Gene therapy using IL-27 ameliorates Sjogren's syndrome-like autoimmune exocrinopathy

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Title:
Gene therapy using IL-27 ameliorates Sjogren's syndrome-like autoimmune exocrinopathy
Physical Description:
Book
Language:
English
Creator:
Lee, Byung Ha
Carcamo, Wendy C.
Chiorini, John A.
Peck, Ammon B.
Nguyen, Cuong Q.
Publisher:
Arthritis Research and Therapy

Notes

Abstract:
Introduction: Sjögren’s syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and lacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that TH17 cells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive inflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of TH17 activity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS. Methods: Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or LacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and IL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to quantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA) staining, and stimulated saliva flow rates were used to profile SjS disease severity. Results: Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited long-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice injected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed that rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF, lower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva flow rates. Conclusions: These data support the concept that IL-27, when provided exogenously, can induce a suppressive effect on SjS development and thus may be an effective therapeutic agent for regulating TH17 pro-inflammatory activity in autoimmune diseases where the TH17 system has been shown to play an important role in their pathogenesis.
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http://arthritis-research.com/content/14/4/R172

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University of Florida
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University of Florida
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http://arthritis-research.com/content/14/4/R172
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fl(+) origin TR







ApR pTR-UF14

6421bp


ColE1 ori


TR
bGH poly(A)


WPRE


SmaI (87)


CMV ie enhancer
Chiken b-actin promoter
Exonl
Chimeric intron


I IRES


fl(+) origin TR


SnaI (98)
CMV ie enhancer
Chicken b-actin promoter
Exonl
*V \Chimeric intron


rAAV2-IL27
6568 bp


CoIEI on A
hGFP F64LIS65T on1 (362)i
TR -
Simal(3631( )
bGH poly(A)
Sall (3322)


NotI (2610)


