Transcriptional landscapes of emerging autoimmunity: transient aberrations in the targeted tissue’s extracellular milieu...

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Title:
Transcriptional landscapes of emerging autoimmunity: transient aberrations in the targeted tissue’s extracellular milieu precede immune responses in Sjögren’s syndrome
Series Title:
Arthritis Research and Therapy
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Mixed Material
Creator:
Nicolas Delaleu
Cuong Q Nguyen
Kidane M Tekle
Roland Jonsson
Ammon B Peck
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Arthritis Research and Therapy
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Abstract:
Introduction: Our understanding of autoimmunity is skewed considerably towards the late stages of overt disease and chronic inflammation. Defining the targeted organ’s role during emergence of autoimmune diseases is, however, critical in order to define their etiology, early and covert disease phases and delineate their molecular basis. Methods: Using Sjögren’s syndrome (SS) as an exemplary rheumatic autoimmune disease and temporal global gene-expression profiling, we systematically mapped the transcriptional landscapes and chronological interrelationships between biological themes involving the salivary glands’ extracellular milieu. The time period studied spans from pre- to subclinical and ultimately to onset of overt disease in a well-defined model of spontaneous SS, the C57BL/6. NOD-Aec1Aec2 strain. In order to answer this aim of great generality, we developed a novel bioinformatics-based approach, which integrates comprehensive data analysis and visualization within interactive networks. The latter are computed by projecting the datasets as a whole on a priori-defined consensus-based knowledge. Results: Applying these methodologies revealed extensive susceptibility loci-dependent aberrations in salivary gland homeostasis and integrity preceding onset of overt disease by a considerable amount of time. These alterations coincided with innate immune responses depending predominantly on genes located outside of the SS-predisposing loci Aec1 and Aec2. Following a period of transcriptional stability, networks mapping the onset of overt SS displayed, in addition to natural killer, T- and B-cell-specific gene patterns, significant reversals of focal adhesion, cell-cell junctions and neurotransmitter receptor-associated alterations that had prior characterized progression from pre- to subclinical disease. Conclusions: This data-driven methodology advances unbiased assessment of global datasets an allowed comprehensive interpretation of complex alterations in biological states. Its application delineated a major involvement of the targeted organ during the emergence of experimental SS.

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University of Florida
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University of Florida
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innate immunity (darkened = dependence on susceptibility regions) adaptive immunity (darkened = dependence on susceptibility regions) transcriptional landscape of salivary glands targeted by autoimmunity preto subclinical stable subclinical to overt autoimmunity subclinical phase Sjgren’s syndrome Emergence of Autoimmunity in C57BL/6.NODAec1Aec2 mice timeline from 4 to 16 weeks of age Transcriptionallandscapesofemerging autoimmunity:transientaberrationsinthe targetedtissue ’ sextracellularmilieuprecede immuneresponsesinSjgren ’ ssyndromeDelaleu etal. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174 http://arthritis-research.com/content/15/5/R174

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RESEARCHARTICLEOpenAccessTranscriptionallandscapesofemerging autoimmunity:transientaberrationsinthe targetedtissue ’ sextracellularmilieuprecede immuneresponsesinSjgren ’ ssyndromeNicolasDelaleu1*,CuongQNguyen2,KidaneMTekle3,RolandJonsson1,4andAmmonBPeck2AbstractIntroduction: Ourunderstandingofautoimmunityisskewedconsiderablytowardsthelatestagesofovertdisease andchronicinflammation.Definingthetargetedorgan ’ sroleduringemergenceofautoimmunediseasesis, however,criticalinordertodefinetheiretiology,earlyandcovertdiseasephasesanddelineatetheirmolecular basis. Methods: UsingSjgren ’ ssyndrome(SS)asanexemplaryrheumaticautoimmunediseaseandtemporalglobal gene-expressionprofiling,wesystematicallymappedthetranscriptionallandscapesandchronologicalinterrelationships betweenbiologicalthemesinvolvingthesalivaryglands ’ extracellularmilieu.Thetimeperiodstudiedspansfrom pre-tosubclinicalandultimatelytoonsetofovertdiseaseinawell-definedmodelofspontaneousSS,theC57BL/6. NODAec1Aec2 strain.Inordertoanswerthisaimofgreatgenerality,wedevelopedanovelbioinformatics-based approach,whichintegratescomprehensivedataanalysisandvisualizationwithininteractivenetworks.Thelatterare computedbyprojectingthedatasetsasawholeon apriori -definedconsensus-basedknowledge. Results: Applyingthesemethodologiesrevealedextensivesusceptibilityloci-dependentaberrationsinsalivarygland homeostasisandintegrityprecedingonsetofovertdiseasebyaconsiderableamountoftime.Thesealterations coincidedwithinnateimmuneresponsesdependingpredominantlyongeneslocatedoutsideoftheSS-predisposing loci Aec1 and Aec2 .Followingaperiodoftranscriptionalstability,networksmappingtheonsetofovertSSdisplayed,in additiontonaturalkiller,T-andB-cell-specificgenepatterns,significantreversalsoffocaladhesion,cell-celljunctions andneurotransmitterreceptor-associatedalterationsthathadpriorcharacterizedprogressionfrompre-tosubclinical disease. Conclusions: Thisdata-drivenmethodologyadvancesunbia sedassessmentofglobaldatasetsanallowed comprehensiveinterpretationofcom plexalterationsinbiologicalstates.Itsapplicationdelineatedamajor involvementofthetargetedorganduringtheemergenceofexperimentalSS.IntroductionCommontoautoimmunediseasesisalongandclinically silentphase.Asaconsequence,affectedindividualsarediagnosedonlyafterimmunesystem – mediatedfunctional deficienciesoftheaffectedtissuesresultinovertdisease [1,2].Hence,owingtotheunavailabilityofhumanspecimensreflectingsubclinicaldiseasestages,understanding ofthemolecularbasisofautoimmunityisskewedtoward lateandovertdiseasephases.Toconclusivelyassignetiologicalrelevancetoanybiologicalprocessalteredinsuch specimensisdifficult,consideringthecausalitydilemma.Nevertheless,stratifyingthechronologyofthese eventsiscrucialinestimat ingwhethergeneticpredispositiontodevelopaspecificautoimmunedisease mightalsoinvolvegenesassociatedwithtissuedevelopmentandhomeostasisorifthegenesexclusivelycluster inprocessesassociatedwit hspecificphasesofinnate adaptiveimmunematuration[3-5]. *Correspondence: nicolas.delaleu@k2.uib.no1BroegelmannResearchLaboratory,DepartmentofClinicalScience,University ofBergen,LaboratoryBuilding,JonasLiesvei65,5021Bergen,Norway Fulllistofauthorinformationisavailableattheendofthearticle 2013Delaleuetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174 http://arthritis-research.com/content/15/5/R174

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Oneapproachtodelineate,andtoacertainextentstratify,themoleculareventsassociatedwithsubclinicalphases ofautoimmunediseasesistheuseofadequateexperimentalmodels[6,7].Forthispurpose,asuitableexperimental strainmust,incorrespondencewithhumans,developits relevantautoimmunephenotypeoveranextendedperiod oftimeandinthecontextofitsgeneticbackground. C57BL/6.NODAec1Aec2 micefulfillthesecriteriaasa modelofprimarySjgren ’ ssyndrome(SS)becausethey develop,intheabsenceofotherinflammatoryconditions, allmajorfeaturesrelevanttothediagnosisofSSin humansspontaneouslyandoveraperiodofseveral months[7,8]. Withaprevalenceof0.1%to0.3%inthetotalpopulation,SSisconsideredarelativelycommonautoimmune disease.Itmainlyinvolvestheexocrineglands.Nearlyall patientscomplainaboutpersistentsymptomsofdry mouth,andmanypresentwithhyposalivation.Severe diseaseoutcomesalsoincludedisablingfatigueanddevelopmentofnon-Hodgkin ’ slymphoma.Todate,all therapiestestedhavebeenineffectiveinreversingthe courseofSS[9,10].Similartopatientswithsystemic lupuserythematosus,asubpopulationofindividualswith SSexhibitatype1IFNsignature,suggestingthataviral agentmaybeinvolvedintriggeringthedisease[11].As aconsequence,studiesdesignedtodiscovergeneticassociationshavefocusedeitheroninnateimmunity[12] orongenesthatmightexplainthedominantroleofB cellsinthepathogenesisofSS[10].Unfortunately,these studieshaveyettoyieldresultsthatallowestimationof anindividual ’ sriskofdevelopingSS. Histologicalevaluationsofminorsalivaryglands(SGs) obtainedfrompatientswithSScommonlyshowfocalinflammationthatmaycoincidewithepithelialcellatrophyandthepresenceofadiposetissueandfibrosis. Morphologically,theseglandsmayalsodisplaystructural disorganization,includinglossofcell – cellandcell – extracellularmatrix(ECM)adhesion[13,14].However, organizingthesefindingschronologicallyandconclusivelyasetiological,pathogenicorbystanderprocesses hasnotyetbeenpossible[9]. Thus,theaimofthisstudywastodelineatethetranscriptionallandscapeassociatedwiththeextracellular milieu(EM)oftheSGsduringspontaneousemergenceof experimentalSS.Theglobalscopeofouraimfavorsintegrationoverreductionandisideallybasedonadatadrivenapproachthatensuresimpartialinterpretationof datasetsasawhole.Forthispurpose,wedevelopeda noveldataanalysispipelinethatcombinesgenesetenrichmentanalyses(GSEAs)[15],leadingedge(LE)analyses [15]andMarkovclusteralgorithm(MCL)clustering[16] foranalysisofbiologicalstates.Thissetofdataanalyses formedthebasisforcomputationofinteractivenetworks withintheCytoscapesoftwaresuite(NationalInstituteof GeneralMedicalSciences,Bethesda,MD,USA)[17]and designofanadvancedvisualizationmethodology.By exploitingthisapproach,wesoughttosignificantlyimproveourabilitytoanalyzesuch “ -omics ” datasetscomprehensivelyandsystematicallyand,inturn,tominimize theintroductionofpersonalbias.MethodsAnimalsC57BL/6.NODAec1Aec2 andC57BL/6malemicewere bredandmaintainedunderspecificpathogen-freeconditionsattheDepartmentofPathologymousefacilityatthe UniversityofFlorida,Gainesville,FL,USA.Todissectthe SGs,micewerekilledbycervicaldislocationafterdeep anesthetization.Allprocedureswereapprovedbythe UniversityofFlorida ’ sInstitutionalAnimalCareandUse Committee(protocolsB317-2007and2008011756).IsolationofRNAfromsalivaryglandsTotalRNAwasisolatedaccordingtotheprotocoldescribedindetailelsewhere[18].Whenthemicewere4,8, 12and16weeksofage,theSGsfreeoflymphnodeswere excisedinparallelfromfiveC57BL/6.NODAec1Aec2 and fiveC57BL/6mice,thensnap-frozeninliquidnitrogen. TotalRNAfromeachmousewasisolatedconcurrently usingtheRNeasyMiniKit(QIAGEN,Valencia,CA, USA),thenRNAconcentrationsandpuritieswereevaluatedusingUVspectroscopy.Theratioofabsorbance (260nmand280nm)oftheRNAsamplesaveraged1.976. Subsequently,eachsamplewashybridizedseparatelyona GeneChipMouseGenome4302.0Arrayand3 IVTExpressKit(Affymetrix,SantaClara,CA,USA)accordingto themanufacturer ’ sinstructions(annotation:build32;6 September2011).MicroarrayswereassessedusingAffymetrixExpressionConsoleSoftware1.1withoutchanging thedefaultsettings(Affymetrix),andthedataqualitywas deemedadequateforfurtheranalyses.SubmissionofdatatoGeneExpressionOmnibusAllthedatasetsreportedhereinhavebeendeposited andarepubliclyavailableintheGeneExpressionOmnibus[GSE15640,GSE36378].VerificationofmicroarraydataInadditiontotheexperimentsperformedtovalidatethe qualityofthemicroarraydatapresentedpreviously [19,20],verificationexperimentswereexpandedtoinclude groupsofgenesinaccordancewiththespecificaimsof thisstudy.Real-timepolymerasechainreactions(PCRs) werecarriedoutusingtheExtracellularMatrix&AdhesionMoleculesPCRArray(PAMM-013Z;SABiosciences, Valencia,CA,USA)andthePI3K-AKTSignalingPCR Array(PARN-058Z;SABiosciences)accordingtothe instructionsprovidedbythemanufacturer.ThesearraysDelaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page2of19 http://arthritis-research.com/content/15/5/R174

