From Department of Medical Biochemistry and Biophysics, Division of Medical Inflammation Research,

Karolinska Institutet, Stockholm, Sweden

IDENTIFICATION OF GENETIC VARIANTS AND THEIR IMPLICATIONS IN

AUTOIMMUNITY

Ulrika Norin

Stockholm 2017

All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Eprint AB 2017 © Ulrika Norin, 2017 ISBN 978-91-7676-561-6

Identification of genetic variants and their implications in autoimmunity

THESIS FOR DOCTORAL DEGREE (Ph.D.)

By

Ulrika Norin

Principal Supervisor: Professor Rikard Holmdahl Karolinska Institutet Department of Medical Biochemistry and Biophysics Division of Medical Inflammation Research Co-supervisor(s): Dr Liselotte Bäckdahl Karolinska Institutet Department of Medical Biochemistry and Biophysics Division of Medical Inflammation Research Docent Johan Bäcklund Karolinska Institutet Department of Medical Biochemistry and Biophysics Division of Medical Inflammation Research

Opponent: Professor Marie Malissen Centre d'Immunologie de Marseille-Luminy Examination Board: Professor Helena Erlandsson Harris Karolinska Institutet Department of Medicine, Solna Division of Rheumatology Professor Göran Andersson Sveriges lantbruksuniversitet Department of Animal Breeding and Genetics Division of Molecular Genetics Professor Mikael Karlsson Karolinska Institutet Department of Microbiology, Tumor and Cell Biology

Pulling the puzzles apart Questions of science Science and progress Nobody said it was easy No one ever said it would be so hard The Scientist by Coldplay

ABSTRACT Autoimmunedisordersstarttodevelopwhenthebody’simmunesystemrecognizesorgansandtissuesasforeignandinitiatesuncontrolledimmunereactionsagainstthem.Mostofthesedisordersareregardedascomplexwithbothenvironmentalandgeneticfactorscontributingtodiseasedevelopment.CurrenttreatmentofautoimmunedisorderssuchasRheumatoidarthritis(RA)isassociatedwithlackofefficacy,developmentofresistanceandseriousside-effectsandaccentuatestheneedfordevelopmentofnewtherapeutics.Improvedunderstandingoftheunderlyinggeneticpathwaysthatconveypathogenicityinarthritisiskeytodiscovermoreefficientandsafetherapies.Theheterogeneticnatureofautoimmunediseasesandtheinteractionwithenvironmentalfactorsdelaysthediscoveryofsusceptibilitygenesinhumans,whichsuggeststheuseofanimalmodelswherebothgeneticbackgroundandenvironmentcanbecontrolled.Inthisthesiswehaveusedratmodelstoidentifygenesthatregulatetheinductionofautoimmunearthritis.Instudyone,weidentifythegeneencodingEndophilinA2asamajordeterminantinregulatingtheinductionofautoimmunityandshowthattheEndophilinA2mediatedprotectionisregulatedviaTcellresponsiveness.Instudytwo,weinvestigatetheroleoftheVav1gene,previouslyassociatedtomultiplesclerosis,foritsroleinarthritisinratsandhumansandshowthatnaturalvariantsintheVav1generegulateTcelldependentarthritis.Instudythree,wedeterminebyfunctionalstudiesthattheincreaseinreactiveoxygenspeciesconveyedbytheNcf1gene,isresponsibleforreducedarthritisseverityseeninNcf1congenicrats.InstudyIV,weusehighresolutionmappinginaratheterogeneousstocktoidentifygenesregulatingexpressionofcellsurfacemoleculesandfrequencyofdifferentleukocytesinblood.Bycombininganimalstudiesandhumandatawehaveinthisthesisidentifiednewgenesinvolvedinthepathogenesisofarthritis,whichfurtherillustratestheheterogenicnatureofRAandthesharedperipheraltolerancepathwaysregulatingdifferentautoimmunedisorders.Furthermore,theresultsinthisthesishavedemonstratedthevalueofusinganimalstudiestoidentifygenesandpathwaysrelevanttohumandisorders.

LIST OF SCIENTIFIC PAPERS I. SpontaneousmutationrevealsEndophilinA2asamajorregulatorof

autoreactiveTcellsandapotentialnewtargetinautoimmunedisease.UlrikaNorin,CarolaRintisch,FlorianForster,LiesuMeng,DianaEkman,JonatanTuncel,KatrinKlocke,JohanBäcklund,MinYang,KlementyShchetynsky,HannaAxelsson,MartinHaraldsson,ThomasLundbäck,MariaBergquist,LeonidPadykov,IngerGjertsson,PietrodeCamilli,NorbertHubner,LiselotteBäckdahl,RikardHolmdahlManuscript

II. VAV1regulatesexperimentalautoimmunearthritisandisassociatedwithanti-CCPnegativerheumatoidarthritisAndréOrtliebGuerreiro-Cacais,UlrikaNorin,AlexandraGyllenberg,RasmusBerglund,AmennaiDanielBeyeen,RheumatoidArthritisConsortiumInternational,ElisabethPetit-Teixeira,FrançoisCornélis,AbdelhadiSaoudi,GilbertJ.Fournié,RikardHolmdahl,LarsAlfredsson,LarsKlareskog,MajaJagodic,TomasOlsson,IngridKockum,LeonidPadyukovGenesImmun.2017Jan;18(1):48-56

III. PositioningofaPolymorphicQuantitativeTraitNucleotideintheNcf1GeneControllingOxidativeBurstResponseandArthritisSeverityinRatsMalinHultqvist,OutiSareila,FredrikVilhardt,UlrikaNorin,LinaM.Olsson,PeterOlofsson,UlfHellman,RikardHolmdahlAntioxidantsandredoxsignaling,2011,14,2373-2383

IV. Combinedsequence-basedandgeneticmappinganalysisofcomplextraitsinoutbredratsAmelieBaud,RoelHermsen,VictorGuryev,PernillaStridh,DelythGraham,MartinWMcBride,TatianaForoud,SophieCalderari,MargaritaDiez,JohanOckinger,AmennaiDBeyeen,AlanGillett,NadaAbdelmagid,AndreOrtliebGuerreiro-Cacais,MajaJagodic,JonatanTuncel,UlrikaNorin,ElisabethBeattie,NganHuynh,WilliamHMiller,DanielLKoller,ImranulAlam,SamreenFalak,MaryOsborne-Pellegrin,EstherMartinez-Membrives,ToniCanete,GloriaBlazquez,EliaVicens-Costa,CarmeMont-Cardona,SiraDiaz-Moran,AdolfTobena,OliverHummel,DianaZelenika,KathrinSaar,GianninoPatone,AnjaBauerfeind,Marie-ThereseBihoreau,MatthiasHeinig,Young-AeLee,CarolaRintisch,HerbertSchulz,DavidAWheeler,KimCWorley,DonnaMMuzny,RichardAGibbs,MarkLathrop,NicoLansu,PimToonen,FransPaulRuzius,EwartdeBruijn,HeidiHauser,DavidJAdams,ThomasKeane,SantoshSAtanur,TimJAitman,PaulFlicek,TomasMalinauskas,EYvonneJones,DianaEkman,ReginaLopez-Aumatell,AnnaFDominiczak,MartinaJohannesson,RikardHolmdahl,TomasOlsson,DominiqueGauguier,NorbertHubner,AlbertoFernandez-Teruel,EdwinCuppen,RichardMott&JonathanFlint.NatureGenetics.2013Jul;45(7):767-75

ADDITIONAL PUBLICATIONS

Publicationsnotincludedinthethesis.Effectsbyperiodontitisonpristane-inducedarthritisinrats.ErikssonK,LönnblomE,TourG,KatsA,MydelP,GeorgssonP,HultgrenC,KharlamovaN,NorinU,JönssonJ,LundmarkA,HellvardA,LundbergK,JanssonL,HolmdahlR,Yucel-LindbergT.JTranslMed.2016Nov3;14(1):311.Genomesandphenomesofapopulationofoutbredratsanditsprogenitors.BaudA,GuryevV,HummelO,JohannessonM;RatGenomeSequencingandMappingConsortium.FlintJ.SciData.2014Jun10;1:140011NaturalpolymorphismsinTap2influencenegativeselectionandCD4:CD8lineagecommitmentintherat.TuncelJ,HaagS,YauAC,NorinU,BaudA,LönnblomE,MaratouK,YtterbergAJ,EkmanD,ThordardottirS,JohannessonM,GillettA;EURATRANSConsortium.,StridhP,JagodicM,OlssonT,Fernández-TeruelA,ZubarevRA,MottR,AitmanTJ,FlintJ,HolmdahlR.PLoSGenet.2014Feb20;10(2):e1004151FinemappingofthearthritisQTLPia7revealsco-localizationwithOia2andtheAPLEClocus.RintischC,KelkkaT,NorinU,LorentzenJC,OlofssonP,HolmdahlR.GenesImmun.2010Apr;11(3):239-45.