ApR



















Saline LacZ IL-27
pre-disease stage


Saline LacZ IL-27
clinical-disease stage


Treatment




Full Text

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RESEARCHARTICLE OpenAccessGenetherapyusingIL-27amelioratesSjgren s syndrome-likeautoimmuneexocrinopathyByungHaLee1,2,WendyCCarcamo2,JohnAChiorini3,AmmonBPeck2,4,5andCuongQNguyen4,6*AbstractIntroduction: Sjgren ssyndrome(SjS)isasystemicautoimmunediseasecharacterizedbydecreasedsalivaryand lacrimalglandsecretions,resultinginseveredrymouthanddryeyes.RecentstudieshavesuggestedthatTH17 cellsanditssignaturecytokineIL-17areinvolvedintheunderlyingpathogenicmechanismsleadingtodestructive inflammationandautoimmunity.Inthepresentstudy,weexaminedwhetherIL-27,anaturalinhibitorofTH17 activity,coulddown-regulateorreverseSjSinC57BL/6.NODAec1Aec2 mice,amodelofprimary-SjS. Methods: Recombinantserotype2adeno-associatedviral(AAV2)vectorsexpressingeither IL-27 (rAAV2IL2 7)or LacZ (rAAV2LacZ )wereinjectedinto6or14week-oldC57BL/6.NODAec1Aec2 mice.ChangesinIL-27,IL-17,and IL-10cytokinelevelsinperipheralbloodweredeterminedbyELISAs,whileflowcytometryanalyseswereusedto quantifycytokine-positivesplenocytes.Histologicalassessmentofsalivaryglands,anti-nuclearautoantibody(ANA) staining,andstimulatedsalivaflowrateswereusedtoprofileSjSdiseaseseverity. Results: MicesystemicallytreatedwithintravenousrAAV2IL27 injectionsateither6or14weeksofageexhibited long-termelevatedlevelsofserumIL-27withconcomitantlyreducedlevelsofIL-17comparedwithserafrommice injectedwithrAAV2LacZ orsalineoutto20weekspost-inoculation.Mostimportantly,diseaseprofilesrevealed thatrAAV2IL27 treatmenthadlittleeffectonlymphocyticfocus(LF)scores,butresultedinstructuralchangesinLF, lowertitersofANAswithchangesinstainingpatterns,andalesssevereclinicaldiseaseasdeterminedbysaliva flowrates. Conclusions: ThesedatasupporttheconceptthatIL-27,whenprovidedexogenously,caninduceasuppressive effectonSjSdevelopmentandthusmaybeaneffectivetherapeuticagentforregulatingTH17pro-inflammatory activityinautoimmunediseaseswheretheTH17systemhasbeenshowntoplayanimportantroleintheir pathogenesis.IntroductionInterleukin27(IL-27),alongwithIL-12,IL-23,andIL-35, isanovelcytokineoftheIL-6/IL-12family.Itiscomposedoftwosubunits:IL-12 p40-relatedEpstein-Barr virus-inducedgene3(Ebi3)proteinandIL-12p35-related p28protein(p28)[1].Theorphancytokinereceptor WSX-1(TCCR)andglycoprotein-130(gp130)makeup theheterodimericsignaltran sducingreceptorforIL-27 [2].IL-27actsonCD4+Tcellsandplaysapivotalroleas bothapro-andanti-inflammatorycytokine.Asaproinflammatorycytokine,IL-27activatesThelper1(TH1) responsesintheearlyphasesofimmunity,inwhich secretionofinterferon-gamma(IFNg )isoneofthekey inflammatorymediatorsinautoimmunity.Themechanismappearstobetheactivationofsignaltransducerand activatoroftranscriptio n1(STAT1)[3].Asanantiinflammatoryprotein,IL-27suppressesIL-2,antagonizingIL-6functionandactivatingexpressionofsuppressor ofcytokinesignaling(SOCS )protein(s)[4].Instudies withWSX-/-receptorknockoutmice,abnormalsignal transductionofIL-27showedhyper-productionofvariouspro-inflammatorycytokinessuchastumornecrosis factor-alpha(TNFa )andIL-6whenchallengedby Trypanosomacruzi or T.gondii [5,6].Moreover,IL-27 cansuppresstheexpressionof forkheadboxP3-positive (Foxp3+)regulatoryT(Treg)cellsandactasanegative regulatorofhumanneutrophilfunction[7,8].Recent *Correspondence:nguyenc@ufl.edu4CenterforOrphanAutoimmuneDisorders,CollegeofDentistry,University ofFlorida,Gainesville,FL32610,USA FulllistofauthorinformationisavailableattheendofthearticleLee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 2012Leeetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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studiesalsoconfirmedthatIL-27hasanti-tumoreffects [7,9]. IL-27iswellknownforitsinhibitoryeffectsonretinoic acid-relatedorphanreceptorgammat(ROR g t),thetranscriptionfactorforTH17cells,byactivatingbothT-bet, thetranscriptionfactorforTH1cells,andtheSTAT1 pathway,thusinhibitingexpressionofIL-17A(commonly referredtoasIL-17)[10].Inaddition,WSX-1-deficient miceshowedgreatersusceptibilityforexperimentalautoimmuneencephalomyelitis(EAE)incomparisonwith wild-typecontrolmiceandexhibitedincreasedlevelsof IL-17[11].MorerecentreportshavedescribedthecapacityofIL-27tosuppressTH17cellsbyinhibitingTH17 celldifferentiation,therebyreducingseverityofTH17mediatedautoimmunediseases[11,12]. Genedeliveryusingrecombinantadeno-associatedvirus (rAAV)-basedvectorshasbeenshowntoconveylongtermgeneexpressionsintreatedhosts[13-16].Previous studiesofgenetherapyusingAAVhavealsoprovenits safetyandabilitytoelicitminimalinflammatoryresponses incomparisonwithothertypesofgenedeliveryagents [17-20].Nevertheless,todate,nostudyusingtherAAV systemhasreportedaroleforIL-27inSjgren ssyndrome (SjS).Thus,weexaminedtheeffectsofIL-27treatmenton SjSdiseaseofC57BL/6.NODAec1Aec2 micewhendeliveredeitherat6weeksofage(pre-disease)orat14weeks ofage(clinicaldisease).Resultsreportedhereindicatethat IL-27,apotentinhibitorofTH17celldevelopment,may beausefulreagentfortreatingSjS.MaterialsandmethodsAnimalsC57BL/6.NODAec1Aec2 micewerebredandmaintained underspecificpathogen-freeconditionsintheanimal facilityofAnimalCareServicesattheUniversityofFlorida (Gainesville,FL,USA).DevelopmentoftheC57BL/6. NODAec1Aec2 mouseanditsSjS-likediseasephenotype isdescribedelsewhere[21].Briefly,C57BL/6.NODAec1Aec2 mousewasdevelopedbyintroducingtwo geneticregions,oneonchromosome1(designated Aec2 ) andthesecondonchromosome3(designated Aec1 ), derivedfromtheNOD/LtJmouse,intotheSjS-non-susceptibleC57BL/6Jmouse.Allanimalsweremaintainedon a12-hourlight-darkscheduleandprovidedfoodandacidifiedwater adlibitum .Attimesindicatedinthearticle, micewereeuthanizedbycervicaldislocationafterdeep anesthetizationwithisoflurane,andtheirorgansandtissueswerefreshlyharvestedforanalyses.Allexperiments andanalysesdescribedinthisarticlewereperformedwith maleandfemalemice.Thebreedingandtheuseof C57BL/6.NODAec1Aec2 miceforgenetherapystudyas describedherewereapprovedbytheUniversityofFlorida sInstitutionalAnimalCareandUseCommitteeand InstitutionBiosafetyCommittee.RecombinantAAV2-IL27(rAAV2IL27 )vectorconstructionTheserotype2adeno-associatedviralvector(AAV2) wasdesignedtocontainacytomegalovirus(CMV)/ chickenb -actinhybridpromoter ,aninternalribosome entrysite(IRES),andtheinvertedterminalrepeat sequences(pTR-UF14)(FigureS1AofAdditionalfile1). TofullyrecapitulatethefunctionalityofmouseIL-27 cytokine,arAAV2IL27 vectorwasconstructedby insertingthegenesencodingthetwosubunitsofIL-27 (Ebi3andp28)intoapTR-UF14vector. Ebi3 and p28 weregeneratedbypolymerasechainreaction(PCR) frommurinecDNAbyusingtwopairsofprimers( Ebi3 : forward5 -AAACTAGTAGGTCCTTCCCTGGGGCCAGGT-3 andreverse5 -TTTGATATCAAGGATCCAGTCCCTCTTCAG-3 and p28 :forward5 -AAAAA GCGGCCGCATGGGCCAGGTGACAGGA-3 and reverse5 -TTTGTCGACTTAGGAATCCCAGGCTGAGCC-3 ).Ebi3andp28subunitsflankedbyIRES componentwereconstructedallowingtheco-expression ofIL-27subunitsdrivenbythesamepromoter(Figure S1BofAdditionalfile1).WeusedAAV2vectorencodingbetagalactosidase(rAAV2LacZ )asanAAV2controlvector,whoseconstructionwaspreviouslyreported [22].FunctionalactivityofIL-27expressedfromtherAAV2IL27 vectorrAAV2IL27 anditsbackbone(pTR-UF14)plasmids weretransfectedseparatelyintoHEK293cellsbyusing TransIT-293Transfectio nReagent(MirusBioLLC, Madison,WI,USA)inaccordancewiththeinstructions ofthemanufacturer.Aftertransfection,cellpelletand culturemediawerecollectedatdifferenttimepointsup to48hours,andexpressionofthetwosubunitsofIL-27 wasmeasured.Ebi3wasdeterminedbyWesternblotting byusinganti-mouseEbi3antibody(SantaCruzBiotechnology,SantaCruz,CA,USA),andp28wasdetectedby enzyme-linkedimmunosorbentassay(ELISA)byusinga QuantikinekitformouseIL-27p28(R&DSystems, Minneapolis,MN,USA).Biologicalactivitywasdeterminedbyflowcytometry.Culturesupernatantsfrom rAAV2-IL27oremptyAAV2-t ransfectedHEK293cells werecollected(mock).C57BL/6Jsplenocyteswereincubatedwithmock,IL-6/transforminggrowthfactor-beta (IL-6/TGFb )foroptimalTH17differentiation,andIL6/TGFb withsupernatantsofAAV2-IL27transfected cellsat37Cfor5days.IL-17+cellsweremeasuredby flowcytometry.PackagingofrecombinantAAV2(rAAV2)vectorparticlesTogeneraterAAV2viralparticles,adenoviralhelper packagingplasmidpDGwasused.Plates(15cm)withless than40%confluentHEK293Tcellswereco-transfected witheitherpAAV2LacZ (controlvector)orrAAV2IL27Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page2of14

