvirus-inducedgene3 (EBI3),is mainlyproducedbyactivatedantigen-presentingcellsAPCs[35].

IL-27signalsthroughareceptorcomplexconsistingofthecommon IL-6receptorchain,gp130,andtheuniqueIL-27receptoralpha chain(IL-27RaorWSX-1)thatishomologoustoIL-12R␤2ofIL- 12receptor [35,36].Based onthestructural homologybetween IL-12andIL-27andtheirreceptors,IL-27wasinitiallydescribed asa proinflammatorycytokinethatcouldinduceT_H1differenti- ation,which was consistentwiththeability of IL-27toinduce T-bet(Tbx21),themastertranscriptionfactorforthegeneration ofT_H1cells.Subsequentwork,usingbothT_H1andT_H2associated pathogens,establishedthatIL-27suppressesTHcells(TH1,TH2and TH17cells)functionsinvivo,asIl27ra^−/−miceshowedenhanced Tcellfunctions(reviewedin[37]).However,themechanismby whichIL-27-inducedinhibitionofTcellfunctionswasnotunder- stooduntilthediscoverythatIL-27caninduceIL-10production fromCD4⁺Tcells.

3. IL-27controlsTcellresponses

3.1. RegulationofTH1andTH2differentiation

WhileIL-27inducesT-betandexpressionofIL-12R␤2innaïve CD4⁺Tcells,IL-27signalingisnotmandatoryforTH1differentia- tionasillustratedbymicelackingtheIL-27Rsubunit(Il27ra^−/−) that can mount adequate T_H1 responses to eliminate intracel- lular pathogens [38–40]. Moreover, Il27ra^−/− mice die due to uncontrolledimmunopathology and severetissue inflammation associatedwithexaggeratedTcellresponsesandenhancedproduc- tionofIFN-␥andTNF-␣[38–40].IL-27wasalsoreportedtocontrol thegenerationofTH2cells.IL-27treatmentduringStrongyloides venezuelensisinfectiondecreasesT_H2responsesagainstthepara- siteandtreatedmicefailedtodevelopintestinalmastocytosisand exhibitedamarkeddelayinparasiteexpulsion[41].Furthermore,

Fig.1.IL-27inhibitionofdifferentiatingTH17cells.OndifferentiatingTH17cells,IL- 27inhibitstheexpressionoftranscriptionfactorsRor␥tandRor␣,therebyimpairing thesecretiontheTH17-relatedcytokines,IL-17A,IL-17F,IL-22andGM-CSF.

intranasal administration of IL-27inhibits OVA-inducedairway hyperresponsivenessandinflammationinOVA-sensitizedanimals [41].Atthetranscriptionallevel,IL-27hasbeenshowntosuppress themasterTH2transcriptionfactorGATA-3[41].Recently,genome- wideassociationstudy(GWAS)hasshownthatasinglenucleotide polymorphism(SNP)intheIL-27p28genewasassociatedwithan increasedsusceptibilitytoasthma[42]orCOPD[43]andIL-27has beenproposedasapotentialtreatmentforbronchialasthma.

3.2. InhibitionofTH17celldifferentiation

InadditiontoinhibitingbothT_H1andT_H2development,IL-27 preventsthedevelopmentofT_H17cellsinvitroandinvivo.Il27ra^−/− miceareoverlysusceptibletoEAEcomparedtowild-typemiceand presentanincreasedaccumulationofT_H17cellsinthedraining lymphnodesandintheCNS[44].Inthismodel,neutralizationof IL-17inIl27ra^−/−miceduringEAEdiseasecourseattenuatedtheir diseasephenotype[44].Accordingly,recombinantIL-27treatment decreasesthediseaseincidenceandseverityinEAEwiththeinhi- bitionofdevelopmentofTH17cells[45].Similarly,Il27ra^−/−mice chronicallyinfectedwithToxoplasmagondiidevelopedsevereneu- ropathologymediatedbyCD4⁺Tcells,associatedwithincreased TH17celldevelopment.IL-27inhibitstheproductionofIL-17by BMNCsfromchronicallyinfectedmicestimulatedwithIL-23[46].