CONTENTS

1 Introduction……………………………………………………………………………………………12 Autoimmunedisorders…………………………………………………………………………..22.1Tolerancemechanismstopreventtheinductionofautoimmunity………….42.1.1Centraltolerance………………………………………………………………………………….52.1.2Peripheraltolerance…………………………………………………………………………….63 RheumatoidArthritis……………………………………………………………………………..83.1Clinicalfeaturesanddiagnosis…………………………………………………………………83.2Treatment……………………………………………………………………………………………....93.3PredisposinggeneticfactorsinRA…………………………………………………………103.4PredisposingenvironmentalfactorsinRA……………………………………………..113.5ExperimentalmodelsofRA……………………………………………………………………113.5.1Collagen-inducedarthritis…………………………………………………………………..123.5.2Pristane-inducedarthritis……………………………………………………………………123.5.3Glucose-6-phosphateisomerase-inducedarthritis…………………………….134 Geneticdissectionofcomplexdisorders………………………………………………144.1Identifyingdiseasecausinggenesinexperimentalcrosses…………………….144.2Identifyingdiseasecausinggenesinhumans…………………………………………165 PresentInvestigations………………………………………………………………………….175.1PaperI…………………………………………………………………………………………………..175.2PaperII………………………………………………………………………………………………….185.3PaperIII…………………………………………………………………………………………………195.4PaperIV…………………………………………………………………………………………………206 Concludingremarks………………………………………………………………………………217 Futureperspectives………………………………………………………………………………228 Acknowledgement……………………………………………………………………………….239 References……………………………………………………………………………………………26

LIST OF ABBREVIATIONS RA RheumatoidArthritisMS MultipleSclerosisMHC Majorhistocompabilitycomplex TCR TcellreceptorPTM Post-translationalmodificationsROS Reactive-oxygenspeciesmTEC medullarythymicepithelialcellsDC dendriticcellMQ macrophageAPC antigen-presentingcellAIRE autoimmuneregulatorTRA Tissue-restrictedantigenTGFb TransforminggrowthfactorbetaIL-10 Interleukin10GI gastrointestinalRF RheumatoidfactorACPA anti-citrullinatedproteinantibodyDMARD Disease-modifyingantirheumaticdrugPIA pristane-inducedarthrtitisCIA collagen-inducedarthrtitisGPIA glucose-6-phosphateisomerase-inducedarthritisQTL quantitativetraitlociSNP singlenucleotidepolymorphismCNV copynumbervariationGWA genome-wideassociationHS heterogeneousstockMb MegabasepairsKb Kilobasepairs

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1 INTRODUCTION Tokeepussafefrominfectionsandcancerourimmunesystemhasevolvedtorecognizeandneutralizeantigensforeigntous,suchaspathogensandalteredself-antigenspresentincancercells.Firstlineofdefenseincludestheskinbarrierandthemucousmembranesthatpreventpathogensfromenteringourbodiesandtheinnateimmunesystem.Theinnateimmunesystemisarapidactingdefensesystemthatcontainscellsliketheneutrophils,macrophagesanddendriticcellsandthecomplementsystem.Thesehaveevolutionaryconservedmoleculesandreceptorsthatrecognizestructuresforeigntous,likeinfectiousagentsfrombacteriaorviruses,toensureimmediateneutralizationandclearanceofthepathogens.Thesecondlineofdefenseistheadaptiveimmunesystem,whichincludesthelymphocytes,BcellsandTcells.Unliketheinnateimmunesystem,thelymphocytesfunctionlikeanimmunologicalmemoryanduponrecognitionofapathogentheywouldbecomeactivatedandasubsetofthecellswillbecomelong-livedmemorycellsthatcanrespondquicklyiftheinfectionwastohappenagain.Becausetheyarelong-livedtheresponseneedstobetightlyregulatedsinceawrongfullydirectedresponsetowardsforexampleaself-antigencouldhavedetrimentalconsequencesasseeninindividualswithautoimmunity.Toensurethattheresponsetoacertainantigeniscorrect,theinnateandtheadaptiveimmunesystemcommunicateviasurfacemoleculesandcytokinesinordertodistinguishbetweenpotentialharmfulandsafeevents.

InthisthesisIhavestudiedsomeofthegenesinvolvedininthisintricateinterplayoftheimmunesystem.UsinganimalmodelsforcommonautoimmunedisorderssuchasRheumatoidarthritisandmultiplesclerosiswehavedeterminedthefunctionalimplicationthegeneshaveinregulatingtheimmuneresponseandhowdisturbancesinthisregulationcanleadtoautoimmunity.InpaperIandIIweinvestigatethefunctionalimpactthatTcellshaveonautoimmunityandinpaperIIIhowreactiveoxygenspeciescanaltertheautoimmuneresponse.Understandingthemechanismsofthesepathwaysiskeytodevelopbetterandsafertherapiesforautoimmunedisorders.

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2 AUTOIMMUNE DISORDERS Autoimmunityisaconditionwherethebodymountsanattackagainstthebody’sowntissuesandorgansandischaracterizedbythepresenceofautoantibodiesandTcellsreactivetoself-antigens.AutoimmunityaffectsalargeproportionoftheWesternpopulationwithanestimatedprevalenceof7.6–9.4%1.Autoimmunedisorderscanbeorganrestrictedasinthyroiditis,typeIdiabetesandmultiplesclerosisorsystemic,affectingseveralorgans,suchassystemiclupuserythematosusandrheumatoidarthritis.Thereisastronggeneticcomponenttoautoimmunedisordersseenasfamilialaggregationofautoimmunityinaffectedindividuals.However,predispositionisnotonlydeterminedbythegeneticmake-upofanindividualbutincludeenvironmentalfactorsaswell.Thus,theyareconsideredcomplexdisorderswheremanyfactorsdetermineifanindividualwilldevelopautoimmunity.Theinductionofcomplexdisorderscanbeillustratedusingthethresholdliabilitymodel2.Hereasetofgeneticfactorsandenvironmentalfactorswillcontributetothediseasepathogenesisandthesubsequentcontributionofallfactorswilldetermineifanindividualwillcrossthethresholdandmanifestwithaclinicaldisease3,Fig.1.

Figure1.Thresholdmodelforautoimmunedisorders.A)Normallydistributedgeneticliabilityforanautoimmunedisorderinapopulation.AdaptedfromHaegert3B)Expandedmodelofsusceptibilityincludingenvironmentalfactorswhichinfluencediseasesusceptibilityinindividuals..

Geneticandenvironmentalfactorscanincreasetherelativeriskofdevelopingdiseasebuttheycanalsohaveaprotectiveeffect4.Thephenomenonthatautoimmunedisordersarecomplexisfurtherillustratedbythefactthatmonozygotictwins,althoughsharinganidenticalgenome,donotnecessarilyhavethesameriskofdevelopingautoimmunity.Ratherthanbeing100%,theconcordancerateforautoimmunedisordersinmonozygotictwinsrangefrom12%inRAto75%inAnkylosingspondylitis5.Still,therelativeriskofdevelopingautoimmunityishigherinrelativestoaffectedindividualsthanthegeneralpopulation.Thus,thereisastronggeneticlinkinpredisposingindividualstoautoimmunityhoweveradditionalexternaltriggers/factorsarealsoneeded.

Individual A Individual B Individual C Individual D

Dis

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Genetic factorsEnvironmentals factors

Symptomatic

Asymptomatic

Indi

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in p

opul

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Disease liability

Thre

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A B

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Althoughthetargetedorganandsubsequentfunctionaloutcomeisdifferentbetweendifferentautoimmunedisorderstheysharecommonpredisposinggeneticpathwaysandautoimmunedisordersaggregatewithinfamilies6.Themostdominategeneticcontributoristhemajorhistocompatibilitycomplex(MHC).GenesencodingMHCmoleculesarebyfarthemostassociatedgenestoautoimmunedisorders.ThepurposeoftheMHCmoleculesistopresentpeptidestoTcells,eitherbypresentingextracellularantigensasisthecaseoftheMHCclassIImoleculesorintracellularantigensasintheMHCclassImolecules.DifferentMHCgenesassociatetodifferentautoimmunedisordersandcouldindicatethatthepresentationofaspecificantigenonacertainMHCmoleculeortheexpressionofacertainMHCmoleculesinfluencethetargetedresponsetoaspecificorgan7.Othersharedlociregulatingpredispositiontoautoimmunityhasalsobeenidentified.NotablymanyofthemaffectingTcellmediatedimmunefunctions,implicatingTcellsasmajordeterminantofautoimmunepredisposition8.Directtargetingofoneofthesegenes,theCTLA4gene,encodingthecytotoxicT-lymphocyte-associatedprotein4iscurrentlybeingusedinthetreatmentofautoimmunedisorders9.