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wereanalyzedusingRT2ProfilerPCRArrayData Analysissoftware(SABioscie nces)tocalculatethefold changesingeneexpressionoccurringwithintherespective timeperiods.Thesedataweresubsequentlyplotted againstthevaluesyieldedbytheGeneChipMouse Genome4302.0Arrayand3 IVTExpressKitarray (Additionalfile1:FiguresS1AtoS1C)andsubjectedto correlationanalyses(Additionalfile1:FigureS1D).DataanalysispipelineAflowdiagramofthedataanalysispipelineisdepicted inFigure1.NormalizationofmicroarrayprobecellintensityfilesProbecellintensityfiles(.CEL)werequantile-normalized andunderwentgeneralbackgroundcorrection.Control metricsweregeneratedandpassedforeacharray(Robust MultichipAnalysisperformedwithAffymetrixExpression ConsoleSoftware1.1;Affymetrix).Genescoveredbymultipleprobesonthemicroarraychipwerecollapsedto genesbyselectingtheprobeyieldingthehighestsignal (J-Express2009software;MolMineAS,Hafrsfjord, Norway).Rankingdifferentialgeneexpressionbetweentwo biologicalstatesToidentifycoordinatedandsignificantchangesbetween thetwochronologicallyclosesttimepointsfromeach strain,thecollapsedgenelists(21,673genes)were rankedbasedontheobservedrelativedifferenceupon performingsignificanceanalysisofmicroarrays(SAM) inJ-Express2009.SAMmakesnoassumptionaboutthe distributionofthedataandeffectivelyintroducesanonarbitraryfoldincreasecriterion,thussupersedingthe introductionofasubjectivefold-changethreshold.These rankedlistswereloadedintotheGSEAv2.07database (BroadInstitute,Cambridge,MA,USA).CompilationofgenesetsforgenesetenrichmentanalysisAgeneset(GS)isan apriori -definedgroupsofgenes compiled,curatedandannotatedtoreflectonespecific traitthatitsmembersshare,suchastheyareallcollagens [15].ThefirstGSscompiledforthisstudywereextracted fromthefollowingbioinformaticsresourcesasdescribed previously[22]:(1)GeneOntology(GO)( n =12,467),(2) NationalCancerInstitute(NC)( n =219),(3)PFAM(PF) ( n =4,147)and(4)KyotoEncyclopediaofGenesand Genomes(KE)( n =225).UsingtheWhichGenes1.0GS Network Clustering MCL clustering based on -log of CONNECTIVITY Ranking Differential Expression Collapsing probes to genes SAM based ranking of the chronologically closest time-points GS Enrichment Analysis 7871 GS retained (>10 & <1000 genes); 1000 GS permutations Normalization for Agerelated SG Development Exclusion of GS showing the same trend in both strains during the same time period (FDR q <0.05; nom. p -value<0.005; TAGS 50%) SG Global Gene ExpressionC57BL/6.NODAec1Aec2 (disease prone) & C57BL/6 (asymptomatic controls) n=5 per strain per time point (04-, 08-, 12-, 16-weeks) GS Resources n =21855 Gene Ontology (GO)12467 9 1 2 ) C N ( I C N 7 4 1 4 ) F P ( M A F P KEGG pathways (KE)225 Biocarta pathways (BI)249 Reactome pathways (RE)943 Transcription factor targets (TF)615 MicroRNA targets (MI)793 Genebands (GB)393 Data analysis g n i d l i u b k r o w t e N s e c r u o s e r & n o i t i s i u q c a a t a D Requirements for NodesFor EM-related GS (ancestor either GO_0071944, GO_0031012, GO_0005911): (FDR q <0.05) For EM-associated GS: 1st degree neighbor of a EMrelated GS ( 8% overlap in the LEs of the EM-related GS and the EM-associated GS) FDR q <0.05; nom. p -value <0.005; TAGS 50% Leading Edge (LE) Analysis to each GS computation of LE metrics (TAGS, LIST, SIGNAL) Flagging LE genes located in Aec1 and Aec2 Computation of a CONNECTIVITY overlap between the LE genes of all GSs paired one on one Requirements for Edges CONNECTIVITY score 0.08 (corresponding to 8% overlap in the LEs the two GSs) Figure1 Schematicrepresentationofthedataanalysisanddatavisualizationpipeline. Designedforunbiasedmappingofalterationsinthe transcriptionallandscapeoftheextracellular milieu(EM).CONNECTIVITY,degreeofoverlapinLEmembersbetweentheGSstheedgeisconnecting;F DR, falsediscoveryrate;GS,geneset;KEGG,KyotoEncyclopediaofGenesandGe nomes;LE,leadingedge;LIST,percen tageofgenesintherankedgenelist before(forpositiveenrichmentscore(ES))orafter(fornegativeES)thep eakintherunningES(indicationofwhereinthelisttheenrichmentscorei s attained);MCL,Markovclusteralgori thm;NCI,NationalCancerInstitutedatabase;PFAM,Pfam proteinfamiliesdatabase[21];SAM,significancea nalysisof microarrays;SG,salivarygland;SIGNAL,ESstrengththatcombinesLISTandTAGS;TAGS,percentageofgenehitsbefore(forpositiveES)orafter(for negativeES)thepeakintherunningES,(indica tespercentageofgenescontributingtoES). Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page3of19 http://arthritis-research.com/content/15/5/R174

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buildingtool[23],wecompiledtheremainingGSsfrom (5)BioCartapathways(BC)( n =249),(6)Reactomepathways(RE)( n =943),(7)transcriptionfactor(TF)binding motifs(mouseorthologswereinferredfromhumangenes) ( n =615),(8)microRNAbindingmotifsasdefinedinthe miRDBdatabase(http://mirdb.org/miRDB/)(MI)( n = 793)and(9)closegenomiclocalization(Ensemblgenesin bandsresource)(GB)( n =393).AllGSs( N =21,855)were downloadedbetween14and20August2011.Runninggenesetenrichmentanalysisandidentification ofleadingedgegenesGSEAaidsinovercomingtheanalyticalchallengesposed bypleiotropy,asgenesareassignedtoGSsthatrepresenteachoftheirtraits,andbythefactthatbiological processescommonlydependonacoordinatedchangein theexpressionofseveralgenes[15].Statisticalanalyses areperformedforeachGSbyassessingtheexpression patternformedbyitsmemberswithintheentiredataset (21,673genes).Thus,anasymmetricaldistribution skewedsignificantlytotheoverexpressedendofthe rankedlistsignifiessignificantenrichment.Incontrast, suchanasymmetricaldistributionindicatessignificant depletionincaseswheretheexpressionpatternofthe GSisskewedsignificantlytotheunderexpressedendof therankedlist.Thisstepofcomputationalinterpretation basedon apriori -definedandconsensus-basedbiologicalknowledgewithoutsettingarbitrarycutoffs,such asfoldchangeorsignificancelevel,preventstheintroductionofbiasandincreasestherobustnessandcomparabilityofresults.GSEAwasperformedforGSslarger than10andsmallerthan1,000(7,871of21,855GSs retained).Permutationnumberwasdeemedadequateat 1,000iterations,anddefaultvalueswereusedforall otherparameters. LEanalysisidentifiesthegenesofeachGSthatappear intherankedlistatorbeforethepointatwhichthe runningsumreachesitsmaximumdeviationfromzero. Hence,genesassignedtoaGS ’ sLE(LEgenes)arethe genesaccountingfortheindividualGSsignificantenrichmentordepletionsignal[15].LEanalyseswerecomputedafterGSEAusingGSEAv2.07.Subtractionofalterationsassociatedwithage-related salivaryglanddevelopmentTonormalizeforchangesingeneexpressionassociated withnormalSGactivities,wediscardedGSsthatyielded significantenrichmentordepletioninbothstrainsin parallelandoverthesameperiodoftime(falsediscovery rate(FDR)<0.05,nominal P -value<0.005,TAGS 50%in C57BL/6mice)fromallsubsequentanalyses(Figure2A; parallel).Reciprocalchangesoverthesameperiodoftime (forexample,enrichedinC57BL/6.NODAec1Aec2 while depletedinC57BL/6mice)wereretainedand,together withGSsuniquelyalteredinC57BL/6.NODAec1Aec2 mice(Figure2A;exclusive),selectedfornetworkbuilding.NetworkbuildingNetworkanalysisisthestudyofasystemthatisdepicted asconnections(thatis,edges)betweendiscreteobjects (thatis,nodes).TodefinetheEM,GSsthatyieldedaFDR lessthan0.05andhadeitherGO_0071944(cellperiphery), GO_0031012(ECM)orGO_0005911(cell – celljunction) asanancestorintheGOtreewereselectedandqualified asEM-relatedGSs. GSsconnectedbyanedge( 8%ofsharedLEmembers) toanEM-relatedGSwerequalifiedasanEM-associated processwhentheypassedthesignificancecriteria(FDR <0.05,nominal P -value<0.005,TAGS 50%).Definingthe forcesofattraction,thedegreeofoverlapinLEmembers betweentheGSsalsodeterminedtheirpositioninthenetworkcomputedusingtheedge-weighted,springforce – directedlayoutinCytoscape2.8.2[17].Cytoscapeisan opensourcesoftwareplatformutilizedforvisualizing complexnetworksandintegratingthesenetworkswith anytypeofattributedata[17].Theconnectivityparameter, definedbythedegreeofoverlapinLEmembersbetween theGSs,couldtherebyalsobeappliedasanedgeweight forthesubsequentMCLclustering[16]computedwithin Cytoscape.TheclustersidentifiedbytheMCLaredefined bysimulatingthestochasticflowwithinthenetworks[16].ResultsExtentofalterationsacrossthethreetimeperiodsApplicationofthedataanalysispipelineoutlinedin Figure1revealedthatthemostthematicallydiversealterationsspecificforC57BL/6.NODAec1Aec2 mice,involvingthemostEM-relatedGSsandEM-associated GSs(Figure2B),occurredbetween4and8weeksofage. Thesamewastrueforthenumberofgenesaccounting fortheGSs ’ significantenrichmentordepletion,thatis, LEgenes(Figures2Cand2D). Significantenrichmentat8weeksofageinvolved79 GSsthatdependedoncoordinatedupregulationof481 LEgenes.Interestingly,43%oftheseGSsweresimultaneouslybecomingdepletedinage-matchedC57BL/6mice (Figure2A;reciprocal).Overthesameperiodoftime, downregulationof359LEgenesledtosignificantdepletionof29GSs(Figures2Band2C).Between8and 12weeksofage,asingleGSwasbecomingdepleted (Figures2Aand2B)inconjunctionwithdownregulation of12LEgenes(Figures2Cand2D).Thetransitionfrom 12to16weeksofage,whichchronologicallycoincided withtheonsetofovertSS-likediseaseinC57BL/6. NODAec1Aec2 mice,wasmarkedbyenrichmentof12 GSscomprisingatotalof182LEgenes,aswellasdepletionof15GSsasaconsequenceofdownregulationof 227LEgenes(Figures2Band2C).Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page4of19 http://arthritis-research.com/content/15/5/R174