Finallyin theabsenceof IL-27duringmurineflu infection,flu- specificTcellresponsesareskewedtowardsTH17[47].

AboveobservationsclearlyindicatedthatIL-27isnegativereg- ulatorofdevelopmentofT_H17cells.However,themechanismby whichIL-27inhibitsthedevelopmentofT_H17cellsisnotclearly understood.AccumulatingdatasuggestthatIL-27utilizesmultiple mechanismstoinhibitthedevelopmentofT_H17cells(Figs.1and2).

During T_H17 cell differentiation, IL-27 directly suppresses the expressionofbothROR␥t,themastertranscriptionfactorofTH17 cells[48]andROR␣[49](Fig.1).IL-27inhibitsexpressionofROR␥t in T_H17 cells both in mouse and man [48]. Interestingly, IL-27 decreasesthe expression of GM-CSF and thereby dampens the

Fig.2.IL-27inhibitionofcommittedTH17cells.IL-27inducesthedifferentiationofTr1cellsthatinhibitTH17cellsinanIL-10-dependentmanner.IL-27p28monomers interferewithIL-6cytokinesignalingthroughgp130andtherebyinhibitthemaintenanceofTH17cellsandtheirIL-17secretion.IL-27furtherinducesT-betexpressionthat drivesIFN-␥productionandpromotestheconversionofTH17cellsintoTH1cells.

pathogenicityofT_H17cells[16].ByblockingGM-CSFsecretionand inhibitingbothROR␣andROR␥texpression,IL-27interfereswith TH17celldifferentiationatseverallevels,explainingitspotentabil- itytosuppresstheinductionofT_H17cells.

WhetherIL-27candirectlysuppresseffector/memoryT_H17cells orfullydifferentiatedTH17cellsisstilldebated.Indeed,TH17main- tainedincultureforatleasttworoundsbecomeunresponsiveto IL-27asIL-27failstoinhibittheexpressionofROR␣andROR␥tin thesecells[49].However,IL-27couldmodulateeffector/memory T_H17cellsusingdifferentstrategies.AmongthetwoIL-27cytokine subunits,EBI3isconstitutivelyexpressedbutIL-27p28secretionis transcriptionallyregulated.IL-27p28monomerscaninterferewith theIL-6-mediatedproductionofIL-17bypreventingIL-6signal- ingthroughgp130,suggestingthatIL-27p28monomerscouldalso beexploitedinregulatingTcellresponses[50].IL-27p28thuslim- itsthegenerationandmaintenanceofTH17cellsinvivowithout directlyinterferingwithT_H17transcriptionalprogram(Fig.2).Fur- thermore,ithasbeenproposedthatT_H17couldbeconvertedinto TH1cellsthatarepresumablylesspathogenic[51,52].Oneputative mechanismbywhichIL-27couldconvertsT_H17intoT_H1cellsmay bebyinducingtheexpressionofT-betthatdrivesIFN-␥expression andreducestheexpressionofIL-17(Fig.2).However,thishypothe- sisbywhichIL-27mayincreaseT_H17plasticityhasnotbeenproven experimentally.

3.3. InductionofTr1cells

IL-27,whileinhibitingTGF-␤-inducedFoxp-3⁺ Tregs,induces IL-10⁺, IFN␥⁺ Tcells that are immunosuppressive, a phenotype inlinewiththepreviouslydescribedTr1cells[32–34,53,54].The roleofIL-27ingenerationofIL-10-producingTr1cellswasfur- theremphasizedinvivo.IL-27treatedMOG-specificsplenocytes losetheirability totransfer EAEinanIL-10dependentmanner [33].Furthermore,duringfluinfection,IL-27generatesregulatory TcellsthatinhibitTH17cellsbysecretingIL-10andIFN-␥.Inthe absenceofIL-10,flu-specificTcellresponsesdevelopedastronger T_H17 component[47].Furthermore,ithasbeenshown thatTr1 cellscaninhibitTH17cellsinvivoinanIL-10dependentmanner

duringmurinecolitis[55](Fig.2).Akintowhathasbeenobserved inmurineTcells,activationofnaïvehumanTcellsinthepresence ofIL-27similarlyinducesTr1cellsthatproducebothIFN-␥and IL-10[56].