Astheenvironmentaroundusshapestheimmunesystemanditsresponse,thefactthatenvironmentalfactorscaninfluencethepredispositiontoautoimmunedisordersislogical10.Thisnotionismaybebestexemplifiedinceliacdiseasewheretheadditionofgluteninthedietofgeneticallysusceptibleindividualsleadstoanautoreactiveresponsetotransglutaminase-2andinductionofTcelldrivendestructionofthesmallintestines.Exclusionofglutenleadstoanimmediatecessationoftheautoreactiveresponseandthelesionsoftheintestinesheal11.

Latelytheimportanceofimmunesystemhomeostasisinfluencedbythemicrobiota12,andparticularlythemicroflorainthegastrointestinal(GI)tract,hasbeengettingmuchattention.IncreasingevidenceshowsthatthepresenceofdifferentcommensalbacteriacanshiftthebalancebetweenregulatoryanddiseasedrivingTcells.Thedirectinfluenceofthegutmicroflorainthedevelopmentofautoimmunityhasbeenshownbyuseofthesegmentedfilamentousbacteria(SFB)intheK/BxNmousemodel.HeretheyshowedthatintroductionofasinglepathogenintogermfreemiceincreasedthenumberofdiseasedrivingTh17cellsandledtotheinductionofautoreactiveresponses13.Anothermechanismbywhichinfectionscancauseautoimmunitycanbeexplainedbymolecularmimicry14.Hereanantigenfromapathogenisstructurallysimilartoanantigenpresentintheendogenousbodyandthusuponinfectiontheimmunereactionwillbemisguidedtowardsaself-antigenandleadtodestructionofthetargettissue.

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Molecularmimicryhasbeensuggestedtotriggerautoimmunityinavarietyofdisordersforexamplecytomegalovirusleadingtocross-reactivityinType-Idiabetes15andcross-reactiveimmuneresponsetostreptococcalMproteinandcardiacmyosininrheumaticheartdisease16.

Insummary,theoverallinductionofautoimmunityisdeterminedbygeneticandenvironmentalfactorsanddependentofimmune-reactivity,antigenrecognitionandtissuemodulationoftheimmuneresponse.Theautoimmuneresponsecanbeseenyearsbeforeanysignsofclinicaldisease17andtheidentificationofthedysregulatedimmuneresponseearlyoniskeytoimprovetheoutcomeforthepatients.

Figure2Developmentofautoimmunity.Environmentalexposureingeneticallypredisposedindividualleadtoanalteredimmunereactivitytowardsself.Subsequenttissuedamageleadsclinicaldiagnosis.AdaptedfromChoandFeldman18

2.1Tolerancemechanismstopreventtheinductionofautoimmunity

Inordertopreventanaberrantautoreactiveimmuneresponse,theimmunesystemhasdevelopedanumberofmeanstoinducetolerancetowardsself-antigens.InthisthesisweshowthatT-cells(papersIandII)haveaspecificroleinregulatingthedevelopmentofautoimmunity.Toeliminateself-reactingTcellstwodistinctprocesseshaveevolved,centraltolerancethatoccursinthethymusduringTcelldevelopmentandperipheraltolerancewhichoccuroutinthetissues.Disruptionineitherofthepathwayscanleadtoautoimmunity.

Dis

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Time

Clinical diagnosis

Genetic architecture

Environmental factors

Altered immune response

Auto-antibody production

Tissue damage

Cumulative structural damage

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2.1.1Centraltolerance

Mostoftheself-reactingandpotentiallydangerousTcellsaredeletedduringdevelopmentinthethymus.Thisprocessneedstobecarefullybalancedtoincludedeletionofhighaffinityself-reactingTcellswhileensuringthereisenoughvariation(Tcellclones)toallowfordetectionofforeignpathogens.ItisestimatedthatoutofalltheTcellprogenitorsinthethymusonly5%eventuallymatureintoTcellsandentertheperiphery19.

ThematurationstagesofthedevelopingTcellaredividedintotwostagestermedpositiveandnegativeselection.Duringpositiveselection,immaturethymocytesareselectedfortheirabilitytoproduceaTcellreceptor(TCR)thatcanrecognizepeptideboundtoMHCmolecules.AfunctionalTCRwillledtoinductionoffurthersurvivalandmaturationsignalswhileaninabilitytoexpressafunctionalTCRwillleadtodeathbyneglect20.Maturethymocytesarethennegativelyselectediftheyrecognizeself-peptides:MHCwithhighaffinity21.TheseprocessesensurethattheTcellsenteringintotheperipheryareabletorecognizeself-MHC(andthuspossiblepathogenicpeptidespresentedonthem)butnotMHCmoleculesbearingself-antigens.

Thepresentationofself-antigensduringnegativeselectionisdependentonmedullarythymicepithelialcells(mTECs)expressingtissue-restrictedself-antigens(TRAs)andantigen-presentingcells(APCs)loadedwithantigensfromtheperipherymigratingintothethymus22,23.Theimportanceofexpressionofself-antigensduringnegativeselectioninpreventingautoimmunityisillustratedinhumansandmicewithmutationsintheautoimmuneregulator(AIRE)gene.AIREisatranscriptionfactorandregulatestheexpressionofTRAsinmTECsandlossoffunctionofAIREleadstomulti-organautoimmunity24.

Thepresenceofauto-reactiveTcellsinhealthyindividualsimpliesthatnegativeselectionisincomplete25andcouldexplainwhysomeindividualsdevelopautoimmunedisorders.Theincompletenegativeselectioncouldbeduethelackofspecificpost-translationalmodificationofcertainproteinsinthethymus26.Forexample,anincreasedTcellresponsetowardthecitrullinatedversionofcollagentypeIIcomparedtoit’snativeformcanbeseeninRApatients27.Additionally,restrictionsinthepresentationofonlyonesegmentofaproteininthymuswillleadtoanincompletetolerancetowardstotheentireproteinandcanleadtosusceptibilitytoautoimmunity28.

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2.1.2Peripheraltolerance

Duetothelimitedexpressionofself-peptidesinthymustheescapeofself-reactingTcellsisinevitable.Thereforemechanismstokeepself-reactingTcellsundercontrolintheperipheryarecrucial.ThisprocessisregulatedwithintheTcellitselfthroughignoranceandanergyandextrinsicallybyimmunesuppression.FirsttheTcellneedtorecognizetheantigenandthusiftheabundanceofantigenistoolow29oriftheTcellisphysicallyseparatedfromaparticularantigen,liketheantigensinanimmuneprivilegedsiteliketheeye30itwillnotbeactivated.Second,ifanantigenispresentedtoaTcell,theTcellwillalsorequireco-stimulatorymolecules31tobeactivatedoritwillgointoanunresponsivestatecalledanergy.Additionally,ligationofmoleculeslikeCTLA-432orPD-133ontheTcellscanactivelyinduceanergybyinhibitingcelldivisionandcytokinesecretion.Thethirdimportantpathwayoflimitinganautoimmuneresponseisimmunesuppression.SpecializedTcells,calledregulatoryTcells(Tregs),haveacriticalpartinimmunesuppression.ThesearecharacterizedbytheexpressionoftheFOXP3transcriptionfactorandhighlevelsoftheanti-inflammatorycytokinesIL-10andTGFβandareimportantininhibitingautoimmunity34.TheimportantroleofTregshasbeenshowntobeevidentinhumanswiththeIPEX(immunedysregulation,polyendocrinopathy,enteropathy,X-linked)disorder.Here,mutationsthatcausealossoffunctionintheFOXP3transcriptionfactorcausesaggressiveautoimmunityasaresultofdefectsinthefunctionoftheregulatoryTcells35.OthercellssuchastolerogenicAPCsarealsoimportantmediatorsofimmunosuppressionandcanlimitTcellproliferationandactivity36.Highlevelsofanti-inflammatorycytokinesintissuescanalsosuppressofTcellresponses.Forexample,presenceofhighlevelsofIL-10intheGItractkeepstheimmunesystemincheckevenwithahighamountofcommensalbacteriaandblockadeofIL-10leadstocolitisinmice37.