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Majorbiologicalthemesinvolvingtheextracellularmilieu duringemergenceofSjgren ’ ssyndrome Progressionfrompre-tosubclinicaldiseaseoccursbetween 4and8weeksofageThenetworkdisplayingallGSsenrichedby8weeksof age(Figure3),togetherwithinterpretationoftherespectiveLEgenes(Figure4),allowedustoidentifyfour majorbiologicalthemes:(1)activationofpathwayscharacteristicofinnateimmuneresponsestolongdoublestrandedRNAviruses;(2)insulinreceptor(Insr)and insulin-likegrowthfactor1(Igfr1)-mediatedsignaling viaphosphoinositide3-kinase(PI3K)andproteinkinase B(AKT)furtherguidingcellfate,proliferationanddifferentiation;(3)remodelingofepithelialcell – ECManchorageviafocaladhesions(FAs)whosespecificities allowedbidirectionalintegringrowthfactorsignaling pathwaycross-talkaswellasalleffectorprocessesrelatedtocellmotility;and(4)thethreemajorclassesof intercellularjunctioncomplexesengagingincell-cellsignalingviaE-cadherin(CDH1)andinvolvingtransforminggrowthfactor (TGF ). GSsthatweredepletedin8-week-oldC57BL/6.NODAec1Aec2 miceclusteredinthreeindependentnetworks (Figure5A),eachdependingondistinctsetsofLEgenes (Figure5B):(1)decelerationofECMturnover,(2)downregulationofgenesencodinggapjunctionproteinsand (3)lossofpositiveregulationofnerveimpulsesinconjunctionwithdownregulationofgenesencodingmembers ofallclassesofcysteine(Cys)loopneurotransmitter receptorsandfewermetabotropicreceptors. 08wk enriched 08wk depleted 12wk enriched 12wk depleted 16wk enriched 16wk depleted 0 100 200 300 400 500 600 700 Theme 1 Theme 2 Theme 3 Theme 4 no. of LE g enes 08wk enriched 08wk depleted 12wk enriched 12wk depleted 16wk enriched 16wk depleted -5 0 5 10 20 30 40 50 60 70 80 exclusive reci p rocal p arallel no. of signicant gene sets 08wk enriched 08wk depleted 12wk enriched 12wk depleted 16wk enriched 16wk depleted 0 100 200 300 400 500 LE genes not in Aec1 or Aec2 LE genes in Aec1 or Aec2 no. of LE genes 08wk enriched 08wk depleted 12wk enriche d 12wk depleted 16wk enriched 16wk depleted 0 5 10 20 30 40 50 60 70 80 EM-related EM-associated no. of signicant gene setsB A D C Figure2 ExtentofsignificantalterationsduringemergenceandonsetofovertSjgren ’ ssyndrome.(A) NumberofexclusiveGSs(GSs yieldingsignificanceinC57BL/6.NOD-Aec1Aec2miceonly),reciprocalGSs(GSsyieldingsignificanceinC57BL/6.NOD-Aec1Aec2andC57BL/6 mice,butwithoppositetrends)andparallelGSs(GSsyieldingsignificanceinC57BL/6.NOD-Aec1Aec2miceandC57BL/6micewiththesame trend(excluded)). (B) Numberofextracellularmilieu(EM)-relatedandEM-associatedgenesets(GSs)significantlyalteredforeachtimeperiod. (C) Numberofleadingedge(LE)genesunderlyingthechangesdisplayedin (A) ,categorizedwithrespecttolocalizationinsideoroutsidethe susceptibilityregions Aec1 and Aec2 (D) NumberofLEgenesunderlyingeachofthemajorbiologicalthemes. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page5of19 http://arthritis-research.com/content/15/5/R174

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Figure3 (Seelegendonnextpage.) Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page6of19 http://arthritis-research.com/content/15/5/R174

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Stabilizationofsubclinicaldiseasestatebetween8and 12weeksofageGO_0005581(COLLAGEN)wastheonlyGSyielding significanceduringthistimeperiod(Additionalfile1: FigureS2A)andrepresenting12LEgenes(Additional file1:FigureS2B).Transitionfromsubclinicaltoovertdiseasebetween12and 16weeksofageTheGSsenrichedat16weeksofage(Figure6A),in conjunctionwiththeirrespectiveLEgenes(Figure6B), delineatedtwodistinctthemes:(1)emergenceofaneffectorimmuneresponsecharacterizedbyreinforcement oftheIFN signatureandanaturalkiller(NK)cell population,togetherwithformationoftheprimaryimmunologicalsynapseandlatecostimulatorysignalsdeliveringsurvival,proliferationandmaturationsignalsto TcellsandBcells;and(2)resumptionofgenetranscriptionforCysloopreceptorswithacetylcholine(ACh), aminobutyricacid(GABA)andglycine(Gly)binding specificitiesandinitialupregulationofspecificsubsetsof metabotropicreceptors. At16weeksofage,80%ofthedepletedGSs(Figure7A) andalargenumberofLEgenes(Figure7B)showedpartial reversalofthealterationspertainingtoFAsandcell – cell junctionsobservedearlierbetween4and8weeksofage (Figures3and4).Theremaining20%oftheGSs,suchas thoseGSsnotsubjecttoearlieralterations,reinforcedthe cellularcomponent(CC)termsGO_0005923(TIGHT JUNCTION)andGO_0005925(FOCALADHESION).Majorbiologicalthemesdependenttosignificantly differentdegreesongeneslocatedin Aec1 and Aec2ThequantitativecontributionsoftheSS-predisposing genomicregions Aec1 (chromosome3;0to46cM)and Aec2 (chromosome1;29.7to106.1cM)toeachGSare, togetherwiththeLEgeneslocatedinthesesusceptibility regionsperbiologicaltheme,presentedinAdditionalfile 1:FiguresS3toS7.ComparingaverageproportionsofLE geneslocatedin Aec1 and Aec2 perGSandperbiological themeshowedthattheinnateimmunitythemewasleast dependent(mean=1.75%),andthattheadaptiveimmunitythemewasmostdependent(mean=18.71%),onLE geneslocatedin Aec1 or Aec2 (Figure8andAdditionalfile 1:FiguresS3andS6).Regardingthesubclinicalphaseof SS,thegreaterrelianceongeneslocatedinthecongenic regionsofthethemesassociatedwiththeSGs ’ homeostasisandintegritycomparedtoinnateimmunitymayindicatethatthelatteroccursubsequentlyandinresponseto thesetissue-specificalterations(Figure8andAdditional file1:FiguresS3andS4).Detailedannotationofnetworksbasedoninterpretation ofgenesetparametersandleadingedgegenepatternsAsdescribedintheMethodssection,thenumberofLE genessharedbetweenGSsdeterminedtheirposition,interconnectivityandclustermembershipincorrespondencewithallotherGSsofanetwork.Thus,GSsinclose proximitytoeachothersharedistinctsimilaritiesin theirLEgenepatterns.Toresolveredundancies,commonlycausedbyGSsrepresentingcomplexpathways, highlyinterconnectednetworkareasrequireadditional interpretation.ThesameaccountsforlargeGSsannotatedwithtermstoogeneraltoreflectthetruetheme sharedbytheirLEmembers. Thebasisforthiscuratedannotation,writteninitalic typeinFigures3,5A,6Aand7A,isformulateduponanalysisof(1)theLEmembersofeachMCLclusterdisplayed asLEgenecloudsgeneratedbyusingavectorgraphics – capableadaptationoftheWordCloudCytoscapeplugin [24](Figures4,5B,6Band7B),(2)eachGS ’ sLEgenes (Additionalfile2)and(3)currentliterature – basedinteractomemaps(Additionalfile1:FigureS8).Additionalfile3 comprisesthenetworksdisplayedinFigures3-7as infinitelyscalableandelectronicallysearchablevector graphics,therebyallowingthevisualizationofnetwork detail.Transcriptionalchangesunderlyingthemesbeingenriched duringprogressionfrompre-tosubclinicalSjgren ’ s syndrome – likediseaseInFigure4,theLEgenecloudforCluster_01-01,incombinationwiththepercentageofeachpathwaycoveredby itsLEmembers(Additionalfile2;TAGS),pointstoward twopatternrecognitionreceptors,namely,Toll-like receptor3(TLR3)andIFN-inducedhelicaseCdomain – (Seefigureonpreviouspage.) Figure3 Enrichmentsinthetranscriptionallandscapeoftheextracellularmilieuduringtransitionfrompre-tosubclinicalSjgren ’ s syndrome. Genesets(GSs)enrichedat8weeksofagedelineateactivationoftheinnateimmunesystemcoincidingwithsignificantalterations inthetargetedtissue ’ shomeostasisandintegrity.Proportionsofleadingedge(LE)genessharedbetweenGSsdefineddistance,organizationand clusteringoftheGSs.Dashedlines,separatorsbetweenmajorbiologicalthemes;annotationsinitalics,interpretationoftranscriptionalactiv ity inferredfromtheLEgenecloudsdisplayedinFigure4;nodecolor,Markovclusteralgorithm(MCL)clusternumber.Nodeshapes:triangles, extracellularmilieu(EM) – related;circles,EM-associated;nodesize,relativetonumberofdetectedgenesthataremembersofthisGS(reference node=50genes).Nodelabeltypesize,relativetopercentageofgenesbelongingtothisGS ’ sLE(TAGS)(referencenode=75%).Nodeborder: none,alterationofthisGSexclusivetoC57BL/6.NODAec1Aec2 mice;present,reciprocaltrendinC57BL/6mice.Edgecolor:degreeof overlapinLEgenesbetweenthetwoGSsconnectedbythisedge.ECM,ext racellularmatrix;Igfr1,insulin-likegrowthfactorreceptor1; Insr,insulinreceptor. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page7of19 http://arthritis-research.com/content/15/5/R174