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inaccordancewithstandardiz edmethods[23].Clarified celllysateswereadjustedtoarefractiveindexof1.372by theadditionofCsClandcentrifugedat38,000revolutions perminute(rpm)for65hoursat20C.Equilibriumdensitygradientswerefractionated,andfractionswitha refractiveindexof1.369to1.375werecollected.Theparticletiterwasquantifiedbyreal-timePCR,andthevector wasstoredat-80C.InjectionsofvectorsTwodifferentagegroupsofC57BL/6.NODAec1Aec2 micewereusedtotakeadvantageofthepre-diseaseand clinicaldiseasestagesoftheanimal[24]fortheIL-27 genetherapy,therebyaimingtoeitherpreventtheonset ofthediseaseorreversethediseasephenotypeofSjS (6-week-oldmiceforpre-diseasestageand14-week-old miceforclinicaldiseasestage,respectively).Micewere treatedsystemicallywith salineorrAAV2-LacZor rAAV2-IL27,andtherewere10mice(fivemaleandfive female)pertreatmentforeachdiseasestage.Systemic deliverywasdoneviaintravenousinjectionintailveins. Micewereanesthetizedwi thaketamine(100mg/mL, 1mL/kgbodyweight;FortDodgeAnimalHealth,Fort Dodge,IA,USA)andxylazine(20mg/mL,0.7mL/kg bodyweight;PhoenixScientific,St.Joseph,MO,USA) cocktailbyintraperitonealinjection.Then50 Lofvectorsolutioncontaining21010vectorgenome(VG) wasinjected.Controlgroupsreceivedeitherthesame amountofrAAV2-LacZVGorthesamevolumeofsaline.Thedosagewaschosenonthebasisofpublished studiesbyothersforextensivestudyofdosageoptimization[25-29].Allanimalsweresacrificedat20weeks afterdelivery.Atthetimeofeuthanization,systemic transgeneexpressionwasdeterminedbyusingquantitativereal-timePCR(FigureS2ofAdditionalfile2).IntracellularcytokinestainingandflowcytometryanalysisSingle-cellsuspensionsofspleencellswereprepared fromC57BL/6.NODAec1Aec2 miceasdescribedelsewhere[30]andattheagesdesignatedinthetext.In brief,spleenswerefreshlyexplanted,gentlyminced throughstainlesssteelsiev es,suspendedinphosphatebufferedsaline(PBS),and centrifuged(1,200rpmfor 5minutes).Erythrocyteswerelysedbya7-minuteincubationin0.84%NH4Cl.TheresultingleukocytesuspensionswerewashedtwotimesinPBS,counted,and re-suspendedinculturemedia(RPMI1640medium, 10%fetalbovineserum,2mML-glutamine,and 0.05mM b -mercaptoethanol)toadensityof2106cells/mL.Onemillioncellswerepipettedtoindividual wellsofa24-wellmicrotiterplatepre-coatedwithantiCD3(10 g/mL;BDPharmingen,SanDiego,CA,USA) andanti-CD28(2 g/mL;BDPharmingen)antibodies forT-cellactivation.Cellswereincubatedfor5hours withLeukocyteActivationCocktailcontainingGolgiPlug (2 L/mL;BDPharmingen).Collectedcellswerefixed andpermeabilizedbyusingaCytofix/CytopermFixation/ Permeabilizationkit(BDPharmingen).Theflowcytometryacquisitionforintra-a ndextra-cellularstaining wasperformedfollowingstainingwithfluoresceinisothiocyanate(FITC)-conjugatedanti-mouseIL-27(R&D Systems),phycoerythrin(PE)-conjugatedanti-IL-17 (eBioscience,SanDiego,CA,USA),andPE-Cy7-conjugatedanti-mouseCD4(Invitrogen,Carlsbad,CA,USA) forthedetectionofcytokinesfromgene-deliveredmice. ThecellswerecountedbyusingaBDACCURIC6 FlowCytometer(BDBiosci ences,SanJose,CA,USA) andanalyzedbyFlowJosoftware(TreeStarInc.,Ashland,OR,USA).CD3+andCD4+Tcellsweregated, andthecellswereanalyzedforIL-27+orIL-17+cells.CytokinelevelofperipheralbloodMeasurementofIL-27levelsinserasampleswasconductedbyusingaQuantikinekitformouseIL-27p28 (R&DSystems).IL-17,andIL-10levelsaredetectedusing mouseIL-17andIL-10ELISAkits(LifeTechnologies, GrandIsland,NY,USA).Allprocedureswereperformed inaccordancewiththeinstructionsofthemanufacturers. ReadingswerecarriedoutbyusingaModel680MicroplateReader(Bio-Rad,Hercules,CA,USA).Hematoxylinandeosinstainingandhistological assessmentSalivaryglands,lacrimalglands,liver,lung,andkidney weresurgicallyremovedfromeachC57BL/6.NODAec1Aec2mouseatthetimeofeuthanasiaandplaced in10%phosphate-bufferedformalinfor24hours.Fixed tissueswereembeddedinparaffinandsectionedata thicknessof5 m.Paraffin-embeddedsectionswere deparaffinizedbyimmersinginxylene,followedby dehydrationinethanol.The preparedtissuesections werestainedwithhematox ylinandeosin(H&E)dye (HistologyTechServices,Gainesville,FL,USA).H&Estainedsectionswereobservedunderamicroscopefor glandularstructureandleu kocyteinfiltration.Lymphocyticfoci(LF)wereenumeratedbythreeindividualsin blindedfashion.LFweredefinedasaggregatesofmore than50leukocytesquantifiedpereachhistologicalsection,andadjacentsectionswereusedforimmunofluorescentstaining.Stainedsectionswereobservedat6 magnificationbyusingaLeicaMZ8microscope,and imageswereobtainedwithLeicaapplicationsuite(version2.4.0.R1;Leica,Wetzlar,Germany).ImmunofluorescencestainingTissuesectionsweredeparaffinized,rehydrated,and blockedwithhydrogenperoxide.Forantigenretrieval, tissuesectionswereheatedto100CunderpressurewithLee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page3of14