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Inthisthesiswefoundthreedifferentwaysbywhichperipheraltolerancemechanismslimitautoimmunity.InpaperI,areducedantigen-specificresponseoftheperipheralTcellsisdisplayedasaconsequenceofdecreasedsignalingfromtheTCRcomplex,leadingtoprotectionagainstautoimmunity.InpaperIIapolymorphismintheTcellsignalingmoleculeVAV1leadsdecreaseinTcelleffectorfunctionswhichsubsequentlyleadstoareductioninseverityofarthritis.InpaperIIIanincreasedproductionofreactive-oxygenspecies(ROS)byAPCschangethearthritogenicTcellresponse38andleadprotectionofarthritis.

Figure3.PathwaysofcentralandperipheralTcelltolerance.HematopoieticprogenitorsmigratefromthebonemarrowtothethymuswheretheymaturetoTcellsandundergopositiveandnegativeselectionbasedontheirinteractionswithpeptide-MHCmolecules.Self-reactiveTcellsthatfailtoundergodeletionarecontrolledintheperipherybyintrinsicandextrinsicperipheralmechanisms.AdaptedfromWalkerandAbbas39.

Positive selection of self-restricted T cells

Negative selection of self-reacting T cells

XX X

Thymus Periphery

Escape of self-reactive T cells

T cell intrinsic mechanism of peripheral tolerance:• Ignorance• Anergy

T cell extrinsic mechanism of peripheral tolerance:• Tolerogenic APCs• Regulatory T cells• Tissue modulation

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3 RHEUMATOID ARTHRITIS Rheumatoidarthritis(RA)isaninflammatoryautoimmunedisease,characterizedbychronicdestructionofsynovialjointssubsequentlyleadingtolossoffunctionofthejoints.ThetermRheumatoidarthritiswascoinedbySirAlfredGarrodin1850sandisnotadiseaseofthemodernsocietybuthasaffectedhumansforhundredstothousandsofyears40,41.RAaffectsapproximately0.5-1%ofthehumanpopulationtodayandaffectsfemalesthreetimesmoreoftenthanmalesanddiseaseonsetisataround30-60yearsofage.AsinotherautoimmunedisordersbothgeneticandenvironmentalfactorspredisposeindividualstoRAbuttheprecisecauseofRAisstillnotknown.RAprimarilyaffectsthesynovialjointsbutsystemicimmuneresponsesleadtootherextra-articularmanifestationssuchasrheumatoidnodules,pulmonaryandcardiovasculardiseasesandisregardedasystemicdisorder42.IndividualssufferingfromRAhasgreatlyreducedqualityoflifeandtheyhaveashorterlifeexpectancycomparedtothegeneralpopulation43.Asofyetthereisnocureandthuscurrenttreatmentisfocusedontreatingthesymptoms.

3.1Clinicalfeaturesanddiagnosis

RAisdiagnosedaccordingtotheACR/EULARclassificationcriteria(Table1)44andischaracterizedbyleukocyteinfiltrationintothesynovialjointwithsubsequentinflammatoryresponseresultingincartilageandbonedestruction45.Thejointofthehands,thewrists,andsmalljointsofthefeetaremostcommonlyaffectedwithsubsequentinvolvementofthejointsinthehipsandshouldersasthediseaseprogress.RApatientsareroutinelydividedintoserologicalpositiveandnegativepatientsbasedonthepresenceofRheumatoidfactors(RFs)andanti-citrullinatedproteinantibodies(ACPAs).DuetothehighlypredictivevalueofthepresenceofACPAsindiagnosisRAwithasensitivityofaround60%46,ACPApositiveRApatientsareoftendiagnosedandtreatedearliercomparedtoACPAnegativepatients.ThepathogenesisofthetwosubtypesofRApatientsappeartobedifferentintermsofpredisposinggeneticandenvironmentalfactors,discussedinmoredetailbelow,andACPApositivepatientsareoftendescribedtohaveamoreerosivediseasecourse47.

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Co-morbiditiesarecommoninRAandincludeinfections,cardiovasculardisease,malignancies(mostoftenlymphomas)anddepression48.Someareaconsequenceoftheongoingchronicinflammationbutcanalsobepresentbeforeorinconjugationwithclinicalonset.TheincreaseinprematuredeathinRApatientshasbeenlinkedtoco-morbiditiesandespeciallyinfectionsandcardiovasculardisease49.

3.2Treatment

ThereiscurrentlynocureforRAandavailabletherapyforreversingthedestructionofthecartilageandboneismissing.Thus,thestandardtreatmentisfocusedonlimitingtheinflammatoryresponse(Table2)50.Earlyandaggressivetreatmentstrategyforagoodoutcomeisnecessary51andforbettermanagementofthediseaseaschemefortreatmenthasbeenformed52.Initialtreatmentisinitiatedwithdisease-modifyingantirheumaticdrugs(DMARDs)suchashydroxychloroquine,leflunomideandmethotrexateanddependingondiseaseactivityissupplementedwithbiologicalagentssuchasTNFblockersoranti-CD20.Thefirstlineoftreatmentmostcommonlyusedisthefolateantagonist,methotrexate.Itisthoughttoinhibitproliferationofcellsbyinhibitingthesynthesisofpyrimidineandpurine,thebuildingblocksofDNAandRNA.Methotrexatehasalsobeenshowntoinhibitcytokineproductionanddecreaseexpressionofadhesionmolecules53.ThemostcommonlyusedbiologicalagentsaretheTNFαblockers.TNFαcanbefoundinhighlevelsintherheumaticjointsandregulatestheexpressionofothercytokinessuchasIL-1andIL-654.TNFαseemsparticularlyimportantinarthritispathogenesisandtransgenicmiceexpressingcontinuouslevelsofhumanizedTNFαdevelopsspontaneousandchronicarthritis55.TNFαblockadeiseffectiveinamajorityofRApatientsbuthasshowntoincreasetheriskofinfections56.

Table1.ACR/EULAR2010criteriafordiagnosing RA

Score points areshown inparentheses.Ascoreofsix orhigher isrequired forRAdiagnosis

1.Jointinvolvement(0–5)

• Onemedium-to-largejoint(0)• Twototenmedium-to-largejoints(1)• Onetothreesmalljoints(largejointsnotcounted) (2)• Fourtotensmalljoints(largejointsnotcounted)(3)• Morethantenjoints(atleastonesmalljoint)(5)

2. Serology(0–3)

• NegativeRFandnegativeACPA (0)• LowpositiveRForlowpositiveACPA(2)• HighpositiveRFor highpositiveACPA (3)

3.Acute-phasereactants(0–1)

• NormalCRPandnormalESR(0)• AbnormalCRPorabnormalESR(1)

4. Durationofsymptoms(0–1)

• Lessthan6weeks(0)• 6weeksormore(1)

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TableadaptedfromSmolenetal57,AllanGibofsky50andSardarandAndersson58

3.3PredisposinggeneticfactorsinRA

UsingfamilialstudiestheheritabilityforRAhasbeenestimatedtoabout~65%59andsuggestastronggeneticcomponenttoRA.ThesharedgeneticpredispositionbetweenRApatientswasfirstdescribedin1976byPeterStatsny60.ThroughmixedlymphocytereactionsStasnyshowedthatcellsfromRApatientsproducedalowresponsetowardRAstimulatorycellswhereasanormalallogenicresponsewasobservedtowardsnon-RAcontrols.StastnybelievedthatthisreflectedanassociationtotheMHCmoleculeandshowedthatthefrequencyoftheMHCgeneHLA-DR4wasincreasedinRApatients.TheassociationwasindeedlaterconfirmedtobetheHLA-DR461,62.ManyHLA-DRalleleshavebeenassociatedtoRAandledtothesharedepitopehypothesis63suggestingasharedmolecularstructureinTcellrecognitionoftheMHCmolecule.TheassociatedHLA-DRB1,HLA-BandHLA-DPinRAhavesubsequentlybeenshowntoshareaparticularfiveaminoacidsequence,inthepeptidebindinggroveoftheoftheassociatedmolecules64andexplainmostofthegeneticassociationinACPApositivepatients.ItwouldtakemanyyearsaftertheidentificationoftheHLA-DRgenesbeforeanothergenecouldbeconvincinglylinkedandassociatedtoRA,

Table 2. Frequently used therapeutics in RA

Conventional DMARDs Mechanism of actionMethotrexate

Folate antagonist, inhibits cell proliferation, cytokine

production

LeflunomideDihydroorotate dehydrogenase inhibitor, inhibits

pyrimidine synthesis, NFkB activation,TNFa and

matrix metalloproteinases production

Hydroxychloroquine Inhibits B- and T cells activity and cytokine release

SulfasalazineFolate antagonist, inhibits acachidonic acid cascade

TofacitinibJAK1/2/3 inhibitor, inhibits cytokine production

Biological DMARDsAdalimumab- Human monoclonal antibody TNF inhibitor

Certolizumab pegol- F(ab’) fragment of a humanised monoclonal

antibodyTNF inhibitor

Etanercept- IgG–Fc-receptor construct (fusion protein) TNF inhibitor

Golimumab- Human monoclonal antibody TNF inhibitor

Infliximab- Chimeric monoclonal antibody TNF inhibitor

Rituximab- Chimeric monoclonal antibody against CD20 B- cell depletion

Abatacept- IgG–Fc-receptor construct (fusion protein)-CTLA4 Anti-T-cell co-stimulation

Tocilizumab IL-6 inhibitor

Corticosteroids prednisolone Inhibits acachidonic acid cascade

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namelythePTPN22gene65.Sincethenmanylocihavebeenidentifiedusingwholegenomeassociationsstudies(GWAS)66withmostgenesdirectlylinkedtoimmuneregulatoryfunctions.