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CLUSTER 03-01 CLUSTER 04 CLUSTER 03-03 CLUSTER 01-02 CLUSTER 02 CLUSTER 03-02 CLUSTER 01-01 REFERENCE NODEikbkeikbkgmaddmap2k3 map2k4map2k7map3k7mapk14 mapk3mapk8mapkapk3 mef2a mef2cnfkb2pik3c3 pik3r4ppp2cbrelatiraptlr3atf2cd14chukdusp3fosgab1 grb2itgb1junly96dusp1ikbkapikbkbrnf31traf3traf6ube2nube2v1rps6ka3 rps6ka5tab3tbk1tlr4tollipirak1irf3mavs met pak1pik3capik3cbpik3r1pten ptk2 ptk2b ptpn11 pxn raf1 rasa1 sos1 src stat3map2k1map4k2map4k3nfkb1 nfkbiangfprkci prkczrfflripk1tanktraddsmpd2sqstm1stat1 bag4birc2birc3 acta1 crk dock1 pik3r2 pik3r3 cflar dap3 ifih1 ubc nfkbib cyld casp2 casp1 capzbcdk5ctnna1ctnnd1cttndnm1ldnm2dock1gab1ilkilkapiqgap1jupkcnq1keap1lama3lims2lpp luc7l3nexnnumbpak1pdlim7pkd1plecptk2ptprmpxnrfwd2scrib slc22a5slc2a1 slc9a1slco2b1 slco3a1sorbs1st14stx4a stxbp3asyne2tenc1tgfbr1tjp1vav2 adam9afap1ank3ap2a1arrb1 arrb2atp7abcar1cdkn1bgrb2grb7 grlf1itgavitgb1jam2prkar1a prkar1bptk2b ptpra bsnd cadm1 cftrcol17a1ddr1dlc1dlg1dspdstebag9enahepb4.1l5epcamephx2erbb2erbb2iperbb3flnbgit1igsf8 pip5k1crac1spp1srcthbs1tln1ywhaz tesc tmod3 tsc1vaspvclzfp384zyxabcc1actn1arhgap26 arhgef11abi1abi2 actr3akt1arhgef6arhgef7rictortns1tns4trf pdk2pdpk1pik3capip5k1apld1 cbl cblb cblc cd2ap cdc42 cltb cltccrkap1m1ccnd1cdc37 diap1git2 hsp90aa1hsp90ab1 itgb5jun map2k1map2k4 mapk8 mapk8ip3 fat1 fgd4gsninppl1 myo10 myo9bnckap1nme1raf1rock1shc1bcrcapn1 capns1egfrapgef1rasa1 rock2 rrassos1pik3cbpik3r1ptenmapk3mylkarpc1a arpc1barpc3arpc4 arpc5l rdx scyl3 slc39a6 stxbp2acta1waslyes1 akt2 als2 amot parp1 ppp1r12a ppp1r14a cd14 cd81 cyfip1 dbnl pik3r2 pik3r3met capn22900073g15rik arhgap5 prkar2a xpo1 nf2 ppp1r12b synm plcg1 gipc1gm2aitchitgb4itpr2 lamc2lancl2lasp1llgl1macf1 map2k1mapre1mark2myo9bnf2palldpdlim7pip5k1a plekha1rab34rab5a rasa1rdxs100a6sh2b1synj2tesctln1tmod3tmsb15ltpm1trpm7 ndfip1nedd4ppp1r9aprkczrac3shroom2spna2spnb2spnb3tchptpm3vclvps11 vps16 clasp1clasp2 cnksr1ctgfcttncytip daxxdbnldstnepb4.1l5eps8l2ezrfrmd4b inpp5jfermt2fkbp15flnagng12myo1cmyo6sept2actn1actn4 add3capza1capza2cd2apdlc1 acta1afap1amotarhgap1arhgef2bcar1 psen1 psen2 ptk2b scnn1a exoc6 exoc8 tsc1 usp2 utrn acap2 tnfrsf12a add3ahnakamotanxa2 dlc1enah fermt2 flnbhdac4ilkkat2bmyh11 myh14 palld pdlim7 ptpn11sorbs1srcssx2iptcf7l1 tcf7l2tgfbr1 amotl2apcarhgap17arhgef2ash1lcadm1cgncldn23 cldn3cldn4cldn7crb3ctnna1ctnnd1erbb2farp2fgfr1 igf1rinsriqgap1lmo7map3k7 mapk3metmllt4nlkkeap1krit1lama3ldb1lin7cmagi1marveld2micall2mpdz mpp5 mpp7 mtdh oclnpak1 ptk2strnsympksynpotjap1tjp1tjp2tjp3sept7acta1actn1actn4baiap2tmsb15l tpm1 tpm3vclzyxerbb3jupkifc3mpz nf2numbpard3plekha7pmp22prkcicoro1cdlg1dlg5dsc2 dsg2dspgnpatitgb1ptpn6ptprjptprkptprmscribshroom2shroom3slc2a1epcamf11rfzd6 igsf5inadl jam2pkp1 pnnprkcdprkczpvrl2pvrl4rac1rac3 smad2 smad4 snai2 pkd1spna2spnb2spnb3 utrn ccm2 cd9cdc42cdh1crebbp9030425e11rikabi2dstn lancl2 lasp1llgl1ppp1r9a scnn1agja1ehd2 epb4.1 epb4.1l1 epb4.1l2 smad7 tgfb1 tgfb2 tgfb3anxa5wasl yes1 akap7 tln1 stx3itgb4krt14krt5lama3lamb2lamb3 lamc2cldn4col17a1egferbb2erbb3grb2 hras1itgb1 metpik3ca pik3r1 prkcasfn shc1 ywhab ywhag ywhah ywhaz myo1cplecslco2b1 slco3a1 trf akt1 cd9 dlg1 dst erbb2ip pdpk1pik3capik3cbpik3r1pip5k1aplcg1srcvaspzyxabca1atg12atg13 atg16l1atg3 atg4catg5 atg9abecn1 chmp1acln3ctsdeif2ak4 gas6hdac6hspa5jun map1lc3a mll5mtor nbr1npc1 ogtpik3c3 psen1rptorslc2a1 srebf1tnrc6avdr wrn pik3r2pik3r3prkcaprkcd prkczptpre relaadrb2cblexoc1exoc3exoc5 exoc6foxo3grb14 grb2hras1ptpn11 ptprarapgef1 rasa1sgk1shc1 sik2sorbs1sos1trip10adipor1akt1 akt2ctnna1ctnnd1 jup hk1igf1rinsrjak2irs1phiptsc2zfp106 bcar1 eif4ebp2enpp1 inppl1ctsb dlc1 flot1 flot2 arhgdiabcl10dlg1dusp3elf1malt1pawrptprjcblb sept7 epb4.1 1) Innate immunity 2) Insr / Igfr1 signaling 3) Focal adhesion & motility 4) Cell-cell junctions & cytoskeleton organization Figure4 (Seelegendonnextpage.) Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page8of19 http://arthritis-research.com/content/15/5/R174

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containingprotein1(IFIH1),alsoknownasMDA5. BoththesereceptorsarekeymoleculesupstreamofIFN regulatoryfactor3(Irf3)andsignaltransducerandactivatoroftranscription1(STAT1)(Figure4,Cluster_01-01). Alsodelineatedbythisclusterareupregulationof Tlr4 anditscoreceptors Cd14 andlymphocyteantigen96 ( Ly96 ).Thesemay,viatheirupregulatedsignalingcascade, deliverthestrongesttriggerfortheobservedcanonicalactivationofnuclearfactor -light-chain-enhancerofactivatedBcells(NFB)andmitogen-activatedprotein kinase8(MAPK8)[4,25].Inaddition,thegenenerve growthfactor( Ngf ),whichencodesanotherimportantinducerofNFB,wasupregulated,eventhoughNGF ’ scrucialreceptorp75neurotrophinreceptor(p75NTR)[26] wasabsentfromthelistofLEgenesforRE_P75NTRSIGNALSVIANF-KB;TAGS=73%(Additionalfile2). Determiningtheeffectoftheinterconnecting NC_TRAILSIGNALINGPATHWAY;TAGS=63%and BI_METPATHWAY;TAGS=74%GSsismoredifficult becauseofincompletecoverageofthedifferentarmsof theTNF-relatedapoptosis-inducingligand(TRAIL)pathway[27]andthehepatocytegrowthfactorreceptor (MET)pathway[28]bytheirrespectiveLEgenes(Additionalfile2).However,theirinvolvementindetermining cellfateandproliferationisreflectedbytheircentralpositioninFigure3. TheGSsandLEgenesassociatedwithCluster_02 (Figures3and4)suggestthatINSRandIGFR1,viatheir shareddownstreamsignalingcascadeinvolvingPI3K andAKT,upregulatemammaliantargetofrapamycin complex1(mTorC1)andmTorC2.ThemTorsystemin turnispivotalindeterminingcellfate[29].Increased autophagyisinferredbythepresenceofPI3Kpathway membersandseveralautophagy-relatedprotein( Atg ) encodinggenes(Figure4,Cluster_02). Incloseproximity,Cluster_03-01andCluster_03-02 delineatecell-matrixadhesioncomplexesthattransmit regulatorysignalsandmechanicalforces(Figures3and4) [30,31].Cluster_03-01,includingGO_0031581(HEMIDESMOSOMEASSEMBLY);TAGS=73%,definesthe hemidesmosome-mediated,laminin-5-dependentanchorageofepithelialcells ’ intermediatefilamentstothebasal laminaoftheECM.Cluster_03-02represents,inlarge part,signalingpathwaysthatareactivatedbyalterationsin acell ’ simmediatesurroundingsandaretransmittedvia actincytoskeleton – anchoredFAs,suchasNC_SIGNALINGEVENTSMEDIATEDBYFOCALADHESION KINASE;TAGS=68%[31].Thus,31of77genesannotatedintheCCGO_0005925(FOCALADHESION)GS werelocatedinitsLE(TAGS=40%). Matchingtheintegringenes(thatis, Itgav Itgb1 Itgb4 and Itgb5 )withthedominantgrowthfactorreceptorgenes (thatis, Met Insr Igfr1 ,fibroblastgrowthfactorreceptor1 ( Fgfr1 )and Tgfbr1 )intheLEprofilesdisplayedinFigure4 suggeststhatintegrin v5,upstreamoftheenriched integrin-linkedkinasesignaling-associatedGSs,providesa basisforIGFR1-integrincross-talk[32].Similarly, v5and v1mayallowforTGFBR1signalingbycollaborating withintegrinpathways(Figure3)[33]. Tgfb1 Tgfb2 and Tgfb3 ,togetherwiththeTFSmadfamilymember2 ( Smad2 )and Smad4 downstreamof Tgfbr1 andthenegativefeedback – associated Smad7 ,areallpresentintheLE ofCluster_04(Figure4).Thepresenceofosteopontin ( Spp1 ),anotherligandofintegrins v1and v5intheLE ofCluster_03-02(Figure4),indicatesthatFAmaturation mayalsooccurinrelationtoinnateimmunecells. SupportingacriticalroleofFAremodelingduringthis transitionfrompre-tosubclinicalSS-likedisease,calpain1( Capn1 )and Capn2 (Figure4),whichregulatethe dynamicsofFAassemblyanddisassembly,areatthe centerofthetwocalpain-specificGSs(Figure3).In addition,allothereffectorphasesofnon-muscle-cell movementarerepresentedbyGSsandLEgenesofCluster_03-02,Cluster_03-03andtheintercalatedsectionof Cluster_04,respectively(Figures3and4)[30]. Thefourthbiologicalthemeshares14.3%ofitsLE geneswithCluster_03-02describedabove.Thisisdueto molecularsimilaritiesbetweenCCGO_0030055(CELLSUBSTRATEJUNCTION);TAGS=41%andCC GO_0005913(CELL-CELLADHERENSJUNCTION); TAGS=46%.MultipleLEgenesbelongingtotheclaudin andtheoccludingenefamiliesfurtherindicateincreased formationoftightjunctionsatCCGO_0016327(APICOLATERALPLASMAMEMBRANE);TAGS=51% [30].Thesetwotypesofcell – celljunctioncomplexesdependcriticallyonCDH1expressedbyepithelialcells [30].Correspondingly,NC_E-CADHERINSIGNALING INTHENASCENTADHERENSJUNCTION;TAGS= 60%andCDH1anchorage-relatedGSGO_0017166 (VINCULINBINDING);TAGS=60%weresignificantly enrichedandaremappedatthecenterofFigure3.Furthermore,enrichmentofGSGO_0030057(DESMOSOME);TAGS40%[30]delineatesathirdclassof intercellularjunctioncomplexesassociatedwithgenesthat (Seefigureonpreviouspage.) Figure4 AnnotationoftheMarkovclusteralgorithmclusterswiththeirrespectiveleadingedgegeneclouds. Theindividualgenesets (GSs)ofeachMarkovclusteralgorithmclustershowninFigure3werecollaps edintoametanode.Network:metanode colorrepresentstheoriginalcolo r oftheancestorGSsandnodesizeandnodelabelfontsizeareproportiona ltothenumberofGSscollapsedintothismetanode(referencenode=15 GSs).Clusteredleadingedge(LE)geneclouds:Fontcolorrepresentsclu steringoftheLEgenesbasedontheconn ectionsbetweentheoriginalGSs.Fon t sizeisproportionaltothefrequencyofthegenein theLEsoftheGSscollapsedintothismetanode. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page9of19 http://arthritis-research.com/content/15/5/R174