4. MolecularpathwaysinvolvedinIL-27biology

Similartoothertype1cytokinereceptors,IL-27alsoinduces theactivationofJanuskinase/Statpathway.IL-27predominantly inducesthephosphorylationofStat1andStat3.Herewewilldis- cusstheIL-27-inducedsignalingeventsfollowingtheactivation oftheStatsandanalyzetheirrolesininhibitingTH17cellandin inducingTr1celldifferentiation.

4.1. IL-27andStat1activation

4.1.1. Stat1activationbyIL-27repressesT_H17differentiationand inducesTr1cells

TheactivationoftheIL-27specificsubunitWSX-1drivesthe tyrosine phosphorylation of JAK1 that further activates Stat1.

Indeed,JAK1, butnototherJAKs,coprecipitateswiththeWSX1 subunit[57].

TheStat1signalingpathwayisnecessaryforIL-27-inducedT- betexpression[58].T-betnotonlydrivestheexpressionofIFN-␥ butalsoplaysanimportantroleintheinhibitionofTH17cytokines, independentlyofIFN-␥.T-betcanreprogramcommittedTH17cells byrepressing T_H17 gene program,which resultsinfewer tran- scriptsofRorc,il17a,il17f,il23r[59].Thesefindingweresupported bystudiesshowingthatT-betutilizesRunt-relatedtranscription factor1(Runx1),atranscriptionalactivatorthatsequestersRorc awayfromtheregulatoryregionsonRorcpromoter[59].Indeed Runx1bindingsiteislocatedupstreamofT-betbindingsiteonRorc promoter.BysequesteringRunx1,T-betinhibitstheexpressionof ROR␥t,resultingimpaireddevelopmentofT_H17cell[59](Fig.3).

Stat1^−/− and T-bet^−/− miceexhibit an increased number of T_H17cells bothduring systemicinflammationinvivoorduring T_H17cells differentiationinvitro. IL-17production isgreaterin theabsenceofT-betcomparedtotheabsenceofStat1[60].This

Fig.3. ReciprocalregulationofTH17andTr1cellsbyIL-27.Themolecularmecha- nismsbywhichIL-27promotesFoxp3⁻IL-10⁺Tr1celldifferentiationandrepresses TH17celldevelopmentthroughactivationofStat1andStat3activationareshown.

IL-27activatesStat1throughthesubunitWSX1thatinhibitsRor␥texpression throughT-bet-dependentaswellasT-bet-independentpathways.Alternatively,IL- 27canpromoteT-betexpressioninaStat1independentpathwayviaGADD45␥.In addition,IL-27activatesStat3signalingthroughgp130.Stat3inductionthendrives Maftranscription.MaftogetherwithAhrtransactivatesil21andil10promoters.

Ontheotherhand,IL-27inhibitsFoxp3transcriptioninaStat3/Smad3dependent manner.

mayberelatedtothefactthatT-betmightalsobeinducedina Stat1independentmanner.Inthisvein,Owakietal.haveshown that IL-27 induces a Stat1 independent T-bet expression [61].

IndeedIL-27inducestheexpressionofGADD45␥thatfurtherdrives the phosphorylationof p38 MAPK leading to T-bet expression (Fig.3).

IthasbeenfurtherproposedthatStat1couldinhibitROR␣and ROR␥texpressionindifferentiatingT_H17cellsinaT-betindepen- dentmanner(Fig.3).WhileadirectinhibitoryeffectofStat1on ROR␣andROR␥texpressionhasnotbeenruledout,Statscould alsoindirectlyaffectT_H17responsesbypromotingthefunctionof auxiliaryinhibitoryTH17factors.DifferentrepressorsofTH17cells differentiationhavebeenidentified,includingEts-1,whichnega- tivelyregulatesT_H17celldifferentiation[62].Stat1andEts-1have beenshowntobindtogether[63]andmightcooperatetoinhibit TH17celldifferentiationbydirectlyorindirectlyinterferingwith ROR␥tfunctioninT_H17cells.