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10mMcitratebufferfor15minutes.Thesectionswere incubatedwithratanti-mouseB220(BDPharmingen, SanDiego,CA,USA)orgoatanti-mouseCD3(Santa CruzBiotechnology,Inc,SantaCruz,CA,USA)todetect BcellsandTcells,respectively,overnightat4C.The sectionswerewashedtwotimeswithPBS-Tweenand incubatedwithTexasRed-conjugatedrabbitanti-ratIgG (Biomeda,FosterCity,CA,USA)forB220markerand FITC-conjugatedrabbitanti-goatIgG(Sigma-Aldrich,St. Louis,MO,USA)forCD3detectionfor30minutesat roomtemperature.Afteradditionalwashes,theslides weretreatedwithmountingmediumwith4 -6-diamidino-2-phenylindole(DAPI)(VectorLaboratories, Burlingame,CA,USA).Stainedsectionswereobservedat magnificationwithaZeissAxiovert200Mmicroscope,andimageswereobtainedwithAxioVs40software (version4.7.1.0;CarlZeiss,Jena,Germany).Detectionofanti-nuclearantibodiesintheseraAnti-nuclearantibodies(ANAs)intheseraofmicewere detectedbyusinganHEp-2ANAkit(InovaDiagnostics, Inc.,SanDiego,CA,USA).Allprocedureswereperformed inaccordancewiththeinstructionsofthemanufacturer. Inbrief,HEp-2-fixedsubstrateslideswereoverlaidwith appropriatemouseseradiluted1:50,1:100,1:200,1:400, and1:800.Slideswereincubatedfor1houratroomtemperatureinahumidifiedchamber.Afterthreewashesfor 5minuteswithPBS,thesubstrateslideswerecoveredwith Alexa488-conjugatedgoatanti-mouseIgG(H/L)(Invitrogen)diluted1:100for45minutesatroomtemperature. Afterthreewashes,fluorescencewasdetectedbyfluorescencemicroscopyat00magnificationbyusingaZeiss Axiovert200Mmicroscope,andallimageswereobtained withAxioVs40software(version4.7.1.0;CarlZeiss)witha constantexposureof0.3seconds(CarlZeiss).Inthis study,threeindividualsinblindedfashionexaminedpositivestainingpatterns.MeasurementofstimulatedsalivaflowTomeasurestimulatedsalivaflow,eachmousewas weighedandinjectedwithacocktailofisoproterenol (0.2mg/mL)andpilocarpine(0.5mg/mL)in100 Lof salinebyintraperitonealinjection.Salivawascollectedfor 10minutesfromtheoralcavityofindividualmiceby usingamicropipettestarting1minuteafterinjectionof thesecretagogue.Thevolumeofeachsalivasamplewas measured.Baselinesalivaflowrates(SFRs)weremeasured aweekbeforethegenetherapy,andSFRsweremeasured every4weeksafter8weeksofpost-genedeliveryperiods untiltheendoftheexperimentineachgroup.StatisticalanalysesThedataareexpressedasthemeanstandarderrorof themean.Statisticalevaluationwasdeterminedbyusing one-wayanalysisofvariancetestwiththeGraphPad Prismsoftware(GraphPadSoftware,Inc.,LaJolla,CA, USA)orchi-squaredtest.A P valueoflessthan0.05 consideredstatisticallysignificant.ResultsrAAV2IL27 plasmidishighlyefficientinproducinga functionalIL-27product invitroTodeterminefunctionalactivityoftherAAV2IL27 plasmidconstruct,HEK293cellsweretransfectedwitheither therAAV2IL27 oritsbackbone,pTR-UF14plasmid.To controlforbackground,untreatedHEK293cellswereculturedinmediumcontainingthetransfectionreagentsonly (representingamocktransfection).Forty-eighthoursafter transfection,celllysateswerepreparedandculturemedia werecollectedtoquantifyexpressionlevelsbyELISAof bothsecretedandnon-secretedIL-27protein.AspresentedinFigure1A,IL-27p28levelsweresignificantly increasedinlysatesfromcellstransfectedwithrAAV2IL27 (222pg/mL)incomparisonwithlysatesfromeither themocktransfected(24pg/mL)orpTR-UF14transfected (22pg/mL)cells.Similarly,examinationofculturemedia showedelevatedlevelsofIL-27p28(272pg/mL),butIL27p28wasnotdetectedineitherculturemediaofmock transfectedorpTR-UF14transfectedHEK293cells.Analysisofthecelllysatesandculturemediaalsoindicatedthat Ebi3expressionwashighlyincreasedinrAAV2IL27 transfectedcells(datanotshown). Biologicalactivityofreco mbinantIL-27(rIL-27) secretedbyrAAV2IL27 wasdeterminedbyusingculture mediasupernatantscollectedfromtherAAV2IL27 and controlbysupernatantsfrompTR-UF14transfected HEK293cellsorcellsalone.C57BL/6JsplenocytesculturedinmediacontainingIL-6andTGFb supplemented withsupernatantscontainingrIL-27showedsignificant decreasesinIL-17+cellswithconcomitantincreasesin IFNg+cellsincomparisonwithmediasupplementedwith supernatantfromeitherpTR-UF14ormocktransfected cells(Figure1B).Consideredtogether,theseresultsindicatethattheheterodimericrIL-27proteinisproperly expressedbytherAAV2IL27 vectorandisbiologically functionalinsuppressingTH17cells invitro .DecreasedserumIL-17levelsinmicefollowingrAAV2IL27 treatmentTodeterminethesystemiccytokinelevelsofIL-27in C57BL/6.NOD-Aec1Aec2 micefollowingtreatmentwith rAAV2IL27 ,rAAV2LacZ (vectorcontrol),orsaline (negativecontrol),seraoftreatedmicewereexamined 8,12,and20weeksaftergenedelivery.Asindicatedin Figure2A,6-week-oldmiceinoculatedwithrAAV2IL27 exhibitedsignificantincreasesinIL-27levels duringthesubsequent20-weekobservationperiod, resultinginathreefoldincreaseincomparisonwiththeLee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page4of14

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rAAV2LacZ -orsaline-treatedcontrolgroups(91versus34and30pg/mL,respectively).HighlevelsofcirculatingIL-27wereconcomitantlycorrelatedwithIL-17 leveldecreasesobservedatboth12and20weeksafter injection(Figure2C).Interestingly,IL-10levelswere unaffectedbytheincreasedlevelsofIL-27(Figure2E). Similar,yetmorepronounced,resultswereobserved inmiceadministeredrAAV2IL27 at14weeksofage, thetimeofadaptiveimmunityonsetintheC57BL/6. NODAec1Aec2 mice.At8weeksafterinjection,mice injectedwithrAAV2IL27 showeddramaticincreasesin thelevelsofIL-27incomparisonwiththerAAV2LacZ orsaline-treatedcontrolgroups(278versus175and173 pg/mL,respectively),andt heselevelsweremaintained foratleast20weeksafterinjection(Figure2B). Concomitantly,rAAV2-IL27 -treatedmiceshowed reducedlevelsofIL-17atboth12and20weeksafter vectordeliveryincomparisonwiththerAAV2LacZ andsaline-treatedcontrolgroups(Figure2D),indicating thatdecreasedexpressionofIL-17intheseraiscorrelatedwiththeincreasedexpressionofIL-27inthe rAAV2IL27 -treatedmice.Incontrasttomicetreatedat 6weeksofage,IL-10washighlyelevatedat8weeks afterinjectioninmicetreatedwithrAAV2-IL27 inthe clinicaldiseasephase(Fig ure2F).Thesedatasuggesta directnegativeregulationofTH17cellswithacorrespondingupregulationofIL-10production,whichcould furtherinhibitcytokineproductionsfromTH1,TH2,and TH17cells.Thus,thetwodiseasestagesofSjSrespond differentlytoIL-27treatment. Figure1 GenerationoffunctionalIL-27-expressingvector(rAAV2IL27 ) (A) IL-27p28expressionlevelsinthecelllysatesorculturemedia ofmocktransfectionwhichcontaintransfectionreagentsonly(whitebar)orcontrolemptyAAV2plasmid(greybar)orrAAV2-IL27(blackbar) transfectedHEK293cellsat48hoursaftertransfection. (B) BiologicaleffectsofIL-27weregeneratedfromrAAV2-IL27onC57BL/6splenocytesby usingmediaonlyor (a) mock, (b) IL-17differentiationcocktailcontainingIL-6andtransforminggrowthfactor-beta,or (c) IL-17differentiation cocktailwithsupernatantIL-27fromrAAV2-IL27transfectedHEK293cells(sIL-27).Theexperimentwasdoneintriplicateandrepeatedthreetimes forconsistency.Valuesinbargraphsaremean.** P <0.01,rAAV2IL27 groupversusmockorrAAV2transfectedbyone-wayanalysis-of-variance test.IFNg ,interferon-gamma;IL,interleukin. Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page5of14

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Figure2 ChangesincirculatingcytokinesinserafollowingrAAV2IL27 treatment .Tenmice(fivemaleandfivefemale)weretreated systemicallywithsaline(negativecontrol)orrAAV2LacZ (vectorcontrol)orrAAV2IL27 pertreatmentforeachdiseasestage(6-week-oldmice forpre-diseasestageand14-week-oldmiceforclinicaldiseasestage).Eachmousewasinjectedwith21010vectorgenomeofrAAV2IL27 or rAAV2LacZ in50 Lofsalineviaintravenouslineintailveins.Serawerecollected1weekbefore(-1,baseline)and8,12,and20weeksafterthe treatmentofpre-diseaseandclinicaldiseasemicegroups.Levelsofcytokines(IL-27,IL-17,andIL-10)weredetectedbyenzyme-linked immunosorbentassay.IL-27levelsinpre-disease( A )andclinical-disease( B )stages.IL-17levelsinpre-disease( C)andclinical-disease( D )stages. IL-10levelsinpre-disease( E )andclinical-disease( F )stages.Valuesaremeanstandarderrorofthemean( n =7).* P <0.05rAAV2IL27 group versusrAAV2LacZ orsalinegroupsbyone-wayanalysis-of-variancetest;** P <0.01.IL,interleukin;IL-27,rAAV2IL27 -treatedgroups;LacZ,rAAV2LacZ -treatedgroups;Saline,saline-treatedgroups. Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page6of14