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PF_01410 (FIBRILLAR COLLAGEN C-TERMINAL DOMAIN) PF_10582 (GAP JUNCTION CHANNEL PROTEIN CYSTEINE-RICH DOMAIN) GO_0005922 (CONNEXON COMPLEX) GO_0030199 (COLLAGEN FIBRIL ORGANIZATION) PF_00029 (CONNEXIN) REFERENCE NODE GO_0005581 (COLLAGEN) BI_INTRINSIC PATHWAY GO_0005921 (GAP JUNCTION) GO_0008066 (GLUTAMATE RECEPTOR ACTIVITY) GO_0005578 (PROTEINACEOUS EXTRACELLULAR MATRIX) GO_0031012 (EXTRACELLULAR MATRIX) PF_00090 (THROMBOSPONDIN TYPE 1 DOMAIN) GO_0005231 (EXCITATORY EXTRACELLULAR LIGAND-GATED ION CHANNEL ACTIVITY) PF_00093 (VON WILLEBRAND FACTOR TYPE C DOMAIN) PF_00060 (LIGAND-GATED ION CHANNEL) GO_0043205 (FIBRIL) PF_00045 (HEMOPEXIN) GO_0004970 (IONOTROPIC GLUTAMATE RECEPTOR ACTIVITY) GO_0015276 (LIGAND-GATED ION CHANNEL ACTIVITY) PF_01094 (RECEPTOR FAMILY LIGAND BINDING REGION) GO_0005234 (EXTRACELLULAR-GLUTAMATE-GATED ION CHANNEL ACTIVITY) GO_0008328 (IONOTROPIC GLUTAMATE RECEPTOR COMPLEX) PF_00413 (MATRIXIN) GO_0051971 (POSITIVE REGULATION OF TRANSMISSION OF NERVE IMPULSE) GO_0022834 (LIGAND-GATED CHANNEL ACTIVITY) GO_0005230 (EXTRACELLULAR LIGAND-GATED ION CHANNEL ACTIVITY) PF_01391 (COLLAGEN TRIPLE HELIX REPEAT (20 COPIES)) GO_0005583 (FIBRILLAR COLLAGEN) RE_NCAM1 INTERACTIONSPositive regulation of nerve impulses Core components of gap junctions Contact activation pathway (coagulation) Collagens (all structural classes) Cys-loop receptors (all categories) Formation and structural organization of the ECM Mmp-mediated collagenolysis Angioinhibitory signals 1) ECM turnover3) Neurotransmission 2) Gap junctions GO_0043205 (FIBRIL) CLUSTER 01-01 CLUSTER 01-02 CLUSTER 02 REFERENCE NODE CLUSTER 03c1ql2c1ql3c1qtnf1c1qtnf2 c1qtnf3c1qtnf6c1qtnf7 col10a1col11a1 col11a2col12a1col13a1 col14a1col15a1col19a1col1a1col1a2col23a1col24a1col25a1col2a1 col3a1col5a1col5a2col5a3col6a1 col6a2gdnfcol4a1col4a2 col4a3col4a5col4a6f10 f12f2f8f9fga fgbfggklkb1 procserpinc1 col6a3 col6a4 col8a2col9a1col9a2 colec10 colec11 colqeda emid2 emilin1 fcna gm7455 mbl2 scara3 sftpa1 sftpd ecm2 fras1 nell1 nell2 nov pxdn sspo thbs2 vwc2l vwce wisp1 wisp2 foxc2 lmx1b plod3 serpinh1 bmper chrdl1 chrdl2 acan cntn2 calb2gja10 gja5 gja8 gjb2 gjb3 gjb4 gjb5 gjb6 gjc3 gjd2 gjd4 gje1mippanx3 ahsgalbambn amelxangptl4 aspnbmp7clec3b col10a1 col11a1 col11a2 col12a1 col14a1 col15a1col1a1 col1a2col24a1 col2a1col3a1 col4a1col4a2 col4a5col4a6 col5a1col5a2 col5a3col6a1 col6a2col6a3col8a2col9a1 col9a2cpa6cpz crtapdmbt1 dsppecm2 efemp2eln emid2emilin1 emilin3enam epycfbln5fbn1 fbn2fras1 frem1frem2gabra6gpc2gpc3 gpc5gria3grin1grin2bhapln1hapln3hmcn1igf1ihhkeralama1 lama2lama4 lamb1loxl1lpllummamdc2 matn3mepe mfap2mfap4mmp10 mmp11 mmp13mmp14 mmp16mmp19 mmp1a mmp1b mmp2 mmp20mmp23mmp7mmp8 mmp9muc2ndpnepn nid1nid2nov npntntn3oc90olfml2aolfml2boptc otoapaplnprss36ptn pxdnrbp3rptnserac1serpine1sftpa1 sftpdshhslc1a3 smoc2sncasod3sparcsparcl1 spock1spon2tecta tectbtff3tgfbithbs2thbs4thsd4timp1 tnrush2avcan vitvtnwnt1 wnt10awnt10b wnt2wnt2b wnt3awnt8a wnt9awnt9bzg16zp1zp3a930038c07rikacan acheadamts12 adamts16 adamts19 adamts20adamts3adamts5adamts6adamts7 adamts8 adamts9 adamtsl1 adamtsl2bai2bai3c6c8b thsd7a unc5c unc5d wisp1 wisp2 wisp3 thsd1 rspo1 sspo hpxaccn1accn2accn3 accn5chrna1 chrna2 chrna3 chrna5 chrna6 chrna7 chrna9 chrnb1chrnb2chrnb3 chrnb4chrnd chrnechrngclca5cnga4cngb3gabra1gabra2 gabra3gabra4 gabra5gabra6 gabrb1gabrb2 gabrb3gabrd gabrg1gabrg2 gabrg3gabrq gabrr1gabrr2glra1glra2 glra3glra4glrbgria1gria2gria3gria4grid2grik1grik2grik3grik4 grik5 grin1 grin2agrin2bgrin2c grin2dhtr3ahtr3bkcnh2kcnh7kcnj1 kcnj10kcnj12kcnj14 kcnj2kcnj3kcnj4kcnj5 kcnj6kcnj8kcnj9p2rx1p2rx3 p2rx5p2rx6pex5lryr2trpc1trpc2 trpc6 grm1 grm2 grm3 grm5 grm7 grm8gucy2c gucy2e gucy2g npr1 npr3 tas1r1 tas1r2 vmn2r10 vmn2r84 vmn2r88 htr6 ifng il6 itga2 lama2 lgi1 ncs1 nmu nr2e1 oxt oxtr ptgs2 slc1a3 snca tacr1 tnf tnr uts2grin3ashisa9 casrgabbr1gprc2a-rs5 camk2b cartpt cckbr ccl2 drd1a egfr gfap gip cnih2 cnih3 dlg4fbn1 fbn2 mfap2 slc1a3 snca thsd4 1) ECM turnover3) Neurotransmission 2) Gap junctions A B Figure5 (Seelegendonnextpage.) Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page10of19 http://arthritis-research.com/content/15/5/R174

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areupregulatedapproximately8weekspriortotheonset ofSS-likediseaseinC57BL/6.NODAec1Aec2 mice.Transcriptionalchangesunderlyingthemesbeingdepleted duringprogressionfrompre-tosubclinicalSjgren ’ s syndrome – likediseaseInFigure5A,CCtermGO_0031012(EXTRACELLULAR MATRIX)islocatedatthecenterofthefirstmajorbiologicaltheme,becomingdepletedduringthistimeperiod (Figure5A).ItwasthelargestGSthatyieldedsignificance inthisstudy,with159ofits320memberscontributingto itssignificance(TAGS=50%).TheLEgenesgroupedin Cluster_01-01delineatebroaddownregulationofgenes encodingcollagensoftheECM(Figure5B)[34].ContributedbyGSBI_INTRINSICPATHWAY;TAGS=73%and suggestingendothelialcellactivation,thisclusteralsoincludescoagulationfactor – encodinggenes. TheLEgenecloudofCluster_01-02inFigure5Blists genesassociatedwithallcategoriesofspecializedECM proteins[30].Theseincludelaminin(LAM)encodingsubunits,suchas Lama4 and Lamb1 ;proteoglycans,suchas versican( Vcan );andglycoproteins,suchasfibrillin1 ( Fbn1 )and Fbn2 .Genescodingforallmatrixmetalloproteinases(MMPs)capableofdegradingcollagens,aswell asdistinctmembersofthedisintegrinsandmetalloproteinaseswiththrombospondinmotif(ADAMTS) family,alsocontributedtothesignificanceoftheGSs groupedinCluster_01-02(Figure5A).ADAMTSpeptidasescatalyzeprocollagens(forexample, Adamts3 )and inhibitangiogenesis(forexample, Adamts5 Adamts8 Adamts9 and Adamts20 )[35].Genesannotatedas inducersofwingless-typemousemammarytumorvirus (MMTV)integrationsitefamilymembers( Wnt )(for example,Norriedisease( Ndp )),severalWntgenes(for example, Wnt1 )andallWnt1-induciblesignalingpathway proteins( Wisp1 Wisp2 and Wisp3 )[30]completedtheLE ofCluster_01-02.Thesechangescomplementthemarked andbroaddecelerationofECMturnoverasapotential consequenceoftheongoinginnateimmuneresponse and/ordelayedconclusionofdevelopmentalprocessesin theSGsofC57BL/6.NODAec1Aec2 mice. Thesecondthemedelineatesdownregulationofgenes associatedwithGSsannotatinggapjunctioncoreproteins (forexample,PF_00029(CONNEXIN);TAGS=67%) (Figures5Aand5B)andthusrepresentstheonlyclassof cell-celljunctionsnotenrichedat8weeksofage. Thethirdthemeisdominatedbygenescodingfor ligand-gatedionchannelsessentialforneurotransmission(Figure5B,Cluster_03)[36].Thelargestpartof thesegenesencodessubunitsofanionicCysloopreceptors(GABAA12/12,GABAA2/3andGlyR5/5),cationicCysloopreceptorsubunits(serotonin-gated5HT3Aand5-HT3BandnicotinicAChreceptor14/16 subunits),18of20ionotropicglutamatereceptorsubunitsandATP-gatedchannelsP2XpurinoceptorsP2X1, P2X3,P2X5andP2X6,aswellassubsetsofvoltagegatedandacid-sensingpotassiumchannels(forexample, amiloride-sensitivecationchannels1to3(ACCN1to ACCN3)andACCN5).Theremainingclustersofthis genecloudrepresentmainlymetabotropicreceptorsinvolvedinsensoryperception,whereastheLEgenesassociatedwithGO_0051971(POSITIVEREGULATIONOF TRANSMISSIONOFNERVEIMPULSE;TAGS=62%) alsoincludeinflammatorymediatorssuchasIFN tumornecrosisfactor(TNF)andinterleukin6(IL-6),all ofwhichareknowntodecreasethethresholdfornerve impulsegeneration(Additionalfile2)[37].Transcriptionalchangesunderlyingstabilizationof subclinicaldiseasebetween8and12weeksofageLEgenesassociatedwiththecontinueddepletionof GO_0005581(COLLAGEN);TAGS=46%encodeallpeptidechainsforcollagentypeI,themostabundantcollagen oftheECM,andcollagentypeIII(Additionalfile1:Figure S2BandAdditionalfile2).CollagentypeIV,whichhas coverageof67%,isassociatedwithbasalmembranes[34].Transcriptionalchangesunderlyingthemesbeingenriched duringtransitionfromsubclinicaltoovertSjgren ’ s syndrome – likediseaseInCluster_01ofFigure6A,GO_0009897(EXTERNAL SIDEOFPLASMAMEMBRANE);TAGS=33%interconnectstheEM-associatedGSsthatdelineatetheadaptive effectorimmuneresponse.TheLEpatternofintegrins (Cluster_01;Figure6B)sug gestsanincreaseofCDH1 adhesiveintegrin E 7-expressingintraepithelialTcells, whereasCD11c,encodedby Itgax and Itgb2 ,pointstowardantigen-presentingcells(APCs)ofmyeloidorigin (Seefigureonpreviouspage.) Figure5 Depletionsinthetranscriptionallandscapeoftheextracellularmilieuduringtransitionfrompre-tosubclinicalSjgren ’ s syndrome.(A) Genesets(GSs)depletedwhenthemicewere8weeksofagedefinemarkeddecelerationofextracellularmatrix(ECM)turnover andsignificantlydecreasedtranscriptionofgenesassociatedwithgapjunctionformationandneurotransmission.Thelayoutparametersof Figure4AcorrespondpreciselytothelayoutparametersofFigure3.Thereferencenodeallowsestimationofscalinganddirectcomparisonof Figures3,5A,6Aand7AandAdditionalfile1:FigureS2A.Mmp,matrixmetalloproteinase. (B) AnnotationoftheMarkovclusteralgorithmclusters displayedin (A) withtheirrespectiveleadingedgegeneclouds.ThelayoutparameterscorrespondpreciselytothelayoutparametersofFigure4. ThereferencenodeallowsestimationofscalinganddirectcomparisonofFigures4,5B,6Band7BandAdditionalfile1:FigureS2B. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page11of19 http://arthritis-research.com/content/15/5/R174