IL-27hasbeenshown toinduceIL-10expressionfromCD4⁺ Tcells usingboth Stat1 and Stat3pathways(Fig.3).Indeed, in theabsenceof Stat1signaling,IL-27driven IL-10production is decreased.WhileitisclearthattheStat1drivenIL-10secretion isindependentofT-betsignaling,theunderlyingmechanismsstill remainunclear[34].

4.2. IL-27andStat3activation

4.2.1. Stat3activationbyIL-27doesnotenhanceTH17cell differentiation

IL-27utilizesgp130subunitofIL-6receptor complex,which resultsinactivationofStat3 signaling.AgeneticdefectinStat3

signalinginhumans,inhyperIgEsyndrome, resultsindefective TH17 cells and in unrelenting fungal infections, supportingthe criticalroleof Stat3inthegenerationofT_H17cells[64].Atthe firstglance,itispuzzlingthatIL-6andIL-27,whichbothactivate Stat3pathways,haveantagonisticproperties.Ithasbeenproposed that IL-6leadsto afaster and more persistentpattern of Stat3 phosphorylationthatiscrucialtodrivepro-inflammatorysignals downstreamStat3. pStat-3directlybindstoil17a andil17fpro- motersandtransactivatethesegenesbycollaboratingwithother transcriptionfactorslikeIRF-4andROR␥t.Furthermore,theforma- tionofStat1–Stat3heterodimersinresponsetoIL-27ratherthan theformationofmainlyStat3homodimersinresponsetoIL-6or IL-21mayplayaroleinthedifferencebetweenIL-6andIL-27sig- naling.Indeedpreliminarydatafromourlaboratorysupportsthis hypothesis.Inaddition,IL-6activationrapidlyinducesStat3repres- sorSOCS3[65].SOCS3isanessentialnegativeregulatorofStat3 phosphorylationandconstrainsT_H17celldifferentiation[66,67].

WhileIL-27inducesexpressionofSOCS3,IL-27-mediatedinhibi- tionofIL-17productionisindependentofSOCS3[46].Ittherefore seemsunlikelythatIL-27-inducedSOCS3contributestotheinhi- bitionofTH17cells.Instead,theinhibitionofTH17differentiation mightmainlybemediatedthroughStat1andT-betasdiscussed above.

4.2.2. Stat3activationbyIL-27promotesTr1celldifferentiation IL-27-inducedStat3phosphorylationisessentialfortheanti- inflammatoryroleofIL-27,asittriggersIL-10secretionfromCD4⁺T cells[34](Fig.3).SustainedactivationofStat3leadstotheinduction ofthetranscriptionfactorMaf[68].Weandothershaverecently shownthatMafisessentialforIL-10productioninducedbyIL-27 [53].SimilarlytoStat3deficientCD4⁺Tcells,MafdeficientCD4⁺ TcellscannotproduceIL-10inresponsetoIL-27.Ithasbeenfur- thershownthatMafdirectlytransactivatesil10andil21promoters [53].InadditiontoMaf,IL-10productionbyIL-27isregulatedby theligandactivatedtranscriptionfactorArylhydrocarbonreceptor (AhR)thatbindstoMafresultinginacomplexthatinducesboth il10andil21transcription[69].ThefindingofAhRinvolvementin IL-10productionissignificantasitprovidesimpetustodesignAhR ligandsthatcanmodulatetheanti-inflammatorypropertiesofTr1 cellsbothinvitroandinvivo(reviewedin[31]).Theexpression ofthecytokineIL-21isfurtheressentialforIL-27-induced-IL-10 production[53](reviewedin[37]).IntheabsenceofIL-21,IL-10 productionisreducedinTr1cells.IL-21secretioncanbefurther amplifiedbyAhRactivation[69].

4.2.3. Stat3activationbyIL-27andinhibitionofFoxp3

IL-27inhibitsthegenerationofFoxp3⁺Tregs[70].Thefactthat Foxp3⁺TregsexpressIL-27RstronglysuggestedthatIL-27might blockthedevelopmentofthoseregulatorycellsinvitro[71].IL-27 indeedleadstoadecreasedexpressionofFoxp3throughamech- anismthat isatleast partiallydependentonStat3 [70].Smad3 bindingtoFoxp3promoterisimplicatedinFoxp3transcription.