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IntravenousinjectionofrAAV2IL27 negativelyregulates splenicTH17cellfunctionsAsmentionedpreviously,IL-27functionstoinhibitthe activityofTH17cellsbynegativelyregulatingtheproductionofIL-17whiledirectlypromotingtheproductionof IFNg byTH1cells.SincerAAV2IL27 wasadministered intravenously,splenocyteswereisolatedtoexaminethe systemiceffectsofthevectoronTH17andTH1cells. Splenocytesfrommicetreatedateither6or14weeksof agewereisolated20weeksaftertreatmenttoprofilethe levelsofIL-27,IFNg ,andIL-17producedbyspecific T-cellpopulations.AspresentedinFigure3,rAAV2IL27 -injectedmiceshowedrelativeincreasesinthenumberofCD3+CD4+IL-27+cells,determinedbyflowcytometryanalyses,incomparisonwithmicetreatedwith rAAV2LacZ vectororsaline.TheincreasesinIL-27+TcellswereassociatedwithincreasesinIFNg+cells. AlthoughrAAV2IL27 treatmentofmiceat6weeksof ageresultedinincreasednumbersofIL-27+Tcells,there werenodifferencesinIL-17+Tcellsbetweensaline-, rAAV2LacZ -,andrAAV2IL27 -treatedgroups,andthis waspossiblyduetotheoveralllownumbersofIL-27+Tcells.Incontrast,miceinjectedwithrAAV2IL27 at14 weeksofagehaddecreasednumbersofIL-17+Tcells withaconcomitanttwo-tofourfoldincreaseinIL-27+TcellsoverrAAV2LacZ -andsaline-treatedgroups (Figure3).Thesedataareconsistentwiththeconcept thatsystemicdeliveryofIL-27canregulatethelevelsof IL-17+Tcellsinthespleen,buthighlevelsofexogenous IL-27mayneededtosuppressthelevelsofIL-17.Lymphocyticinfiltratesintheexocrineglandsof rAAV2IL 27-injectedmiceLymphocyticinfiltrationpresentinthesalivaryglandsis oneofthemaindiagnosticmarkersforSjS.Inthe C57BL/6.NODAec1Aec2 mousemodel,thedevelopment andonsetofautoimmunityintheexocrineglandsare consideredtheresultsofacinarcellapoptosisthatsignals aninfluxofleukocytesexpressingpro-inflammatory cytokines.Salivaryglandlymph ocyticinfiltratesinitially arecomposedofT-cellclustersfollowedbyrecruitment ofBlymphocytes[31,32].Themigrationofspecific T-cellpopulationssecretingIL-17andIL-23hasbeen showntodirectlycontributetothepathogenesisofthe disease[33].Therefore,todeterminetheeffectof rAAV2IL27 treatmentonlymphocyticinfiltrationinthe salivaryglandsofmiceinjectedat6or14weeksofage withrAAV2IL27 ,freshlyexplanted,formalin-fixed,paraffin-embeddedsalivaryglandsectionswereexamined aftereuthanizatonat20weeksaftertreatment. Representativeimagesofsa livaryglandinfiltrates, showninFigure4,revealthepresenceofLFcomposed ofbothBandTlymphocytes,whereasthenumbersofLF inthesalivaryglandswerescoredforeachexperimental group(Table1).Ascanbeseen,rAAV2IL27-treated miceshowednosignificantreductionsinthenumberof Figure3 rAAV2-IL27-expressingvectoriscapableofnegativelyregulatingsplenicTH17cells .Splenocyteswereisolatedfromeachmouse andstainedforCD3andCD4fluorochrome-conjugatedantibodies.CellswerefurtherintracellularlystainedforIL-27,IL-17,andIFNg .Flow cytometryanalysiswasperformedwithcellsgatedonCD4+populations.Valuesaremeanstandarderrorofthemean( n =7).* P <0.05 rAAV2IL27 groupversusrAAV2LacZ orsalinegroupsbyone-wayanalysis-of-variancetest;** P <0.01.IFNg ,interferon-gamma;IL,interleukin;IL27,rAAV2IL27 -treatedgroups;LacZ,rAAV2LacZ -treatedgroups;Saline,saline-treatedgroups. Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page7of14

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LFinthesalivaryglandsincomparisonwitheither rAAV2LacZ -orsaline-treatedmice,irrespectivelyofthe ageatwhichtreatmentofthemicewascarriedout.In contrast,lacrimalglandsoftherAAV2IL27 -treatedmice werenotedtohaveadecreaseinthenumbersofLFin comparisonwithsaline-treatedmice,whereaslacrimal glandsexplantedfrommicetreatedwithrAAV2LacZ showedslightlylowerLFscores.Furthermore,ingeneral, largerinfiltrateswerefoundintherAAV2LacZ -andsaline-treatedgroupswithhighernumbersofBcellsin comparisonwithrAAV2IL27 -treatedmice.Owingto theintravenousinjectionoftheviralvectors,other Figure4 Lymphocyticinfiltratesinthesalivaryglands .Representativeimagesforhematoxylinandeosin(H&E)andCD3/B220stainingfor saline-,rAAV2-LacZ-,andrAAV2-IL27-treatedgroupsat20weeksaftergenedeliveryinpre-diseaseandclinicaldiseasegroups.H&Eimageswere takenatmagnificationandimmunofluorescencestainingimagesoflymphocyticinfiltrateswithanti-CD3todetectTcells(green)andantiB220todetectBcells(red)weretakenatmagnificationwithaZeissAxiovert200Mmicroscope.IL-27,rAAV2IL27 -treatedgroups;LacZ, rAAV2LacZ -treatedgroups;Saline,saline-treatedgroups. Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page8of14