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CLUSTER 02 REFERENCE NODE CLUSTER 01cd8acd8b1cr2ctla4cxcl10cxcl12cxcl9 cxcr3cxcr4 fasfcer2afcgr2bhcsticam1icam2ifitm1 5830411n06rik aceaqp4btla ccl19ccr4ccr7cd19cd2cd200r4cd247cd3dcd3e cd3g h2-ab1h2-dma h2-dmb2 h2-eb1 h2-m3 h2-oa h2-obh2-q7 h2-q8ifna1ifna5 ifna9il2il4tgtpotshb itgaeitgalitgb2ptprcs1pr1sellsema7aspnstab2thy1cd40cd40lgcd86cga cd209bcd22cd244 cd274cd28ciitaicosicosligh-6 il2rbil2rg il7rlck rag2tnfrsf13b tnfrsf13czap70 itga4itgb7klrg1klrk1ms4a1 cd48cd5 cd69cd74cd79acd79b cd83cd4tnfrsf18 tnfrsf4 tnfrsf9 ulbp1vcam1 aicdabtkmadcam1itgax kcnj3 klrd1 tnfrsf17 tnfsf13b acheanxa9brs3cckbr chrm4chrna1chrna3chrna4chrna5chrna6chrna7chrna9chrnb1chrnb2chrnb3chrnb4chrndchrnechrngdrd1adrd2 drd3gabra2galr2galr3 gpr143gpr83grin1grin2agrin2b grin2dgrin3agrin3bhtr7 kiss1rmc2r mrgpra1 mrgpra4 nmbrnmur1 nmur2npffr2 npy1rntsr1 ntsr2ppyr1 prokr2slc18a3 slc6a11slc6a13sstr1sstr2 sstr4sstr5 tacr1tacr2 tacr3tspo gabra3 gabra4 gabra6 gabrb1 gabrb2 gabrb3 gabrd gabre gabrg1 gabrg2 gabrg3 gabrp gabrq gabrr1 gabrr2 glra1 glra2 glra3 glra4 htr3b 1) Adaptive immunity2) Neurotransmission(partial reversal) B BI_THELPER PATHWAY GO_0009897 (EXTERNAL SIDE OF PLASMA MEMBRANE) GO_0005892 (NICOTINIC ACETYLCHOLINE-GATED RECEPTOR-CHANNEL COMPLEX) GO_0030594 (NEUROTRANSMITTER RECEPTOR ACTIVITY) KE_04672 (INTESTINAL IMMUNE NETWORK FOR IGA PRODUCTION) KE_05320 (AUTOIMMUNE THYROID DISEASE) KE_05340 (PRIMARY IMMUNODEFICIENCY) REFERENCE NODE BI_TCYTOTOXIC PATHWAY GO_0042165 (NEUROTRANSMITTER BINDING) RE_IMMUNOREGULATORY INTERACTIONS BETWEEN A LYMPHOID AND A NON-LYMPHOID CELL PF_02931 (NEUROTRANSMITTER-GATED ION-CHANNEL LIGAND BINDING DOMAIN) PF_02932 (NEUROTRANSMITTER-GATED ION-CHANNEL TRANSMEMBRANE REGION)B-cell activation and differentiation (BAFF-system/Cd40(l)/Icos(l)/Il4/Aicda) Ag-presentation via MHCII Immunological synapse & tertiary signals T-cell activation Interferons (Inf -1, -5,-9, Ifitm1) NK-cell-mediated pos. & neg. regulation of cytotoxic activity Nicotinic acetylcholine receptors (neuronal & muscle type) Neurotransmitter receptor expression (specific for several classes of ligands) ACh-, GABA& Glygated ionotropic receptors 1) Adaptive immunity2) Neurotransmission(partial reversal) A Figure6 (Seelegendonnextpage.) Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page12of19 http://arthritis-research.com/content/15/5/R174

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[38].Thelatterrepresentthemostprobablesourceforthe concomitantincreaseintranscriptionofvariousINF encodinggenes( Inf -1 Inf -5 Inf -9 and Ifitm1) inthe SGsofC57BL/6.NODAec1Aec2 mice(Figures6Aand6B). TheestablishmentofaNKcellpopulationinthetargetedtissuesissupportedbyseveraldistinctLEmembers (Cluster_01;Figure6B).Cytoto xicity-triggeringreceptors NKG2-DtypeIIintegralmembraneprotein( Klrk1 ), Cd244 anditsligandencodedbyUL16-bindingprotein1( Ulbp1 ) representthreekeycomponentsofNKcells ’ effectorpathway.Incontrast,theNKcellreceptorcomplexencodedby killercelllectinlikereceptorsubfamilyDmember1( Klrd1 ) andGmember1( Klrg1 )exertaregulatoryanticytotoxic effect[38,39].TheLEgenes Cd244 and Cd48 ,inconjunctionwith Cd2 andintercellularadhesionmolecule2 ( Icam2 ),mayfurthersuggestregulationofCD8+Tcellsby NKcells.Expressionofmajorhistocompatibilitycomplex (MHC)andMHC-relatedgenes,however,wereskewedtowardupregulationofMHCclassII(MHCII)andMHCII invariantchain( Cd74 )expression(Figure6B). Thechemokinereceptor-ligandprofilecharacterizes emigrationofmultipleAPCandlymphocytepopulations ( Cxcr4 : Cxcl12 ),aswellasreinforcedrecruitmentof T-helpertype1(TH1)cells,NKcellsandplasmacytoid dendriticcells( Cxcl3 : Cxcl9 / Cxcl10 and Ccr7 : Ccl19 ) (Figure6B)[38].ImmunecellhomingmayalsobefacilitatedbyincreasedexpressionofLEgenesthatencode mucosalvascularaddressincelladhesionmolecule1 ( MAdCAM-1 )andlymphocytefunction – associatedantigen1( Lfa-1 ),encodedby Itgal and Itgb2 and Icam1 ICAM1andLFA-1ligationisalsocriticalforCd28dependentT-cellactivation[38].ThepatternofLEgenes encodingcostimulatorymoleculesassignsimportanceto boththeactivatingCd28-dependentpathwayandthe inhibitorycytotoxicT-lymphocyteantigen4(Ctla4) – dependentpathway(Figure6B;Cluster_01).Regardingthe T-cell-associatedcentralcomponentoftheimmunological synapse,T-cellreceptor(TCR)accessoryproteins(for example, Cd3 ),TCRcoreceptors Cd4 and Cd8 andTCRassociatedmolecules(forexample, Cd45 ( Ptprc ))arealso coveredbytheLEgenecloudofCluster_01.Theconcomitantupregulationof Il2 Il2rb and Il2rg ,aswellasthe presenceof Cd69 ,representeffectsdownstreamofT-cell activation[38].RegulatingactivationofT-celleffector lineagesatthisstagemaytherebyinvolvethetwoLEgene B7familymembersBandTlymphocyteattenuator( Btla ) andTnfreceptorsuperfamily18( Tnfrsf18 )(Figure6B). Withrespecttolatecostimulatorysignals, Cd40 : Cd40lg andinducibleT-cellcostimulator( Icos) : IcosL arethe receptor-ligandpairspresentintheLEofCluster_01 (Figure6B).Boththesesystems,togetherwithLE-gene Il4 ,arecriticalformountingeffectiveTH2responses[38]. B-cell-specificgenes(forexample,immunoglobulin heavyconstant ( Igh-6 ), Cd79a Cd79b Cd19 and Cd22 ) arehighlyrepresentedintheLEgenecloudofCluster_01 (Figure6B).Increasedtranscriptionof Tnfrsf13C (thatis, Baffr ), Tnfrsf17 (thatis, Bcma )and Tnfrsf13B (thatis, Taci ), togetherwiththeircommonligand Tnfsf13b (thatis, Baff ), aswellastheactivation-inducedcytidinedeaminasegene ( Aicda) (Figure6BandAdditionalfile2;LEgenelist forKE_04672(INTESTINALIMMUNENETWORKFOR IGAPRODUCTION);TAGS=60%),furtherindicates strongsignalingforsurvival,proliferationanddifferentiationofBcellsinSGsmarkedbyovertdisease[38,40]. Thesecondbiologicalthemeenrichedby16weeksof agepertainstoneurotransmissionandmarksapartialreversalofchangesthatoccurredearlierinthedisease course.Ofthe78LEgenesdefiningenrichmentatthis laterstage(Figure6B;Cluster_02),40werepreviouslyassociatedwithdepletionofGSsconcerningneurotransmissionat8weeksofage(Figure5B;Cluster_03).Reinitiating transcriptionaremainlyACh-,GABA-andGly-gated ionotropicreceptorscodinggenes(Additionalfile2). Overlapswerealsofoundforgenesencodingreceptorsfor dopamine(thatis, Drd1a )andsubstanceP(thatis, Tacr1 ). Uniquetoenrichmentat16weeksofageweregenesencodingformetabotropicreceptorsspecificforACh(that is, Chrm4 )andsomatostatin(thatis, Sstr1 Sstr2 Sstr4 Sstr5 )(Figure6BandAdditionalfile2)[36].Transcriptionalchangesunderlyingthemesbeingdepleted duringtransitionfromsubclinicaltoovertSjgren ’ s syndrome – likediseaseGSs(Figure7A)andtheirLEgenes(Figure7B)depleted anddownregulated,respectively,duringthistimeperiod, predominantlysignifythereversalofpreviousenrichmentsinFAsandcell-celljunction – associatedGSsobservedat8weeksofage(Figures3and4).Pairwise (Seefigureonpreviouspage.) Figure6 EnrichmentsinthetranscriptionallandscapeoftheextracellularmilieuduringtransitionfromsubclinicaltoovertSjgren ’ s syndrome.(A) Genesets(GSs)enrichedat16weeksofagemirrortheestablishmentofapathogenicimmunereactioninthetargetedtissues and,inaddition,reflectpartialnormalizationofpriorneurotransmitterreceptorgene-associatedalterations.Thelayoutparameterscorrespo nd preciselytothelayoutparametersofFigure3.ThereferencenodeallowsestimationofscalinganddirectcomparisonofFigures3,5A,6Aand7A andAdditionalfile1:FigureS2A.ACh,acetylcholine;GABA, -aminobutyricacid;Gly,glycine;MHCII,majorhistocompatibilitycomplexclassII;NK, naturalkiller. (B) AnnotationoftheMarkovclusteralgorithmclustersdisplayedinFigure5Awiththeirrespectiveleadingedgegeneclouds.The layoutparametersofFigure5BcorrespondpreciselytothelayoutparametersofFigure4.Thereferencenodeallowsestimationofscalingand directcomparisonofFigures4,5B,6Band7BandAdditionalfile1:FigureS2B. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page13of19 http://arthritis-research.com/content/15/5/R174

