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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
1
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
ease
sus
ecpt
ibili
ty
Genetic factorsEnvironmentals factors
Symptomatic
Asymptomatic
Indi
vidu
als
in p
opul
atio
n
Disease liability
Thre
shol
d
A B
3
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.
4
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
ease
pat
hoge
nici
ty
Time
Clinical diagnosis
Genetic architecture
Environmental factors
Altered immune response
Auto-antibody production
Tissue damage
Cumulative structural damage
5
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.
7
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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