WhiletheconcordanceratesbetweentwinsinbothACPApositiveandnegativeRApatientshavebeenreportedtobeequal59,manyotherstudiesreportalowerheritabilityintheACPAnegativeindividuals67.Additionally,differentgenetic68andenvironmental69associationssuggeststhatthepredispositioninthetwodifferentsubtypesmightbedifferent.OurstudiesinpaperIIseemstoagreewiththisnotion.HereanassociationwithVAV1isfoundwiththeACPAnegativesubgroupbutnotintheACPApositivegroup.Additionally,wecouldshowthatthegeneregulatesthearthritisseverityinaB-cellindependentarthritismodelbutnotaB-celldependentmodel.

3.4PredisposingenvironmentalfactorsinRA

Smokingisbyfarthemostwell-knownenvironmentalfactorinRA.ThepresenceofACPAs,sharedepitopeallelesandsmokingincreasetherelativeriskbyupto40%indevelopingRA70.NosuchassociationhasbeenfoundinACPAnegativeRApatientssuggestingthatthetwosubtypesmayhavedifferentenvironmentaltriggers.SomeadditionalfactorshavebeenassociatedtoincreasedriskofRAincludeperiodontitis71andmicrobiotainthegut72whileothersuchasalcoholintakeandhighbirth-weightmightdecreasetheriskofdevelopingRA73.Interestingly,occupationalexposuretomineraloils,andinparticularhydraulicoil,havebeenshowntobeassociatedtoRA74.Aknownconstituentofhydraulicoilispristane75thesameoilusedtoinducearthritisinrats.

3.5ExperimentalmodelsofRA

RAisaheterogeneousdiseaseandtherearemanyanimalmodelsformimickingdifferentaspectsofthediseasedevelopmentandtheycanbespontaneousorinduced58,76.Animalmodelsaregreattoolsforinvestigatingthecauseandconsequenceofdifferentgeneticandenvironmentalfactorsinarthritispathogenesis.Pathwaysthatregulatearthritisdevelopmentaresharedamongspeciesandarthritisregulatinggeneticlocifoundinrodentsoverlapwithregionsfoundinhumanstudies,liketheMHCregion,provingtheirusefulnessinidentifyingnewtargetsfortherapeutics.Forexample,thedevelopmentofthenewIL23/12bi-specificantibodyintreatingautoimmunedisordersisbasedonafindinginmice77.Theneedforanimalmodelsintestingnewtherapeuticsisalsoofimportance78.InthisthesiswehaveusedthreedifferentanimalmodelsforRA;collagen-inducedarthritis(CIA),pristane-inducedarthritis(PIA)andglucose-6-phosphateisomerase-inducedarthritis(GPIA).

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3.5.1Collagen-inducedarthritis

CIAisbyfarthemostcommonlyusedmodelforRAandcanbeinducedinmice79,rats80andnon-humanprimates81.ImmunizationwiththecartilagerestrictedproteincollagentypeIIemulsifiedineitherincompleteorcompleteFreundsadjuvantleadtoimmuneresponsedirectedtowardsthejointsandsubsequentarthritis.InductionofCIAelicitsleukocyteinfiltrationtothesynovium,whichleadstosynovitisandpannusformationandcartilageandbonedestruction.BothB-andTcellsarerequiredfordiseaseinduction,reviewedin82andserumtransfercaninducearthritisinbothmiceandratsindicatingastrongcontributionofantibodiesinCIApathogenesis8384.LikeinRA,CIAisalsohighlydependentoftheMHC85butalsonon-MHCgenes86.ImmunereactivitytocollagentypeIIisalsofoundinRApatientsbothwithantibodyresponsesandauto-reactiveTcells87,88.

3.5.2Pristane-inducedarthritis

Asingleinjectionatthebaseofthetailofthemineraloilpristane((2,6,10,14tetramethylpentadecane))induceachronicrelapsingarthritisinrats.LikeinRA,immunizationcauseasymmetricaljointinflammationwithintwelvedayswithinfiltratingleukocytesintothesynoviumsubsequentsynovialinflammationandproductionofrheumatoidfactors89.Whyimmunizationwithpristaneinducesarthritisisnotknownbutisthoughttobedependentonthepolyclonalactivationofself-reactiveCD4+Tcells.ContrarytoCIA,PIAcannotbetransferredbyserumandisthoughttobelessdependentonBcells90.However,auto-antibodyresponsestowardshnRNP-A2andcollagentypeIXhavebeenidentified91,92.Auto-reactiveresponsestotheseantigenshavealsobeenfoundinRApatientsandcouldindicatecommonmechanisticpathwaysinthetwodisease93,94.VerylowamountsofpristanecausearthritisandtheincidenceisonehundredpercentintheDArats95.UsingblockingantibodiesPIAhasbeenshowntobedependentofTcellsandcanbeadoptivelytransferredbyMHCclassIIrestrictedCD4+Tcells96.PIAcanalsobeinducedinmicehoweveritappearstohaveadifferentinductionpathway.Toinducearthritis,theoilmustbeinjectedintraperitonealandthearthritisstartsmuchlaterataround50daysandincludeslupuslikesymptoms97,98.

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3.5.3Glucose-6-phosphateisomerase-inducedarthritis

InductionofarthritisbyGPIwasdiscoveredasaconsequenceofaTCRtransgenicmouseintrogressedintotheNODmouse99.ThetransgenicmouseproduceslargeamountsofantibodiestowardsGPIandthediseasecanbetransferredbyserum,thismodeliscalledK/BxNmodel100.WhyanimmunereactiontowardsGPI,whichisaubiquitousprotein,leadstoarthritisisthoughttobebecauseGPIisdepositedinlargeamountsinthejointsandantibodiestowardsGPIelicitcomplementactivationandsubsequentimmunereactionlocallyinthejoint101.InourmodelweimmunizemicewithapeptidefromtheorthologoushumanproteinemulsifiedincompleteFreundsadjuvant.Thisleadstomonophasicarthritiswithhighincidence.ItisafastmodelwithdiseaseonsetarounddaytenandhasbeenshowntobedependentonbothBandTcellsasshowninB-andT-cellknockoutmice102.GPIappeartoberelevanttoRApathogenesisandautoantibodiestowardstheproteinandbefoundinRApatients103.

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4 GENETIC DISSECTION OF COMPLEX DISORDERS

ThetheorythatcertaintraitsareinheritedindistinctpatterswereinitiallyillustratedbyGregorMendelin1850swherehecouldshowthatcertainphenotypictraitsofpeaswereinheritedfromparentalplantstodaughterplantsineitherrecessiveordominantform.WhilethetraitsMendelinvestigatedwereduetosinglegenes,thisisnotthecaseforcomplexdisorderslikeRA.Heremanygenesofsmalleffectsizescontributetothephenotype.Identificationofdisease-regulatinggenesarefurthercomplicatedbygene-geneandgene-environmentinteractions.Regionsthatareassociatedwithaparticularphenotype,commonlyentitledquantitativetraitloci(QTL),canbeidentifiedbylinkageandassociationstudies.Inlinkagestudies,co-segregationofaparticulartraitwithgenomiclociinfamiliesofaffectedandunaffectedindividualsareidentified.Inassociationstudies,thefrequencyofanalleleorgenotypeinlargerpopulationsofunrelatedaffectedandnon-affectedindividualsiscompared.Ifthegenotypeisover-representativeinaffectedindividualstheinvestigatedgeneticmarkerisbelievedtobeassociatedtothetrait.Therearedifferenttypesofgeneticvariantsandpolymorphismsthatcanaffectgenesandregulateadiseasephenotype.Inthisthesiswedescribeaninsertionofatransposon,whichreducestheexpressionofageneandcodingsinglenucleotidepolymorphisms(SNPs)thatalterthefunctionoftheencodedproteins.Othergeneticvariantssuchascopynumbervariations(CNVs),asseeninregulationoftheNCF1geneinhumans104,arealsoimportantinregulatinggenefunction.Usingdifferentlinkageandassociationstudies,wehaveidentifiedfourdifferentgenesinvolvedinimmuneregulatoryphenotypes.