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GO_0030055 (CELL-SUBSTRATE JUNCTION) BI_INTEGRIN PATHWAY GO_0005925 (FOCAL ADHESION) GO_0034329 (CELL JUNCTION ASSEMBLY) NC_SIGNALING EVENTS MEDIATED BY FOCAL ADHESION KINASE GO_0007044 (CELL-SUBSTRATE JUNCTION ASSEMBLY) GO_0005924 (CELL-SUBSTRATE ADHERENS JUNCTION) GO_0016328 (LATERAL PLASMA MEMBRANE) GO_0005923 (TIGHT JUNCTION) GO_0005901 (CAVEOLA) GO_0005911 (CELL-CELL JUNCTION) GO_0043296 (APICAL JUNCTION COMPLEX) KE_04530 (TIGHT JUNCTION) GO_0005913 (CELL-CELL ADHERENS JUNCTION) GO_0016327 (APICOLATERAL PLASMA MEMBRANE) REFERENCE NODEApicolateral plasma membrane & tight junctions Cell-cell adherens junctions Focal adhesion maturation1) Focal adhesion & cell-cell junctions (partial reversal) GO_0005901 ( CAVEOLA ) REFERENCE NODE CLUSTER 01atp1b3 cav1 cdh13 cln3 dlc1 gnaq hdac6 hk1 igf1r insr ldlr lipe myof ptrf slc27a1 slc2a1 smpd2 src tsc22900073g15rikactg1 actn4akt1akt2amotl1b230120h23rikcask cdk4cgncgnl1cldn1cldn10 cldn23 cldn3cldn4cldn7 cldn8clic4crb3csnk2a1 csnk2a2csnk2bctnna1cttn pik3ca ptpn21pxnrfwd2 sdc1sdc4syne2 tenc1 tgfb1i1tln2tns4 trip6vclzfp384ankrd23anxa5arhgef2b4galt1cdc42bpacdh1col17a1dag1dpp4dsc2gja1gjb1gjb2jupmyo1cpard3pard6a pard6gppp2cappp2cb ppp2r1appp2r1b ppp2r2cppp2r2dprkcdprkce prkchprkczprkd1ptk2ptprjptprkldb1lin7cmagi1 magi3marveld2 micall2mpp5mpp7oclnp2rx7itga6itgb3itgb4jublims1 lpp mdc1nexnpak1parvapgm5pkp3plekha7pvrl2shroom2 erbb2ipfhl2 flnbgit1 hic2ilkirf2itgb1itgb5jun map2k1map2k4mapk1mapk3mapk8ppp1r12b slc5a1spna2ssx2ipstx3sympksynpotiam1tjp1tjp2 tjp3epb4.1l1 epb4.1l2 exoc3exoc4f11rgnai1gnai3 hras1igsf5 inadl jam2krasllgl2 cdh13cln3dlc1gnaqhdac6 hk1igf1r insrldlr lipemyof ptrfslc27a1slc2a1smpd2srctsc2 dstebag9 enahepb4.1l5fn1gnpat ctnnb1ctnnd1 cxadrepcamerbb3krt14krt5lama3lamb3 lamc1 lamc2 pdpk1plec arhgap26 arhgef11arhgef7cald1cav1crklitga1rab13sgsm3smad3taok2tesk2 tns1tsc1 keap1kifc3kirrel2310057j16rikadd3 pkp1pmp22braf capn2 ccnd1 crk dock1 grb7 grlf1 myh10 myh11 myh14 myl12b myl9 nras rac1raf1rgnef rock2 arhgap6cd151cd9mllt4bcr capn1 capns1 mapk9 nck2 pcdh1 tmem47 akap7 dlg5 rras2 map2k2 fzd6 atp1b3 epb4.11) Focal adhesion & cell-cell junctions (partial reversal) A B Figure7 (Seelegendonnextpage.) Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page14of19 http://arthritis-research.com/content/15/5/R174

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comparisonoftheoverlappingGSsrevealedthat,onaverage,54%ofLEgenescontributingtodepletionat16weeks ofagealsocontributedtotheseGSs ’ priorenrichmentat 8weeksofage.ThehighestpercentageofLEmembers followingthispatternwasidentifiedforNC_SIGNALING EVENTSMEDIATEDBYFOCALADHESIONKINASE, with79%,andthelowestpercentagewasfoundfor GO_0043296(APICALJUNCTIONCOMPLEX),with 43%(Additionalfile2).TheLEgenesnotincludedin theseLEoverlapsdidnotdefineadditionalbiological themes,butinsteadcontributedpredominantlytotheincreasedaveragecoverageoftheEM-relatedGSsat 16weeks(TAGS45%)comparedto8weeksofage(TAGS 38%)(Additionalfile2).DiscussionAlthoughthetechnologyforgeneratingglobalgeneexpressionprofileshasmatured,analysisandinterpretation ofthesedatasetsstillposegreatchallenges.Thisisalso truewithregardtodelineatingtheunderlyingbiological andchronologicalcomplexchangesinbiologicalstates, suchascovertstagesofautoimmunityinanorgansubsequentlytargetedbyanautoimmunedisease.Thus,the possibilityofassessingallrelationshipsamongallcomponentsofabiologicalsystemsimultaneouslywithanintegratedandstandardizedconceptsuchastheone presentedhereinmeetsacleardemand[41]. Withrespecttotheimmunesystem – specificfindingsof thisstudy,consideringthepr esenceofIFNsignaturesinpatientswithSS[11],enrichmentofinnateimmuneresponse pathwaysat8weeksofageinC57BL/6.NODAec1Aec2 micewasanticipated.Inadd ition,themolecularbasis underlyingtheeffectorimmuneresponseatdiseaseonset mimickedallmajoraspectsofsialadenitisdescribedinpatientswithSS[9].Furthervalidatingourfindingsisthat C57BL/6.NODAec1Aec2 mice,duringtheirspontaneous (Seefigureonpreviouspage.) Figure7 DepletionsinthetranscriptionallandscapeoftheextracellularmilieuduringtransitionfromsubclinicaltoovertSjgren ’ s syndrome.(A) Genesets(GSs)depletedat16weeksofagesignify,toamajorextent,thepartialreversalofenrichmentsinFAsandcell – cell junction-associatedGSsobservedat8weeksofage.ThelayoutparametersofFigure6AcorrespondpreciselytothelayoutparametersofFigure3.The referencenodeallowsestimationofscalinganddirectcomparisonofFigures3,5A,6Aand7AandAdditionalfile1:FigureS2A. (B) Annotationofthe MarkovclusteralgorithmclustersdisplayedinFigure6AwiththeirrespectiveLEgeneclouds.ThelayoutparametersofFigure6Bcorrespondprecise ly tothelayoutparametersofFigure4.ThereferencenodeallowsestimationofscalinganddirectcomparisonofFigures4,5B,6Band7BandAdditional file1:FigureS2B. ** *** * * * 1) Innate immunity 2) Insr / Igfr1 signaling 3) Focal adhesion & motility 4) Cell-cell junctions & cytoskeleton organization 1) Deceleration of ECM turnover 2) Gap junctions 3) Neurotransmission 1) Collagen I & III 1) Adaptive immunity 2) Neurotransmission (partial reversal) 1) Focal adhesion & cell-cell junctions (partial reversal) 0 5 10 15 20 2508wk enriched 08wk depleted 12wk depleted 16wk enriched 16wk depleted % of LE genes in Aec1 & Aec2 per GS per theme Figure8 Degreeofdependenceofthemajorbiologicalthemesongeneslocatedin Aec1 and Aec2 (meanSEM). Proportionofleading edge(LE)geneslocatedinthedisease-causingcongenicloci Aec1 and Aec2 /geneset(GS)/majorbiologicalthemewithinthesamediseasephase werecomparedusingone-wayanalysisofvarianceandTukey ’ s posthoc test. P -values<0.05wereconsideredsignificant(* P <0.05,** P <0.01, *** P <0.0001).Columnsrepresentthemeans,anderrorbarsrepresentthestandarderrorsofthemean.ECM,extracellularmatrix;Igfr1,insulin-like growthfactorreceptor1;Insr,insulinreceptor. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page15of19 http://arthritis-research.com/content/15/5/R174

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andslowdevelopmenttowardovertdisease,displayedalterationsinbiologicalpathwaysthat,ifknockedoutoroverexpressedfrombirthonahealthygeneticbackground,induce aspectsofSS.ThesemodelsofSSincludemicedeficientfor NFBfeedbackregulation(C57BL/6. I B M/M)andmice transgenicfor Baff [7]. Directcomparisonoftheresultspresentedhereinwith theconclusionsformulatedsubsequenttoanalysesusing conventional “ topgenelist ” approaches[19,20]definesthe addedvalueofthissystemsbiology – basedmethodologyas follows.(1)FocusingonthetranscriptionallandscaperelatedtoandassociatedwiththeEMseemedadequateto map,initsentiretyandinastandardizedfashion,thealterationsintheSG ’ sdecision-makingprocessesassociated withtheemergenceofautoimmunityinthismodel.EmphasizingtheEMpreventedthemappingofsomeof previouslydocumenteddownstreameffectsinducedvia signalstransmittedbytheEM,however[19,20].(2)The earlyactivationoftheinnateimmunesystemdescribed hereinrepresentsacrucialfeaturewhichhasnotpreviouslybeenreportedinthecontextofthisdataset.(3) Byapplyingthismethodology,thedatasetcouldbe interpretedinsignificantl ymoredetail,whichsubsequentlycouldbecombinedtopresentamorecomprehensivepicture. ThetranscriptionallandscapeoftheEMoftissuestargetedbyautoimmunitydescribedhereinopensanovel andintegrativeperspectiveonthedevelopmentofautoimmunediseasesthatmightbeofmoregeneralrelevance (Figure9)[42].Asafirststep,itwillbeimportanttoinvestigatehowstrongly,inotherexperimentalmodelsof autoimmunity,theLEgenesdiffer.Thechronologicalinterrelationshipsandmajorbiologicalthemesidentified hereinmaybethesame,however.Thisknowledgemay proveespeciallycriticalwhenaimingtodelineate,ona systemslevel,themechanismsofactionandthetargeted organ ’ sstatesubsequenttoexperimentalimmunomodulatoryintervention[43]. -20 0 20 40no. o f LE g e nes in Aec1 & Aec2 481 2 16 -200 0 200 400 no. of LE genes aberrant salivary gland physiology autoantibodies focal inflammation hyposalivation age in weeks Immunity Insr / Igfr1 signaling Focal adhesion & motility, cell-cell junctions & cytoskeleton organization ECM turnover Neurotransmission Figure9 Summaryoftheinterrelationshipsbetweenthemajorbiologicalthemesdefiningthealterationsintheextracellularmilieu duringtheemergenceofSjgren ’ ssyndrome. Timepointsarealignedincorrespondencewiththechronologyofthedevelopmentofmajor featuresofSjgren ’ ssyndromeinC57BL/6.NODAec1Aec2 mice[8].Plottedonthe y -axisisthecumulativenumberofleadingedge(LE)genes (solidlines)associatedwithenrichmentanddepletionoftherespectivesummarizedbiologicalthemeovertime.Theright y -axisrepresentsthe sameparameter,butfortheLEgenesspecificallylocatedinthedisease-causingcongenicloci Aec1 and Aec2 (dashedlines) Thesummarized biologicaltheme Immunity comprisesthethemesInnateimmunity(Figure3)andAdaptiveimmunity(Figure6).Thesummarizedbiologicaltheme Focaladhesionandmotility cell – celljunctionsandcytoskeletonorganization comprisesthethemesFocaladhesionandmotility(Figure3),Gap junctions(Figure5)andFocaladhesionandcell-celljunctions(Figure7).Thesummarizedbiologicaltheme Insr/Igfr1signaling comprisesthe themeInsr/Igfr1signaling(Figure3).Thesummarizedbiologicaltheme ECMturnover comprisesthethemesECMturnover(Figure5)andCollagen IandIII(Additionalfile1:FigureS2).Thesummarizedbiologicaltheme Neurotransmission comprisesthethemesNeurotransmissioninFigures5 and6.ECM,extracellularmatrix. Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page16of19 http://arthritis-research.com/content/15/5/R174