Ithasbeenproposedthat IL-27-inducedpStat3binds toa gene silencerregion(enhancerII)inaconservedregionofFoxp3gene thatreducestheacetylationintheregionofSmad3bindingsite anddecreasesthebindingofpSmad3toFoxp3promoter[72].This resultsinadecreasedaccessibilityandbindingofSmad3toFoxp3 promoterandtherebydecreasesFoxp3transcription(Fig.3).IL-27 impacts Foxp3⁺Treg development and function in vivo. Indeed micethatoverexpressbothIL-27subunits,IL-27p28andEBI3,have decreased number of Foxp3⁺Tregs and developed spontaneous inflammationsimilartomicethatlackFoxp3⁺Tregssuchasthe scurfy Foxp3 mutant mice or IL-2^−/− mice [73]. Interestingly, IL-27transgenicmiceare deficientin IL-2.Thoseresultsare in accordancewithanotherrecentstudyshowingthatIL-27inhibits Foxp3⁺Treg in vivo in a murine T cell transfer colitis model.

Il27ra^−/−deficientTcellstransferredanattenuateddiseasedueto alargerpercentageoftransferredcellsexpressingFoxp3compared towild-typeTcells[74].

5. Therapeuticimplications

5.1. IL-27confersprotectionagainstmultiplesclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease affectingthe centralnervous systemresulting ininflammation, demyelizationandaxonalloss.It isacommonneurologicaldis- order, which attacks young adults. T_H17 cells were shown to contributeto MSdevelopment [75]. By contrast,IL-27protects against autoimmuneinflammation in themouse model EAE as exemplifiedbyIl27ra^−/−micewhichdevelopanacceleratedEAE diseasecoursecomparedtoWTcontrolsandshowincreasedlevels ofTH17cellsintheCNS[44].Furthermore,dailyintrathecaltreat- mentwithIL-27duringEAEalleviatesthediseaseanddecreases boththeinflammationinthebrainand thenumberof infiltrat- ingTH17cells[45].SimilarlyinaTcelladoptivetransfermodel, pre-treatmentofautoreactiveCD4⁺TcellswithIL-27leadstoa reductionoftheirpathogenicityinanIL-10dependentmanner[33].

Interestingly,IL-27wasalsoshowntomediatetheprotectiveeffect ofBonemarrowstromalcells(BMSCs)thatpreventEAEinmiceand suppressIL-17production[76].

SupportforIL-27inregulatingautoimmunetissueinflammation hasalsobeenprovidedinhumans.Theimmunomodulatorydrug IFN-␤,usedinthefirstlineoftreatmentforMS,hasbeenshown toinduceIL-27productionfromdendriticcells (DCs).Interferon (IFN)-␤,amemberofthetypeIinterferonfamily,isanapproved treatmentforrelapsingremittingMS(RRMS)thatreducestherate of relapses by 30%. While the therapeutic mechanisms of IFN-

␤remain poorly understood,recent studies indicate that IL-27 contributestoits regulatoryproperties bothin mouse[77] and human[78,79].OnelimitationofIFN-␤treatmentisthat20–50%

ofpatientsfailtorespondtotherapy thusdelayingachangein thetreatmentstrategyof thosepatients. While thepresenceof neutralizingantibodies(Nabs)againstIFN-␤inthebloodhasbeen proposedtocorrelatewithtreatmentfailure[80],aproportionof non-responderpatientsdonotdevelopNabs,limitingtheuseof NabstopredicttheresponsetoIFN-␤therapy[81].IL-27secretion fromPBMCfromRRMSpatientshasbeenproposedasapredictive factorofclinicalresponsetoIFN-␤treatment.Indeed,PBMCiso- latedfromRRMSpatientsthatrespondtoIFN-␤treatmentsecrete moreIL-27whenexposedinvitrotoIFN-␤thanPBMCisolatedfrom

“non-responder”patients[78].Finally,othertherapiesproposedfor treatingMS,suchasStatins,whichinadditiontotheircholesterol- loweringactivityhaveanti-inflammatoryproperties,wereshown toincreaseinvitroIL-27secretionfromhumanmonocytesofMS patients[82].