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organssuchasliver,lungs,andkidneywerealsoexaminedforlymphocyticinfiltration.However,nosignificant pathologicalchangeswereobservedbetweenmice belongingtothedifferenttreatmentgroups(datanot shown).Theseresultsindicatethatsystemicinjectionof rAAV2IL27 hasapositiveimpactonLFscoresinthe lacrimalglands,especiallywhentreatmentisinitiatedat 6weeksofage,butappearstohavelittleornoimpacton LFscoresinsalivaryglands.IntravenousinjectionofrAAV2IL-27 treatmentaltersthe anti-nuclearantibodyprofileinC57BL/6.NODAec1Aec2 micePatientswithSjSoftendevelopANAs,specificallyantiRo(SS-A)oranti-La(SS-B)orboth.ANAsthatgenerallyshowaspeckedstainingpatternwhenHEp-2cells areusedarehighlyprevalentinSjS,andthetitersof suchANAsareclinicallyimportantfactorsindiagnosing thedisease.ToidentifythepresenceofANAs,seracollectedfromC57BL/6.NODAec1Aec2 micetreatedat either6or14weeksofagewithrAAV2IL27,rAAV2LacZ ,orsalineweretestedforANAreactivitybyusing HEp-2cells.Antibodytiterswereexaminedatdilutions of1:50,1:100,1:200,1:400,and1:800.Aspresentedin Figure5A,serafrommiceinjectedwithrAAV2IL27 at eitherageshowedcomparativelyweakornonuclear speckledantibodystainingontheHEp-2cells(shownat adilutionof1:400),whereasmiceinjectedwithAAV2LacZ orsalineshowedstrongANAstaining,andthis waspredominantlyanuclearspeckledpattern.QuantificationofthenuclearspeckledANAtitersclearly revealedthat,inmicetreatedateitherage,thetiters presentinrAAV2IL27 -treatedmiceweresignificantly lowerthaninmicereceivinginjectionsofrAAV2LacZ vectororsaline(Figure5B,C).Thus,theseresultssuggestthatincreasedlevelsofIL-27inserafollowing rAAV2IL27 treatmentdirectlyaffectproductionorprofilesofautoantibodies(orboth)inSjSmice.MeasurementofstimulatedsalivaflowratesTocompareSFRsintheC57BL/6.NODAec1Aec2 mice injectedwithrAAV2IL27,rAAV2LacZ ,orsaline, pilocarpine/isopreterenol-stimulatedsalivacollections werecarriedoutevery4weeksstarting8weeksafter treatment,andeachvalueequatedtotheaveragebaselineSFRscollectedfromthemice1weekpriortobeginningtreatment.AspresentedinFigure6,micetreated witheitherrAAV2IL27 orrAAV2LacZ at6weeksof ageexhibitedarelativelyrapidlossinSFRs.Miceinthe rAAV2IL27 treatmentgroup,however,showedaslight recoveryofSFRswithtime,whereasmiceinthe rAAV2LacZ treatmentgroupremainedlow.As expected,micereceivingsalineshowedaprogressive lossinSFRs.Theseresultssuggestthattheviralvectors perse caninfluencetheSFRsandthatIL-27maybe abletoreversetheeffect,albeitweakly. Incontrasttotheaboveresults,micetreatedwitheither rAAV2IL27 orrAAV2LacZ at14weeksofageexhibited differentresponsestothetreatments(Figure6B):mice receivingtherAAVLacZ vectoragainexhibitedarapid lossofSFR,whereasmicereceivingtherAAV2IL27 vectorslowlyshowedminorimprovementsintheirSFRs. MicereceivingsalinebeganshowingaslowerSFRafter about8weeksaftertreatment(or22weeksofage),consistentwithdiseaseinthismodel.Theseresultsindicatethat IL-27mightimpactthedelayedrecoveryofSFRswhen treatmentisdonepriortotheonsetofdiseaseandmay partiallyrestoresalivasecretionandglandularfunctionat alaterstage.DiscussionThisstudywasundertakentoexaminetheeffectsofIL-27 genetherapyinSjSbyusingtheC57BL/6.NODAec1Aec2 mousemodelofprimarySjS.SjSischaracterizedbyhyposalivation,autoantibodiesinsera,andlymphocyticinfiltrationinthesalivaryglands.Previousfindingsalsosuggested arolefortheIL-23/TH17/IL-17Asysteminbothpatients withSjSandanimalmodels[33,34].Therefore,we hypothesizedthatIL-27wouldhaveatherapeuticrolein treatingSjSandshowpotentialasatherapeuticforthe humandisease.Theresultsofthepresentstudygenerally supportourhypothesis,showingthatIL-27overexpression bytherAAV2-IL27 vectorinC57BL/6.NODAec1Aec2 miceincreasedsalivasecretionanddecreasedANA Table1Quantificationoflymphocyticinfiltratesinthesalivaryglandsofgene-deliveredmousegroupsDiseasestage Salivaryglands Lacrimalglands Treatment LF MeanaLF Mean Saline5/6b(83%)c2.31.5 4/6(67%) 2.81.3 Pre-disease Lac-Z 7/7(100%) 3.02.4 6/7(86%) 1.50.7 IL-27 6/7(86%) 4.32.2 2/7d(29%) 2.01.4 ClinicaldiseaseLac-Z7/7(100%)3.42.44/7(57%)2.51.0 IL-27 7/7(100%) 3.41.3 2/7(29%) 4.55.0aMeanlymphocyticfoci(LF)scoreofeachglandinmousestandarderrorofthemeanperhistologicalsalivaryglandsection;bnumberofmice;cpercentageof mice;dP <0.05bychi-squaredtest.IL-27,rAAV2IL27 -treatedgroups;LacZ,rAAV2LacZ -treatedgroups;saline,saline-treatedgroups.Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page9of14

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formationdespitealackofdifferencesinLFscores betweenrAAVIL27 -andrAAV2LacZ vector-treated miceorsaline-treatedcontrolsinjectedat6or14weeksof age.Interestingly,rAAV2IL27 treatmentattheearlyclinicaldiseasestageappearedtoshowabetterresponsethan treatmentatapre-diseasestage. TheroleofIL-27hasbeenstudied,buttheunderlying mechanismofIL-27inautoi mmunedisease,specifically SjS,isstillunknown.AlthoughIL-27haspro-inflammatoryfunctions,whichareembodiedinactivationofIFNg productionfromTH1cells,manyrecentstudiesare showinganti-inflammatoryeffectsofIL-27against Figure5 Nuclearspeckledanti-nuclearantibodystainingpatternprofile (A) Representativeimagesofnuclearspeckledanti-nuclear antibodyatadilutionof1:400.Serawerecollectedinthepre-diseasegroupat6weeksofage(beforetreatment)and26weeksofageor20 weeksaftertreatment.Intheclinicaldiseasegroup,serawerecollectedat14weeksofage(beforetreatment)and34weeksofageor20weeks aftertreatment.ImageswerecapturedatmagnificationwithaZeissAxiovert200Mmicroscopewithaconstantexposureof0.3seconds. (B) Nuclearspeckledtitersinpre-diseasetreatedmice. (C) Nuclearspeckledtitersinclinicaldiseasetreatedmice.Threeindividualsinblinded fashiondeterminedautoantibodytiters.Valuesaremeanstandarderrorofthemean( n =7).* P <0.05rAAV2IL27 groupversusrAAV2LacZ or salinegroupsbyone-wayanalysis-of-variancetest.IL-27,rAAV2IL27 -treatedgroups;LacZ,rAAV2LacZ -treatedgroups;Saline,saline-treated groups.Thesesymbolshavenospecificmeaningsbecausethelegendsonthex-axisdefinedthem.However,ifwemustdefinethesesymbols, areyouokwith?Pre-diseasestage:6weeksoldmice(invertedtriangle),saline-treatedmice(circle),rAAV2LacZ -treatedmice(square),rAAV2IL27 treatedmice(triangle).Clinical-diseasestage:14weeksoldmice(invertedtriangle),saline-treatedmice(circle),rAAV2LacZ -treatedmice(square), rAAV2IL27 -treatedmice(triangle). Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page10of14