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Thechronologyoftheetiopathologydefinedhereinestablishesseveralimportantpoints.First,longbeforeovert experimentalSS,susceptibilityloci-dependentandpartiallytransientalterationsassociatedwiththetargetedtissue ’ shomeostasisandintegrityformedthebasisforan innateimmunereaction.Thelatter,incontrast,was dependentpredominantlyongenesdescendingfrom theasymptomaticC57BL/6strainthatservedasageneticbackgroundforthegenerationofC57BL/6.NODAec1Aec2 mice.IfaroleofgenesgoverningtheSGs ’ homeostaticstateatsuchanearlystageofautoimmunity canbeconfirmed,thesegenesmayindeedcruciallycontributetoanindividual ’ sriskofdevelopingSS[5].Second, thelong-lasting,stablesubclinicaldiseasestatemayelucidatenoveldiagnosticstrategiesforidentificationofSSat anearlierstateandtherebyenabletimelyimmunomodulatorytreatment[44].Third,majorthemesthatdefinedthis stablesubclinicaldiseasewereabandonedconcomitantly withtheonsetofovertdisease.Thispermitsspeculation aboutwhetherthesetransientalterationsmayrepresent processesinitiatedbytheSGstoresolveenvironmental challengesortocompensatefordevelopmentaldeficiencies,primarilywithoutinvolvementoftheadaptiveimmunesystem.Fourth,LEgenepatternsassociatedwith costimulatorysignalsrevealedbotheffectorandregulatory ligand-receptorpairs ’ beingpresent,indicatingthateffectoraswellasimmunoregulatoryprocessesgovernthe onsetofovertdisease[45]. Althoughglobaldatasetsareseldomadequatetodefine theroleofasinglegeneorprotein,theisolatedstudyof individualcomponentsinturnislimitedintermsofelucidatinghowpropertiesofbiologicalsystemsemergeasa resultofcoordinatedinteractionsbetweenitsnumerous membersandprocesses[41].Totakefulladvantageofthe unbiasednatureof “ -omics ” datasets,ourconceptintegratesdataanalysisbyrelyingextensivelyonbioinformaticsresourcesforcompilationofconsensus-based, apriori definedbiologicalknowledgewithaninteractivemodelfor datainterpretationbasedonnetworkscomputedentirely fromexperimentaldata.Importantly,thisconceptistransferabletoglobaldatasetsofanynatureandachievesan importantreductioninthenumberofarbitrarycutoffsset atthestageofdataanalysis.Italsodiminishessignificantly theamountofpersonalbiascommonlyintroducedduring theprocessofdatainterpretation[46]andovercomesthe confinesoflistsandmatrices,whichhaveclearlimitations inconveyinglargeamountsofcomplexdataandinterrelationships[47]. Obviously,tobasesuchmappingsonadditionaldimensions,suchasglobalproteinsynthesisorposttranscriptionalmodificationprofiles,wouldsignificantly improvethevalidityofsuchanalyses.Theywillbecome morefeasibletechnologicallyandeconomicallyinthefuture[48].Inthisstudy,wehavecomputedameaningful basisthathasallowedustoformulateconclusionsin agreementwiththegeneralityofouraim.Inthefuture, assigningspecificweightstotheindividualgenesbased ontheiruniquenessorimportancetoaspecificGSmay furtherstandardizeandfacilitatethefinalstepsofdata interpretation.Inthemeantime,itisimportanttoprovideadditionaluser-friendlygraphicallayoutsofthenetworks,suchastheonespresentedherein,toenablethe readertoscrutinizetheauthors ’ detailedinterpretation ofthenetworks.ConclusionsByadheringtotheprinciplesofsystemsbiologyand adaptingbioinformatics-basedmethodologiesanddata visualizationtosuitouraims,thisstudyhasdelineateda novelperspectiveonthechronologyandinterplaybetweentheSGs ’ EMandtheroleoftheinnateandadaptive immunesystemsduringtheemergenceofspontaneous, experimentalSS(Figure9).Thetimelinedefinedherein highlightstheimportanceofgenesgoverningthetarget tissue ’ shomeostaticstateinestablishingastablesubclinicaldiseasestatelongbeforetheclinicalmanifestationof SS.Formulatingconclusionsinagreementwiththegeneralityofouraimwaspossibleonlyafterhavingdeveloped andappliedtheintegrateddataanalysisanddata visualizationpipeline,whichisalsopresentedherein.This data-drivenapproachadvancessystematicandimpartial interpretationofglobaldatasetsonthebackgroundof standardized,consensus-based, apriori -definedbiological knowledge.Itiswidelyapplicabletothefieldsofimmunologyandrheumatologyandwillgreatlyfacilitateanalysis ofcomplexalterationsinbiologicalstatesonasystems level,suchaschangesinducedasaconsequenceofexperimentaltreatmentinterventions.AdditionalfilesAdditionalfile1: FigureS1. Verificationofthemicroarraydata. Figure S2. Stabilizationofthesubclinicaldiseasestatebetween8and12weeks ofage. FiguresS3toS7. Contributionsofgeneslocatedinsusceptibility regions Aec1 and Aec2 toenrichmentsanddepletionsassociatedwith transitionfrompre-tosubclinicalSjgren ’ ssyndrome(SS)andtoonset ofovertSS,respectively. FigureS8. Interactomemodelbasedonthe leadingedgemembersassociatedwithenrichmentsinthetranscriptional landscapeoftheextracellularmilieuduringtransitionfrompre-to subclinicalSjgren ’ ssyndromebetween4and8weeksofage. Additionalfile2: Allnodeparameters,LEgenesandLEgenes locatedinsusceptibilityregions Aec1 and Aec2 intabularform. Additionalfile3: ThenetworksdisplayedinFigures3-7asinfinitely scalableandelectronicallysearchablevectorgraphics. Abbreviations ACh: Acetylcholine;ADAMTS:Adisintegrin-likeandmetalloproteasewith thrombospondinmotifs; AICDA :Activation-inducedcytidinedeaminasegene; AKT:ProteinkinaseB;Atg:Autophagy-relatedprotein;BTLA:B7family memberB-andT-lymphocyteattenuator;Capn:Calpain;CDH1:E-cadherin; Ctla4:CytotoxicT-lymphocyteantigen4;ES:Enrichmentscore;FBN1:Fibrillin; Fgfr1:Fibroblastgrowthfactorreceptor1;GABA: -aminobutyricacid;Delaleu etal.ArthritisResearch&Therapy 2013, 15 :R174Page17of19 http://arthritis-research.com/content/15/5/R174

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Gly:Glycine;Icam:Intercellularadhesionmolecule;Icos:InducibleT-cellcostimulator;IFIH1:Interferon-inducedhelicaseCdomain – containingprotein1; IFN:Interferon;Igfr1:Insulin-likegrowthfactorreceptor1;IgH6:Immunoglobulinheavychain,type ;IL:Interleukin;Insr:Insulinreceptor; IRF3:Interferonregulatoryfactor3;ITG:Integrin;KLRD1:Killercelllectinlike receptorsubfamilyDmember1;KLRG1:Killercelllectinlikereceptor subfamilyGmember1;KLRK1:Cytotoxicity-triggeringreceptor;NKG2-D:Type IIintegralmembraneprotein;LAM:Laminin;LFA-1:Lymphocytefunction – associatedantigen1;Ly96:Lymphocyteantigen96;MadCAM1:Vascular addressincelladhesionmolecule1;MAPK8:Mitogen-activatedproteinkinase 8;MET:Hepatocytegrowthfactorreceptor;MMP:Matrixmetalloproteinase; MMTV:Mousemammarytumorvirusintegrationsitefamilymember; mTorC:Mammaliantargetofrapamycincomplex;NDP:Norriedisease;NFB: -light-chain-enhancerofactivatedBcells;Ngf:Nervegrowthfactor; PI3K:Phosphoinositide3-kinase;P2X:ATP-gatedchannelsP2Xpurinoceptor; p75NTR:p75neurotrophinreceptor;Smad:Smadfamilymember; Spp1:Osteopontin;STAT:Signaltransducerandactivatoroftranscription; TAGS:Percentageofgenehitsbefore(forpositiveenrichmentscore)orafter (fornegativeenrichmentscore)peakinrunningenrichmentscore,which indicatespercentageofgenescontributingtoenrichmentscore; Tgf :Transforminggrowthfactor ;TLR:Toll-likereceptor;TNF:Tumor necrosisfactor;TNFRSF:Tumornecrosisfactorreceptorsuperfamily; TRAIL:Tumornecrosisfactor – relatedapoptosis-inducingligand;Ulbp1:UL16 bindingprotein1;Vcan:Versican;Wisp:Wingless-typeMMTVIntegrationsite family1-inducible-signalingpathwayprotein;Wnt:Wingless-typeMMTV Integrationsitefamily. Competinginterests Theauthorshavenofinancialornonfinancialcompetingintereststodeclare. Authors ’ contributions NDconceivedandimplementedthedataanalysisanddatavisualization pipeline,analyzedandinterpretedthedata,createdallthefiguresandwrote themanuscript.CQNtookpartindesigningthestudy,carriedouttheanimal experimentsaswellastheremainingexperimentsinthelaboratory,andwas involvedindraftingthemanuscript.KMTconceivedandsuccessfullyadapted theWordCloudplug-inandwasinvolvedindraftingthemanuscript.RJcontributedtotheconceptionofthestudy,wasinvolvedinrevisingthemanuscriptandcoordinatedtheproject.ABPconceivedthestudy,plannedthe animalexperiments,participatedinanalyzingandinterpretingthedataand wrotethemanuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements WethankDrBDelaleu-Justitzforcarefulrevisionofthemanuscript.This studywasfundedbytheResearchCouncilofNorway(ND,KMTandRJ),the BergenMedicalResearchFoundation(NDandRJ),theBroegelmann Foundation(NDandRJ),HelseBergen(NDandRJ)andNationalInstituteof DentalandCraniofacialResearch(NIDCR)grantR01DE-014344(toABP), NationalInstitutesofHealthgrantAI0819529(toABP)andNIDCRR01 DE-018958(toCQN). Authordetails1BroegelmannResearchLaboratory,DepartmentofClinicalScience,University ofBergen,LaboratoryBuilding,JonasLiesvei65,5021Bergen,Norway.2DepartmentofInfectiousDiseasesandPathology,CollegeofVeterinary Medicine,UniversityofFlorida,2015S.W.16thAvenue,Gainesville,FL32610, USA.3ComputationalBiologyUnit,Uni-Computing,UniResearchAS,Thormhlensgate55,5008Bergen,Norway.4DepartmentofRheumatology,Haukeland UniversityHospital,JonasLiesvei65,5021Bergen,Norway. 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