4.1Identifyingdiseasecausinggenesinexperimentalcrosses

Experimentalcrossesofinbredstrainsofsusceptibleandresistantstrainscanbeusedtoidentifydisease-regulatinggenes.Asuccessfuloutcomeofusinganimalsforidentificationofdiseaseregulatinggenesthatareimportantinthehumanconditiondependsonboththephenotypiccoherencebetweenhumanandanimalandthephenotyperegulatinggeneticvariabilityintheanimalsused.InpaperI-IIIweusedF2crossestoidentifygenomicregionsthatregulatesusceptibilitytoarthritis.Hereoffspringofonesusceptibleandoneresistantstraincalledfilial1generation(F1)arebredtogethertoproduceasecondfilialgeneration(F2).EachoffspringintheF2generationwillhaveauniquemixofsusceptibleandresistantgenomiclocispreadacrossthegenome,duetorecombinationeventsduringmeiosis.Usingtheinformationofmarkersthatarepolymorphicbetweentheresistantandsusceptiblestrainandbydetectingthephenotypeineachoftheoffspringonecanperformalinkageanalysis.Toidentifylocithatarelinkedtothetraitthelogarithmofodds(LOD)scoremethodisused105.Itiscalculatedbycomparingtheprobabilitythatagivenmarkeris

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inheritedtogetherwiththetrait,andthusarelinked,withtheprobabilityofobservingthesamelinkagebychance.DuetothelimitednumberofrecombinationeventsinaF2cross,thediseaseregulatinggenomiclocicoverlargegenomicregionscontainingmanygenesandthusfurtherisolationofthegeneticlociincongenicstrainsthroughbackcrossingisusuallyneededinordertoidentifythedisease-regulatinggene106.

Figure4.F2crossesandcongenicstrainsusedtoidentifydisease-regulatinggenes.

Nevertheless,toassignaphenotypetoasinglegene,knownaspositionalcloning,usingonlycongenicstrainsisdifficultandtimeconsumingandtheuseoffunctionalstudiesandgeneticallymodifiedanimalsmightbeneeded.Usingthiscombinedapproach,wecouldpositionallyclonetheSH3gl1geneinpaperIandtheROSregulatingnucleotideintheNcf1geneinpaperIII.

AsstandardF2geneticcrossesonlyincludethegeneticvariationoftwoparentalstrainsandhavelowmappingresolution,weutilizedaheterogeneousstock(HS)inpaperIV.TheNIH-ratHSwasestablishedusingeightfoundingratstrains(BN/SsN,MR/N,BUF/N,M520/N,WN/N,ACI/N,WKY/N,andF344/N).ToproduceanHSarandombreedingschemewassetupforabout60generationscreatingamosaicoffoundervariantsallowingthefine-mappingofQTLs.IntheHSratsusedinpaperIVthemappingresolutionforaQTLwasestimatedtolessthan3Mb107andcouldpotentiallyinsomegenomicregionsallowtheidentificationofasinglegeneresponsibleforaphenotype.

Resistant Susceptible F2 cross

x

x F1

x F2

Congenic strain Donor Recipient

Generation 1 (50% donor)

Generation 2 (25% donor)

Generation 3 (12.5% donor)

Generation 10 (< 0.5 % donor)

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4.2Identifyingdiseasecausinggenesinhumans

Identificationofgenesregulatingdiseaseinhumanswasoriginallybasedonfamiliallinkagestudieswhereregionslinkedtodiseaseusuallyrangefrom2-10Mbinspan.Duetothelowresolutiontheidentificationofgenesusingfamiliallinkagestudieshasnotbeenefficaciousincomplexdiseases.Thus,genome-wideassociationstudies(GWAs)wherelociof10-100kbcanbeidentifiedhasbeenfavoredinstead108.Here,onetakesadvantageofthefactthatpolymorphismsarenotinheritedindependently,buttogetherinlinkagedisequilibrium(LD)blocks.AroundonemillionSNPscalledtaggedSNPs,representingdifferentLDblocksacrossthegenomeisselectedandgenotypedinaffectedandnon-affectedindividuals.DuetothenumberofmarkersusedandnumberofindividualsinGWASstudies,associationsneedtobecorrectedformultipletestingtoexcludefalsepositives.Thus,highp-valuesof>10^-8isneededforgenome-widesignificans.AlthoughmanylocihavebeenidentifiedusingGWAS,alargepartofheritabilitycalledthemissingheritability,cannotbeaccountedfor109.Forexample,theheritabilityinRAisestimatedtobe~65%fromstudiesintwinshoweverinarecentGWASstudy,theidentifiedlocicanonlyaccountfor50%oftheheritability66.Thiscouldbeduetomissingrarevariantsbutalsothefactthatthegeneticmarkersused,SNPs,mightnotpickupothertypesofimportantvariantssuchasCNVsorinsertionsanddeletions.Correctcategorizationofthephenotypestudiedisalsoimportantasthepossibilityofmixedtypesofdisordersmightmaskapositiveassociation.ThisisillustratedinpaperIIwheretheneedforstratificationbasedonserologyisnecessarytoidentifyassociationtotheVAV1gene.

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5 PRESENT INVESTIGATIONS

5.1PaperI

SpontaneousmutationrevealsEndophilinA2asamajorregulatorofautoreactiveTcellsandapotentialnewtargetinautoimmunedisease

InthisstudyweidentifiedEndophilinA2(EA2),apreviouslyunknownanduniquetarget,fortreatmentofautoimmunediseases.TheimportanceofthegeneinimmuneregulatorypathwaywasdiscoveredasaconsequenceofaspontaneousmutationthatoccurredinourcolonyofratsrenderingthenormallyhighlysusceptibleDAratresistanttoinductionofarthritis.ThemutationinhibitstranscriptionofthegeneencodingEA2makingtheratanaturalknockout.Tocross-speciesconfirmEA2’sroleinprotectionagainstarthritisweusedgeneknockouttechnologyinmouse.ThegeneencodingEA2isexpressedin,andaffects,manydifferentleukocytes.However,thearthritisprotectionseeninEA2deficientanimalsismainlymediatedviaTcells.TheTcelldependencywasshownthroughpristane-primedCD4+TcelltransferexperimentsandthereconstitutionofTcellknockoutmicewithEA2deficientandwildtypethymocytesandsubsequentarthritisinduction.InvestigatingtheEA2effectonTcellsinlightofitsmolecularfunction,wesawthattheEA2deficientTcellsareunabletointernalizetheirTcellreceptortothesameextentandthusareunabletoproliferateatthesameratecomparedtonormalwildtypeTcells.ThereducedTcellfunctionleadstoadecreaseinautoreactiveTcellsandresultsinunsuccessfulinductionandsubsequentblockageofdiseaseprogression.WecouldalsoshowthattheEA2expressionwasincreasedinRApatientsandthusproposeanewinterestingpathwayinRAbywhichtheactivationofTcellscanbemodulatedusinginhibitorsofEA2.

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5.2PaperII

VAV1regulatesexperimentalautoimmunearthritisandisassociatedwithanti-CCPnegativerheumatoidarthritis

TheVav1genehadpreviouslybeendescribedtoregulateseverityinananimalmodelofmultiplesclerosisandfoundtobeassociatedinmultiplesclerosis(MS)110.InthisstudyweusedifferentanimalmodelsforRAandgeneticassociationstudiestoinvestigatetheroleofVav1inarthritis.WeimmunizedDA.BN-R25congenicratsharboringacodingvariantintheVav1geneandDAlittermatesforCIAandPIA.WhilenoeffectcouldbeobservedinCIAasignificantreductioninarthritisseveritywasfoundinPIA.AsdiscussedaboveBcelldependencyinpathogenesisinPIAandCIAseemtodiffer.StudiesondepletionofTcellsbeforeandafterestablisheddiseaseinadjuvantarthritisandCIAfurtherconfirmthisobservation111.WhiledepletionofTcellsduringprimingisbeneficialinbothadjuvantarthritisandCIA,depletionafterestablisheddiseasereducearthritisseverityonlyinadjuvantarthritiswhilenoamelioratingeffectisobservedinCIA.ThissuggeststhataftertheBcellshavebeenprimedbyTcellstheynolongerneedTcellsinordertopropagatethedisease.TheseexperimentstogetherwiththeobservedreductioninTcellproliferationseeninDA.BN-R25rats110explainwhyweonlycouldobserveregulationbyVav1inPIAandnotCIA.IntheRAcase-controlstudiesonlyaweakassociationinthetotalpopulationcouldbeobserved.However,whenstratifyingtheRApatientstoACPApositiveandACPAnegative,astrongerassociationcouldbefoundfortheACPAnegativewhilenoassociationwasfoundinACPApositivepatients.InbothPIAandACPAnegativeRAthediseaseprogressionisthoughtofasbeinglessdependentonantibodiesandinthesediseasesTcellscouldhaveamoreprominentrole.ThustakentogetherourresultsindicatethatVav1regulatesaTelldependentmechanisminarthritis.OurresultsfurtherillustratethecommonpathwayssharedbydifferentautoimmunedisordersandtheheterogeneouspopulationofRApatients.