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microorganismsandeffectorTcells,includingTH17 cells.RecentstudieshavealsoshownthatIL-27is involvedinanti-inflammatoryfunctions,especiallyinterferingwithTNFa orTH17cells(orboth)inautoimmunediseases,includingmult iplesclerosis,rheumatoid arthritis,andsystemiclupuserythematosus[35-38].For thepotentialtherapeuticuseofIL-27,previous invivo and invitro studieshaveshownthatIL-27canbeusedas ananti-tumororanti-viralagentaswellasamodulator ofautoimmunity[39-43].IL-2 7-expressingvectorshave beenshowntoexhibitpotentanti-tumoractivityin malignanciesandmetastatictumors[9,44,45].Therefore, thepresentstudy,usingIL-27inagenetherapyprotocol, providesevidencethatsupportsitspotentialeffectiveness inautoimmunity. Ourpreviousstudy,inwhichanadenoviralvectorwas used,demonstratedthatoverexpressionofIL-17wascapableofrecapitulatingSjSphenotypes(loweredsalivasecretionandincreasedautoanti bodies)innormalC57BL/6J miceand,atthesametime,showeddifferentialimmunologicalorbiologicalresponsesdependingontheageofmice atthetimeoftreatment[34].Byinducinglong-term expressionofIL-27levelsbyusingAAV2,ourdatashowed markedreductionsinIL-17cytokinelevelsinbloodfrom rAAV2IL27 -injectedmiceattwodifferentphasesofdiseaseonset.Interestingly,therewasdelayedexpressionof IL-27inrAAV2-IL27-treatedmiceintheinnateimmune phaseincomparisonwiththeexpressionintheadaptive immunephase.Thisphenomenoncouldbeattributedto theageoftheanimalsatthetimeofvectortreatment. AstudybyBostickandcolleagues[46]showedthatAAV9 transductionbysystemicdeliverycouldbeinfluencedby theageoftheanimals.TheseauthorsfoundthatAAVs exhibiteddifferenttransductionefficienciesandthatadult miceshowedhighertransductionefficiencythannewborn mice.Furthermore,otherstudieshaveimplicatedthe time lag effectofAAV2-mediatedtransgeneexpressioninvarioustissues,specificallymyocardium[47-49].Zincarelliand colleagues[50]categorizedAAV2asalow-expressionand slow-onsetvirusalongwithAAV3,4,and5amongAAV serotypes1to9aftersystemicinjectioninmice.Ourstudy alsoshowedthattheregulationofIL-17couldbedirectly attributedtotheoverexpressionofanIL-27transgene,but thiscouldbetheindirectsystemiceffectofIL-27onother regulatorycytokinesandcelltypes.AlthoughserumIL-27 iselevatedat8,12,and20weeksaftertreatmentinthe 14-weektreatedgroup,serumIL-17ismarkedlydecreased at20weeksaftertreatment.AnumberoffactorscanregulatethefunctionofIL-17,especiallyTH1cellsreleasing IFNg [4],whichwasincreasedintherAAV2IL27 -treated group(Figure3).Also,independentrolesofIL-27subunits, p28andEbi3,mightbecritical.ArecentstudybyStumhoferandcolleagues[51]showedthatIL-27p28alonebinds togp130asapro-inflammat orysignalantagonist. Althoughgp130isthesubunitforIL-27receptorwith WSX-1(IL-27R a ),itisalsoasubunitforIL-6receptor, whichisinvolvedinTH17celldifferentiation.Inaddition, Ebi3,acytokinesubunitofIL-35,isknownforitssuppressionofTH17cells[52].AlthoughrAAV2IL27 vector expressesbothp28andEbi3,thepossibilityofindependent rolesofp28orEbi3couldnotbeexcludedhere.Lastly, othercelltypes(forinstance,Tr-1)canregulateTH17cells. IL-27hasbeenshowntobeabletostimulateTr-1cells, whichreleaseIL-10throughSTAT1andSTAT3signaling [53,54].IL-10isacytokinethatisknownforitsantiinflammatoryfunctionandtheinhibitoryeffectonTH17 cells.However,recentstudiesusingIL-27-overexpressing transgenicmiceshowedanantagonistroleofIL-27onTregcells,anothersourceforIL-10,throughIL-2modulation [55].Thus,IL-27mayhaveaneffectonthebalanceof Figure6 Measurementofstimulatedsalivaflowrates(SFRs) .Oneweekpriortotreatment,salivawascollectedandmeasuredtodetermine thebaselineSFRs.SFRsforeachtimepoint(8,12,16,and20post-genedeliveryperiods)werenormalizedagainstthebaselineSFRsindicatedas zero(0)post-genedeliveryperiods. (A) Genedeliveryinpre-diseasestageat6weeksofage. (B) Genedeliveryinclinicaldiseasestageat14 weeksofage.Valuesaremeanstandarderrorofthemean( n =7).* P <0.05rAAV2IL27 groupversusrAAV2LacZ orsalinegroupsbyone-way analysis-of-variancetest;** P <0.01.IL-27,rAAV2IL27 -treatedgroups;LacZ,rAAV2LacZ -treatedgroups;Saline,saline-treatedgroups. Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page11of14