5.2. IL-27protectsagainstrheumatoidarthritis

Rheumatoidarthritis (RA)is a systemic inflammatory disor- derthatprincipallyattackssynovialjoints.TH17 cellsand IL-17 expressioniselevatedinRAsynovialtissueandfluidmacrophages comparedtocontrols[83,84].ElevatedlevelsofIL-17havebeen reportedin theanimal model of RA, collagen-induced arthritis (CIA),andIL-17neutralizationpreventsbonedestructionsuggest- inga pathological role ofT_H17 cells in thedevelopmentof RA [85].AdministrationofIL-27inmicesufferingfromCIAreduces theseverityofthedisease,asshownbyreducedcellularinfiltra- tioninthejoints,synovialhyperplasia,andjointerosion[84].IL-27 treatmentfurtherdecreasesserumlevelsofIL-6.Inaddition,lym- phocytesisolatedfromspleenand lymphnodeofIL-27-treated

miceproducesignificantlyreduced amountsof IFN-␥and IL-17 whenculturedwithtypeIIcollageninvitrocomparedwithlympho- cytesfromcontrolmice.SimilarresultswereobtainedwhenIL-27 wasectopicallyexpressedinthejoints[86].Thesestudieshigh- lightinthetherapeuticpotentialofIL-27inRA,especiallywiththe feasibilityoflocal,intra-articular,administrationofrecombinant IL-27.

5.3. ControversialroleofIL-27ininflammatoryboweldisease IL-27 is implicated in thepathogenesis of IBD, Crohn’s dis- easeandulcerativecolitis.Genomewidestudieshaveidentified SNPsinthegeneencodingp28subunitassociated withalower expressionofIL-27andearlyonsetinflammatoryboweldisease, whichwouldbeconsistentwithaprotectiveroleofIL-27inIBD [87].TwootherstudieshavefoundtranscriptsforIL-27p28[88]

and Ebi3 [89] tobeoverexpressedin biopsy samplesfromIBD patients.ThefunctionofIL-27hasbeenassessedusingdifferent murinemodelsofIBD.InthemouseIBDmodelofacuteinflam- mation,whichrelies onthepresenceof dextransulfate sodium (DSS)toinduceinflammation,Il27ra^−/−micereceiving5–10%DSS indrinkingwaterweremoresusceptibletodisease[90].Il27ra^−/−

deficientmiceshowedareductioninT_H1IFN␥-producingcellsand anincreaseinT_H17cellsingut-associatedlymphoidtissuepoint- ingtowardsanimportantregulatoryroleofIL-27indampening T_H17cellfunction[90].Inthe2,4,6-trinitrobenzenesulfonicacid (TNBS)-inducedmouseacutecolitismodel,subcutaneousscIL-27 (EBI3andp28subunitsgeneratedasasingle-chainhumanIL-27) treatmentsignificantlyimprovedinadose-dependentmannerthe extentofthelesionsaswellasnecrosis,ulcerationandthickening ofmucosalepithelium.scIL-27suppressedseveralinflammatory cytokinesininflamedcolon,includingIL-17[91].However,inaT celltransfercolitismodel,IL-27wasshowntoexertproinflamma- toryeffectsasitsuppressedinducedTregdevelopmentinvivo[74].

Incontrast,intheDSSmodel,Il27ra^−/−micetreatedwithlower dosesofDSS(0.5%indrinkingwater),wereprotectedcomparedto WTcontrols[92].Theimplicationofdifferentpathogenicorreg- ulatorysubsetsandtheheterogenicityofthemodelsmayexplain thedifferentresponsestoIL-27treatmentinmurinemodelsofcol- itis.However,inmodelswhereT_H17cells areimplicatedinthe developmentofthedisease,theanti-inflammatoryrole ofIL-27 appearstobedominant.Indeed,T_H17cellshavebeenshownto becrucialfor thedevelopmentof TNBS-inducedcolitis asIL-17 receptorA(IL-17RA)knockoutmicedonotdevelopTNBScolitis [93] and IL-17F-deficient micedevelop more severeDSS colitis than controls[94]. A betterunderstanding of thepathogenesis ofIBDshouldprovideadditionalinsightintotheroleofIL-27in colitis.