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5.3PaperIII

PositioningofapolymorphicquantitativetraitnucleotideintheNcf1genecontrollingoxidativeburstresponseandarthritisseverityinrats

TheNcf1generegulatesproductionofROSbythephagocyteNADPHoxidasecomplexandhaspreviouslybeenassociatedwitharthritisseverityinDA.E3-Ncf1congenicrats.InterestinglythearthritisprotectiveallelefromtheE3ratincreasetheproductionofROSwhichmediateprotection.ThreecodingSNPsintheNcf1genedifferedbetweenthesusceptibleDAratandtheresistantE3rat.Tounderstandthemolecularmechanismsunderlyingtheeffectonarthritis,weneededtoidentifythearthritiscausativeSNP.MutatedrecombinantNcf1atthethreedifferentpositionsweretestedfortheireffectonROSproductioninvitro.TheSNPresponsibleforanaminoacidshiftatposition153frommethioninetothreoninewasshowntorestoredtheNcf1mediatedROSproduction.TotestthefunctionalimpactofthisSNPonarthritisdevelopment,inbredratstrainswerescreenedforpolymorphismsintheNCF1gene.Asub-strainoftheWistarratwasidentifiedwithDAallelesattwoofthethreeSNPsfoundtodifferbetweenDAandE3.OnlythethirdSNPcausingtheaminoacidshiftatposition153wasidenticaltotheE3rat.ThegenomicregioncontainingtheWistarallelicversionoftheNcf1genewasisolatedinacongenicstrainandimmunizedforarthritis.SimilartotheE3rat,thenewDA.Wistar-Ncf1congenicalsoshowedreducedarthritisseverityhighlightingthe153snpastheonlydiseaseregulatinggeneticvariant.ThuswecouldfunctionallyprovethatthereducedarthritisseverityobservedinNcf1congenicratswasduetoincreasedROSproductionandregulatedbyasinglenucleotide.

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5.4PaperIV

Combinedsequence-basedandgeneticmappinganalysisofcomplextraitsinoutbredrats

Positionallycloningofgenesisatimeconsumingandacostlyendeavor.InthefourthstudyweutilizedtheNIHheterogeneousstockforhighresolutionmappingof2000ratsoutbredratsandcollected160phenotypestoidentifygenestocomplextraitsinvolvedinforexamplemetabolism,immuneregulationandcardiovasculardisease.EightfounderstrainsoftheHSratsweresequencedandtheirsequenceimputedtohaplotypesinto1400SNPtypedoutbredratsusedinthestudy.Thestrategywasprovensuccessfulandwecouldidentify35causalgenesinvolvedin31phenotypes.

WiththeuseofflowcytometrywestudiesthefrequencyofdifferentleukocytesandexpressionofsurfacemoleculesinbloodofnaïveNIH-HSrats.Ofinteresttoourworkwas

theidentificationoftheTbx21geneinaQTLregulatingtheproportionofCD4+cellswithhighexpressionofCD25.HerethecandidatevariantintheTbx21geneleadstoaglycinetoargininesubstitutionatposition175oftheTbx21andcouldpossiblyaltertheDNA-bindingdomainofthisprotein.TheTbx21genehasbeenimplicatedinthegeneticcontrolofregulatoryTcellspreviouslyandthustheQTLregulatingCD4+CD25highcellsmightrepresentregulatorycells.Furtherinvestigationandisolationofthisvariantisneededtoconcludeanassociationtotheobservedphenotype.

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6 CONCLUDING REMARKS

LackofefficienttherapeuticsinRApatientsleadstohighsocioeconomiccostsandseverelyreducethequalityoflifefortheindividual112.Inaddition,poormanagementofco-morbiditiesandanincreasedriskofinfectionswithcurrenttherapiesleadtoanincreasedmortalityinRApatients.Thus,thereisahighunmetneedinRAandfurtherunderstandingofthediseasepathogenesisisneededtodiscovernewtherapeutics.

Hypothesisfreediscoveryofgenesinvolvedinautoimmunityusingexperimentalcrossesisavaluabletooltodiscovernewtherapies.TheidentificationoftheROSregulatingeffectoftheNcf1geneinautoimmunityhasledtothedevelopmentofROSinducersfortreatingautoimmunedisordersandhavebeenshowntoworkinarthritisinrats113.WehopethatthediscoveryofEA2canleadtonewandmoreeffectivetherapiesinautoimmunedisorderstoo.ByinhibitingEA2thereisapotentialofaddressingmanyoftheunmetneedsinRA.WiththeuseofEA2inhibitorswecouldpotentiallyaddresstwoimportantfeaturesofRA,lymphocyteactivationandtargettissuedestruction.FirstbyinhibitingtheactivationofautoreactiveTcells,whichhavebeenlinkedtothedetrimentalchroniccircleofimmunecellactivation,stoppingtheautoreactiveresponse.Second,throughpublicallyavailabledatarepositories(BioGPS)wehavefoundthatthegeneexpressionofEA2isincreasedinsynoviocytesofRApatientscomparedtohealthycontrolsandindividualssufferingfromosteoarthritis.EA2hasalsobeenshowntoregulatetheendocytosisofmembraneboundmetalloproteinases114,knowntobeimportantfordegradationoftheextracellularmatrix.ThusbytargetingEA2insynoviocytesonecanreducetheirproliferationandinhibittheirinvasivenessleadingtoasubsequentregenerationofthetargettissue.Additionally,RApatientshaveanincreasedriskofmalignancies115anditisstilldebatedwhethercurrenttreatmentscontributingtothis116.KnockingdownEA2incancercellshaveshowntoreduceproliferationandtumorinvasion117thusinhibitingEA2inRApatientsonecouldpotentiallyalsoreducetheprevalenceofmalignancies.

Insummary,theresultsofthisthesisshowthatRAisindeedaheterogeneousdiseaseandidentificationofgenescouldbenefitfromstratification,thereisanoverlapindiseasepathwaysbetweendifferentautoimmunediseasesandthatperipheraltolerancemechanismmediatedbyEndophilinA2,Vav1andNcf1arecrucialinlimitinganautoimmuneresponse.

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7 FUTURE PERSPECTIVES Positionallycloningofgenesusinganimalsisapowerfultooltofunctionallycharacterizeandstudygenesinvolvedautoimmunedisorders.Althoughprovensuccessfulinthisthesis,theidentificationofgenesusingF2crossesandisolationincongenicstrainsisatimeconsumingendeavor.Withtheintroductionofnewtechnologiesinthe‘omicseraandthedropinpricesofsequencing,directstudiesinhumanscomparinggenotypetophenotypewillprobablyreplacemanyoftheanimalstudies.However,theneedforanimalmodelswillnotdecline,asmanyofthefindingsinhumanswillneedfunctionalcharacterizationandvalidationinanimals.Additionally,theneedformanydifferentanimalmodelsmimickingdifferentsubgroups,asevidentinwiththeVAV1gene,willbevaluablewhendevelopingnewtherapeutics.

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8 ACKNOWLEDGEMENT Iguessthisisthemostimportantpartofthethesistomostofmyreaders.Itistometoo!Ihavehadtheprivilegetomeetandworkwithsomanysmart,talentedandhardworkingpeopleduringmyPhDstudiesandithasbeentrulyinspiring.Researchershavethemostexcitingjobintheworldbutalsothemostfrustratingattimeswithfailedexperiments(forwhatsometimesappeartobewithoutanyreasonableexplanation)andwithoutmyfellowscientificfriendsIwouldhavegivenupalongtimeago.I’vemadefriendsforlife.Sothankyouforbeingthereandmakingmyexperiencesospecial.

Tomysupervisors:

RikardHolmdahl,foryourvisionaryapproachtoscience,sharedpassionfornovelfindingsandthefreedomyouhavegivenmetofollowmyideasanddreams(whichIhavetakenfulladvantageof,myapologies).