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Tr-1cellsandTregcellsinthemouseorhumansystem. Therefore,multi-factorialregulationofIL-17mighthave contributedtothedatainthisstudy. OneuniquefeatureofSjSistheformationofleukocyte aggregatesinthesalivaryandlacrimalglands,referredto aseitherlymphoepithelialsialadenitisorLF[56,57].At times,theseLFappearhistologicallytobegerminal-like centersandmaybeindicativeofamorepronouncedand severehypergammaglobulinemia[58].AlthoughLFand LFscoresareimportantcrit eriaforclinicaldiseaseand depicttheleveloflymphocyticinfiltrationsoftheexocrine glands,thescoresoftendonotcorrelatewiththeseverity ofdisease[59,60].Untilrecently,itwasthoughtthatsalivarygland-infiltratingleukocytesweremostlyBlymphocytesandCD4+TH1cells.However,werecentlyreported thatLFcontainsignificantnumbersofCD4+TH17memoryTcellsplusIL-23-producingmacrophagesordendritic cellsorboth[33].Althoughthepresentstudyshowsno differenceinmeanLFscoresbetweenIL-27-treatedand AAV2LacZ vector-orsaline-treatedcontrolsattheinnate oradaptiveimmunephase,otherresearchhassuggesteda poorcorrelationbetweenthismarkerofdiseaseandsalivaryglandfunction[61]. Asmightbeanticipated,onlytreatmentwithrAAV2IL27 attheclinicaldiseasestageshowedastableandgradualincreaseofSFRscomparedwithbaselineSFRsover thecourseoftheexperiment,whereasmicetreatedwith rAAV2IL27 atthepre-diseasestageshowedslightrecoveryofglandularfunctionat20weeksaftertreatment. ThesedataareconsistentwiththeserumIL-17levels, whichweresignificantlydownregulatedat20weeksafter treatmentforbothstudiedgroups.Furtherinvestigations areneededtodeterminetheunderlyingmechanismof howIL-27influencesSFRs,althoughonepossiblemechanismisthedirectsuppressionofIL-17inpromotingtheformationofspontaneousgerminalcenters[62],thereby dampeningpathogenicautoantibodyproduction[63].This conceptappearstobesupportedinpartbythealtered ANAprofileandsignificantdecreaseinautoantibodytiters inrAAV2IL27 -treatedmice.ConclusionsWehaveprovidedaproofofconceptthatexogeneousIL27isapotentialfactorfort reatingSjS.Althoughadditionalresearchinregardtogenedeliverymethodsand optimaldiseasetimepointsareneededtofurthervalidate theuseofrAAVgenetransfer,theuseofmousemodels andsystemicdeliveryofIL-27providesauniquesystem tostudytreatmentofautoimmunityattheclinicaldisease stage,whichisapplicablefortreatingpatientswithestablisheddisease.OurresultssuggestthatIL-27genetherapycouldbeaneffectivetherapeuticstrategytoreverse thesymptomsofSjSandshouldbefurtherinvestigated.AdditionalmaterialAdditionalfile1:FigureS1.GenerationofIL-27expressingserotype 2adeno-associatedviralvector(AAV2-IL27) .A:DiagramofpTR-UF14 vector,whichwasgivenbyDr.SergiZolotuhkin(Departmentof Pediatrics,UniversityofFloridaCollegeofMedicine)fortheback-bone structureofrAAV2-IL27.B:DiagramofrAAV2-IL27.Tofullyrecapitulate thefunctionalityofmouseIL-27cytokine,arAAV2-IL27vectorwas constructedbyinsertingthegenesencodingthetwosubunitsofIL-27 (Ebi3andp28)intoapTR-UF14vector. Additionalfile2:FigureS2.RelativeexpressionofIL-27p28mRNA inliver .Tocomparethetransgeneexpression,liversineachgroupwere collectedattheendofexperiments(20-weekofpostdeliveryperiods) andtotalRNAswereextractedforcDNAsynthesis.PCRprimerswere designedusingIDT sPrimerQuestSM(IntegratedDNATechnologiesInc., Coralville,IA,USA).QuantitativerealtimePCRwasperformedusingthe iCyclerIQTMmulti-colorrealtimePCRdetectionsystem(Bio-Rad Laboratories).TheCtvaluesobtainedwerenormalizedtothoseof18S ribosomalRNA.LevelofIL-27p28mRNAinLacZorIL-27delivered groupwerenormalizedagainstthemRNAlevelinsalinegroup.(values aremeanSEM,*p<0.05rAAV2-IL27groupversusrAAV2-LacZorsaline groupsbyone-wayANOVAtest). Abbreviations AAV2:serotype2adeno-associatedviralvector;AEC:autoimmune exocrinopathy;ANA:anti-nuclearautoantibody;EAE:experimental autoimmuneexcephalomyelitis;Ebi3:Epstein-Barrvirus-inducedgene3; ELISA:enzyme-linkedimmunosorbentassay;FITC:fluoresceinisothiocyanate; gp130:glycoprotein-130;H&E:hematoxylinandeosin;IFN:interferongamma;IL:interleukin;IRES:internalribosomeentrysite;LF:lymphocytic foci;PBS:phosphate-bufferedsaline;PCR:polymerasechainreaction;PE: phycoerythrin;rAAV:recombinantadeno-associatedvirus;rpm:revolutions perminute;SFR:salivaflowrate;SjS:Sjgren ssyndrome;STAT:signal transducerandactivatoroftranscription;TGF:transforminggrowthfactorbeta;TH:Thelper;TNF:tumornecrosisfactor-alpha;Treg:regulatoryT;VG: vectorgenome. Acknowledgements WethankSergiZolotuhkin(DepartmentofPediatrics,UniversityofFlorida CollegeofMedicine)forgenerouslyprovidingtheAAV2vectors(pTR-UF14). WethankTeganNLavoieforhelpingwiththeflowcytometryanalysisused inthisstudy.ThisworkwassupportedbytheSjgren sSyndrome Foundation(CQN)andPublicHealthService(PHS)grantK99DE018958 (CQN)fromtheNationalInstituteofDentalandCraniofacialResearch.ABP andCQNweresupportedbyPHSgrant1R21AI081952fromNational InstituteofAllergyandInfectiousDiseases. Authordetails1DepartmentofOralandMaxillofacialDiagnosticSciences,Collegeof Dentistry,UniversityofFlorida,Gainesville,FL32610,USA.2Departmentof OralBiology,CollegeofDentistry,UniversityofFlorida,Gainesville,FL32610, USA.3MolecularPhysiologyandTherapeuticsBranch,NationalInstitutesof DentalandCranialResearch,NationalInstitutesofHealth,Bethesda,MD 20892,USA.4CenterforOrphanAutoimmuneDisorders,CollegeofDentistry, UniversityofFlorida,Gainesville,FL32610,USA.5DepartmentofPathology, Immunology&LaboratoryMedicine,CollegeofMedicine,Universityof Florida,Gainesville,FL32610,USA.6DepartmentofInfectiousDiseaseand Pathology,CollegeofVeterinaryMedicine,Gainesville,FL32608,USA. Authors contributions BHLperformedvectorconstruction,genetherapy,salivacollections,and diseaseprofilingofthemiceandparticipatedinthedesignofthestudy, dataanalyses,andmanuscriptpreparation.WCCcarriedouthistological analysisandANAstaining.JAChelpedwithAAV2packaging,studydesign, andmanuscriptpreparation.ABPandCQNparticipatedinthedesignofthe study,dataanalyses,andmanuscriptpreparation.Allauthorsreadand approvedthefinalmanuscript.Lee etal ArthritisResearch&Therapy 2012, 14 :R172 http://arthritis-research.com/content/14/4/R172 Page12of14

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Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:21February2012Revised:2July2012 Accepted:24July2012Published:24July2012 References1.PflanzS,TimansJC,CheungJ,RosalesR,KanzlerH,GilbertJ,HibbertL, ChurakovaT,TravisM,VaisbergE,BlumenscheinWM,MattsonJD, WagnerJL,ToW,ZurawskiS,McClanahanTK,GormanDM,BazanJF,de WaalMalefytR,RennickD,KasteleinRA: IL-27,aheterodimericcytokine composedofEBI3andp28protein,inducesproliferationofnaiveCD4 (+)Tcells. Immunity 2002, 16 :779-790. 2.PflanzS,HibbertL,MattsonJ,RosalesR,VaisbergE,BazanJF,PhillipsJH, McClanahanTK,deWaalMalefytR,KasteleinRA: WSX-1andglycoprotein 130constituteasignal-transducingreceptorforIL-27. JImmunol 2004, 172 :2225-2231. 3.VillarinoAV,HuangE,HunterCA: Understandingthepro-andantiinflammatorypropertiesofIL-27. JImmunol 2004, 173 :715-720. 4.BattenM,GhilardiN: Thebiologyandtherapeuticpotentialofinterleukin 27. 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Abstract
Introduction
Sjögren's syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and lacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that TH17 cells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive inflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of TH17 activity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS.
Methods
Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or LacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and IL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to quantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA) staining, and stimulated saliva flow rates were used to profile SjS disease severity.
Results
Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited long-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice injected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed that rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF, lower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva flow rates.
Conclusions
These data support the concept that IL-27, when provided exogenously, can induce a suppressive effect on SjS development and thus may be an effective therapeutic agent for regulating TH17 pro-inflammatory activity in autoimmune diseases where the TH17 system has been shown to play an important role in their pathogenesis.
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Lee, Byung H
Carcamo, Wendy C
Chiorini, John A
Peck, Ammon B
Nguyen, Cuong Q
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