6. Openquestionsandconcludingremarks

While IL-27promotes Tr1 cells, it inhibitsCD4⁺Foxp3⁺Tregs inducedbyTGF-␤.Theseobservationsarereminiscentoftheaction ofAhRligandssuchasFICZthatpromotesTr1cells butinhibits Foxp3⁺Tregs.ThisparadoxicaleffectonregulatoryTcellsmight stemfromdifferentand/orcomplementaryrolesofregulatoryT cells. Tr1 cells but not Foxp3⁺Tregs maydevelop in situ in the inflamedtissueasIL-27canbesecretedbyresidentcells inthe targetorgan,suchasinthebrainduringEAEandMS.Foxp3⁺Tregs cannotinhibithighlypathogeniceffectorTcellsinthetargetorgan [95]buttheyinducetolerogenicplasmacytoiddendriticcell(DC) thatsecreteIL-27thuspromotingTr1cellgeneration[32].Under inflammatory settings, Foxp3⁺Tregscan produce cytokinesthat belongtootherlineages[96,97]andweproposethatTr1cellscould bemorestableand therebyregulatetissue inflammationatthe targetsite.

IL-27controlsinflammationbyinhibitingTH17cellsandbypro- motingthedevelopmentofIL-10-producingregulatoryTr1cells.

Despitetheiroppositeinvivofunctions,Tr1andT_H17cellsharbor strikingsimilarities.First,theyrelyonthetranscriptionfactorsMaf andAhRfortheirgeneration.Second,theyrequireIL-21fortheir growth.Third,theyproduceIL-10.Inthisregard,Ghoreschietal.

showedthatT_H17differentiated withTGF-␤andIL-6 (T_H17(␤)) producedIL-10andwerepoorlypathogenicinvivoincontrastto T_H17cells inducedbyIL-6,IL-1␤andIL-23(T_H17)(23)thatdid notproduceIL-10andwerehighlypathogenic.Inaddition,TGF-␤ inducedTH17expressedhigherlevelsofMafandAhRcomparedto TH17inducedwithIL-1,IL-6andIL-23(23).Thisobservationwould thusbeinlinewithapreviousworksuggestingthattheMaf-driven inductionofIL-10inTH17cellsreducedtheirpathogenicity[98].

SincewehaveshownthattheexpressionofMafandAhRisrequired fortheproductionofIL-10andIL-21inTr1cells,itmightbeinter- estingtoexplorewhetherIL-27couldactuallybeconvertingT_H17 toTr1cells.Wearecurrentlyconductingafunctionaltranscrip- tionalanalysisofTr1(differentiatedwithIL-27)andT_H17(IL-6and TGF-␤)cellsusingacomputationalapproachandawholegenome microarrayanalysistoaddressthisquestion.

In the same line, IL-21 has been ascribed a functional role inpromotingbothT_H17[99,100]andTr1 cells[53].Therole of IL-21during autoimmunedisease suchas EAE is controversial.

WhileinitialstudieshaveproposedthatIL-21R^−/−micepresented alesssevereEAEdisease[100],longerobservationofEAEdisease courseshowedthatIL-21R^−/−micedevelopedamoreseveredis- ease[101,102].BesidesbeingagrowthfactorforTH17cells[103], IL-21maybehaveasananti-inflammatoryeffectbypromotingIL- 10secretionfromdifferentTcellsubtypes.Itremainstobeseen whetherIL-27anditsdownstreamcytokineIL-21canmodulatethe pathogenicityandstabilityofdifferentsubtypesofT_H17cellsthat havebeenfurthertreatedwithIL-23.Inconclusion,IL-27notonly inducesthegenerationofanti-inflammatoryTr1cellsbutbroadly controlsautoimmune responsesbyinhibitingeffector Tcells in varioustargetorgans.

Acknowledgements

TheauthorsaresupportedbygrantsfromSwissNationalScience Foundation(SFGBM)and theNovartis Foundation(C.P.)andthe AgenceNationaledelaRecherche[ANR-10-PDOC-014-01](L.A.).

AAissupportedbyresearchgrantfromCrohn’sandColitisFoun- dationofAmerica,NewYork.Studiesinourlaboratorywerefunded bythe National Institutes of Health [NS030843, AI039671, and AI056299](V.K.K.).

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