LiselotteBäckdahl,foryourinvaluablehelpandmentalsupport,neverlettingmegiveupalwayssaying”youaresoclosenow”insuchaconvincingwaythatIactuallybelievedyou.

JohanBäcklund,foryourloveofskånskaanddiscussionsaboutourlovelyrats.

TotheMIRfamilymembers:

AngelYao-Mattisson,foryourlovingcare,makingsureweallgetacknowledgedonourspecialdaysandyoureffortsintryingtokeepussocial.CarlosandKristina,formakingmefeellikeatinypartinyourfamilyandthedescentdowntotheanimalhousemoreamicable.KatrinKlocke,mylife-fixer,foralwaysbeingreadytolendahand,teachingmehowtomakegreatfiguresandmakingsurewehaveamazingcakesatvariousevent.CarolaRinstisch,forteachingmeallaboutratgeneticsandlayingasolidgeneticfoundationintheDACPproject.DianaEkman,thequeenofbioinformatics,foryourinvaluablehelpwithduringtheHSprojectandtheNGSstudies.FlorianForster,forventuringoutintotheTCRsignalingworldwithme.LiseuMeng,foryourhappyspiritandallyourhelpwiththeanimalsandcontinuingtheGOIgroupinChina.LinaOlsson,forbeingthelab’slinktohumanstudiesandteachingmeallabouthumangeneticsandbeinganoutletforfrustrationduringroughtimes.AngelaPizolla,forallourtalksaboutworkandlifegoingtoandfromthelabandgreattimesinJapanandvästraskogen.MichaelFörster,forallgreatmemoriesandyourenthusiasminlifeandwork.IdaAndersson,forallgreatmommydiscussionsduringmy/ourpregnancyandfuntimesduringmaternityleave.IngridLindh,forhavingafriendfromtheverybeginning,includingmeinlabactivitiesandmakingmefeelathomeinthelabfromthestart.BingzeXu,foryourimpressivespeedyandaccurateexecutionofwhatevermaycomeyourwaye.g.GOIproteinbindingassays.DanielleVaartjees,forkeepingmementallyandphysicallyfitandprocrastinatingInstapicsduringthesiswriting.AnthonyYau,forsharingthetrialsandtribulationofworkingwithratsandratgeneticswithme.ErikLönnblom,foryournever-endingsourceofinterestingconversationtopics.

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JaimeJames,forsharingthesameexcellenttasteinmusicandoursing-a-longs.GonzaloLahore-Fernandez,forthesmart-offchallenge(BTW,IthinkI’mwinning).MikeAuon,forthecontroversialandfundiscussionsaboutanythingandeverything.AmitSaxena,forourjointlysharedpassionofreachingforthestars.ClaraMarquinas,foryourgreatsenseofhumorandbeinganamazingkaraokepartner.ChangrongGe,foryourhelpwiththeGOIcrystalsandbindingassays.VilmaUrbonaviciute,forallyoursmartinputsandsharedideas.MinYang,forourlunchdatesdiscussinglife,loveandscience.SusannWinter,foryourcoolandcalm.EmmaMondoc,forneversayingnowhenIwanttoordersomethinganddiscussionsaboutleadingahealthierlifestyle.PatrickMerkyandSaraLind-Enoksson,foralwayshavingtheWednesdaylunchestolookforwardtoandyourflowcytometryexcellence.MartinaJohannesson,forbeingagenuinelykindpersonandourdiscussionsregardingtheHSproject.BrunoRaposo,foralwaysshowinganinterestinmyworkduringseminars.NandakumarKuttySelva(Nan),foryourphilosophicalinputduringseminarsmakingusthinkofthebiggerpicture.KajsaWing,forexcellentinputwheneverIhaveanissueIwanttodiscuss.Biborka,forallyourgreathelpwiththeanimaladministrationandprovidingthelabmemberswithfinejewelry.Bibo,forexpandingmytasteinChinesefood.Naru,foryourpeacefulways.Dongmei,for”sharing”variouslabequipmentwithme.SusannevandenBerg,formakingsurewekeepthelabinthestateitshouldbeforanefficientandsafeworkplace.JianghongZhong,foryourtirelessnessattitudetowardswork.HuseyinUysalfornicebaklavaafterRamadanandinterestingdiscussions.DorotaKlaczkowska,foryourrelaxedandfunattitude.IaKhmaladze,forfindingafriendintheanimalhouseduringstrangehours.FridaLaulundforstandingyourgroundandnicefikasoutsidethelab.OutiSareila,fornicewineandspaexperiences.CeciliaHagert,forfundiscussions.SimonGuerard,forthemotivatingquickexit.JonatanTuncel,forintroducingmetotheHS-project.SabrinaHaag,formakinglatenightsinthelablesslonely.ChristophandKatarinaKessel,forallthenicetimesatdinnersandparties.MarcusHoffman,forinterestingscientificandnon-scientificdiscussions.MalinHultqvistandPeterOlofsson,forpavingthepathwithyourworkonNcf1anddiscussionsregardingstart-ups.FranziskaLange,forgivingthearrivaltoStockholmafamiliarfeeling.ThereseLindvallandTiinaKelkkafornicetimesinLund.GuotianLuo,forstartinguptheoncologysectionofMIR.ThomasBlom,forallyourhelpandsupportinLund.Animalhousestaff,Isabelle,Evelina,Lina,Lilu,Jinlianfortakingexcellentcareoftheanimalsthroughouttheyears

Tomydream-team:

EveryoneatKIABandmycoachesMarkFarmery,foryourmentoringandencouragementandChristianKrog-Jensen,forgreatinputonmyproject.TheChemicalbiologicalconsortiumSwedenatKI,ThomasLundbäck,forteachingmesomuchaboutproteinchemistryanddrugdiscoveryusingsmallmolecules.MartinHaraldsson,forallyourworkonthemoleculesandourdiscussions.HannaAxelsson,forallyourworkontheGOIassay

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development.TomasNymanatSPF,forallyourhelpwiththeprotein-bindingstudies.

Tomycollaborators:

ProfessorTomasOlssonandyourwonderfulgroupforgreatcollaborationsandfuntimes,inparticularPernilla,Petra,Andre,Maja,Melanie,Amennai,RasmusandNada.LeonidPadyukov,ProfessorIngridKockum,AlexandraGyllenberg,IngerGjertsson,MariaBergquist,TulayLindberg,AnnaKatsandKajaErikssonforgreatcollaborations.TheHSgroupinparticularAlberto,Esther,Regina,Toni,Gloria,Carme,Amelie,ErikWandJonathanforgreatcollaborationsandfuntimes.

Tomyscientificcommunity:

BobHarris,myun-officialmentor,forourdiscussionsonresearchandresearcheducationandyournoBSapproachbutmostimportantlyforthefeelingthatsomeonehadmyback.

PeopleintheDoctoralstudent’sassociationArash,Åsa,Hugo,Marcus,Sonal,Yuan,JayeshandBoardofresearcheducationProfessorsAndersGustavsson,MarianneSchultzberg,LarsAlfredsson,KristinaBroliden,LenavonKochandLennartNilssonforinterestingandimportantdiscussionsregardingresearcheducationin2012.

Mystudents,BeatriceBergström,AnetteFriberg,EllinoreJanssonandMalinLjunggrenforimprovingmypedagogicskillsandhelpingmybecomeabetterteacher.

Tomyfamilyandfriends:

Tomydearestfriendsfortakingmymindoffworkandmakingmylifesospecialandfuneveryday.Minkärafamilj,mammaochpappaföralldenkärleknigerossochattnilärtosskännaempatifördesomhardetsämre.Mamma,förallatelefonsamtaltillochfrånjobbetsenakvällarochnätterochbarnpassningnärvibehövdedetsommest.Pappa,förattdualdriglätossvinnaispelnärvivarsmåsomgjortattjaginterädskämpaiunderlägeochintegermig.Finastesyskonenmankanönskasig,KarinochRickard,förallaroligastundervihartillsammansocherkärlekochuppmuntrangenomlivet,jagharalltidenvänier.MinandrafamiljAgneta,RaymondochÅse,förattniinkluderademigieralivfrånförstabörjanochallhjälpmedVille.

MinälskadeBjörn,vadvorejagutandig.Dugermittlivvärdeochgördetmervackertvarjedag.Dinuppmuntranochtropåmigochminaförmågorhargjortattjagorkatkämpapåmångagångernärjagvillegeupp.Särskilttackföralltditttålamodochstödpåsistatiden.VårfinasonVilhelm,minälskadesolstråle,förattdulyseruppävendengråasteavdagar.Duärredansåklokochjagsermedspänningframemotalltvadduskatadigförilivet.

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