Reduced levels of CCL2 and CXCL10 in systemic lupus erythematosus patients under treatment with prednisone, mycophenolat...

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Reduced levels of CCL2 and CXCL10 in systemic lupus erythematosus patients under treatment with prednisone, mycophenolate mofetil, or hydroxychloroquine, except in a high STAT1 subset
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Arthritis Research and Therapy
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Mixed Material
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Paul R Dominguez-Gutierrez
Angela Ceribelli
Minoru Satoh
Eric S Sobel
Westley H Reeves
Edward KL Chan
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Arthritis Research and Therapy
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Abstract:
Introduction: Our recent data showed that signal transducers and activators of transcription 1 (STAT1), adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), and C-X-C motif chemokine 10 (CXCL10) were significantly elevated in a systemic lupus erythematosus (SLE) cohort compared to healthy donors. High and low STAT1 subsets were identified in SLE patient visits. The present study analyzed the correlation of common treatments used in SLE with the levels of these biomarkers. Methods: Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, of whom 60 had samples from two or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real-time polymerase chain reaction (PCR) assays. Relative expression of interferon signature genes, CCL2, and CXCL10 were determined by the ΔΔCT method. Results were correlated with therapy using prednisone, mycophenolate mofetil, and hydroxychloroquine and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher’s exact test. Results: CCL2 and CXCL10 were significantly higher in untreated patients compared to treated patients, however, in high STAT1 patient visits there is no significant difference between treated and untreated patients’ visits. When comparing linear regression fits of interferon (IFN) score with CCL2 and CXCL10, untreated patients and high STAT1 patients displayed significantly higher slopes compared to treated patients. There was no significant difference between the slopes of high STAT1 and untreated patients indicating that CCL2 and CXCL10 were correlated with type-I IFN in high STAT1 patients similar to that in untreated patients. CCL2 and CXCL10 levels in the high STAT1 subset remained high in treated patient visits compared to those of the low STAT1 subset. Conclusions: Among the biomarkers analyzed, only CCL2 and CXCL10 showed significantly reduced levels in treated compared to untreated SLE patients. STAT1, CCL2, and CXCL10 are potentially useful indicators of therapeutic action in SLE patients. Further work is needed to determine whether high STAT1 levels convey resistance to therapies commonly used to treat SLE and whether STAT1 inhibitors may have therapeutic implication for these patients.

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University of Florida
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University of Florida
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Reduced levels of CCL2 and CXCL10 in SLE patients under treatment with prednisone, mycophenolate mofetil, or hydroxychloroquine, except in a high STAT1 subsetPaul R. Dominguez Gutierrez1, Angela Ceribelli1, Minoru Satoh2,3, Eric S. Sobel2, Westley H. Reeves2and Edward K.L. Chan1* 1Department of Oral Biology, University of Florida, P.O. Box 100424, 1395 Center Drive, Gainesville, FL 32610 0424, USA, 22Division of Rheumatology and Clinical Immunology, Department of Medicine, 3Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, P.O. Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610 0221, USAAdditional file 1 Figure S1 8

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Figure S1 Expression of different biomarkers in high versus low STAT1 populations in both SLE and healthy donors. When analyzing different candidate biomarkers, there was no significant difference between high and low STAT1 UTX patients, with the exception of STAT1 ( D ). Comparing high and low STAT1 HD showed significant difference in STAT1 ( D ), CCL2 ( E ), and CXCL10 ( F ) but no significant difference for anti dsDNA( A ), IFN score ( B ), ADAR ( C ), miR 146a ( G ), pri miR 146a ( H ), and TNF ( I ). Regardless of STAT1 levels, UTX patients were significantly higher in anti dsDNA, IFN score, ADAR, STAT1, CCL2, and CXCL10 than HD ( A F ). Data are presented in box plot. All groups were compared among each other and only significant P values are shown indicating each specific comparison.

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Figure S2Comparison of high and low STAT1 subsets of all treated to untreated SLE patient visits. Data from Figure 1 are replottedto examine significant differences between the high and low STAT1 subsets.

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Figure S3Comparison of high and low STAT1 subsets of PDN treated patient visits to untreated patient visits. Data from Figure 2 are replottedto examine significant differences between the high and low STAT1 subsets.

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Figure S4Comparison of high and low STAT1 subsets of HCQ treated patient visits to untreated patient visits. Data from Figure 3 are replottedto examine significant differences between the high and low STAT1 subsets.

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Figure S5Comparison of high and low STAT1 subsets of MMF treated patient visits to untreated patient visits. Data from Figure 4 are replottedto examine significant differences between the high and low STAT1 subsets.

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Figure S6 Comparison of high and low STAT1 and dosage subsets on expression levels of the various biomarkers in the SLE cohort. Data from Figure 5 are replottedto examine significant differences between the high and low STAT1 subsets.

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Figure S7 Separate analyses of high and low STAT1 effects on CCL2 expression in various combined therapies. Data from Figure 8A are replottedto examine significant differences between the high and low STAT1 subsets.

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Figure S8 Separate analyses of high and low STAT1 effects on CXCL10 expression in various combined therapies. Data from Figure 8B are replottedto examine significant differences between the high and low STAT1 subsets.



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RESEARCHARTICLEOpenAccessReducedlevelsofCCL2andCXCL10insystemic lupuserythematosuspatientsundertreatment withprednisone,mycophenolatemofetil,or hydroxychloroquine,exceptinahighSTAT1 subsetPaulRDominguez-Gutierrez1,6,AngelaCeribelli1,4,5,MinoruSatoh2,3,EricSSobel2,WestleyHReeves2andEdwardKLChan1*AbstractIntroduction: Ourrecentdatashowedthatsignaltransducersandactivatorsoftranscription1(STAT1),adenosine deaminaseactingonRNA(ADAR),C-Cmotifchemokineligand2(CCL2),andC-X-Cmotifchemokine10(CXCL10) weresignificantlyelevatedinasystemiclupuserythematosus(SLE)cohortcomparedtohealthydonors.Highand lowSTAT1subsetswereidentifiedinSLEpatientvisits.Thepresentstudyanalyzedthecorrelationofcommon treatmentsusedinSLEwiththelevelsofthesebiomarkers. Methods: Peripheralbloodleukocyteswerecollectedfrom65healthydonorsand103SLEpatients,ofwhom60 hadsamplesfromtwoormorevisits.TotalRNAwasisolatedandanalyzedfortheexpressionofmRNAand microRNAusingTaqmanreal-timepolymerasechainreaction(PCR)assays.Relativeexpressionofinterferon signaturegenes,CCL2,andCXCL10weredeterminedbytheCTmethod.Resultswerecorrelatedwiththerapy usingprednisone,mycophenolatemofetil,andhydroxychloroquineandanalyzedbyWilcoxon/Kruskal-Wallistest andFisher ’ sexacttest. Results: CCL2andCXCL10weresignificantlyhigherinuntreatedpatientscomparedtotreatedpatients,however, inhighSTAT1patientvisitsthereisnosignificantdifferencebetweentreatedanduntreatedpatients ’ visits.When comparinglinearregressionfitsofinterferon(IFN)scorewithCCL2andCXCL10,untreatedpatientsandhighSTAT1 patientsdisplayedsignificantlyhigherslopescomparedtotreatedpatients.Therewasnosignificantdifference betweentheslopesofhighSTAT1anduntreatedpatientsindicatingthatCCL2andCXCL10werecorrelatedwith type-IIFNinhighSTAT1patientssimilartothatinuntreatedpatients.CCL2andCXCL10levelsinthehighSTAT1 subsetremainedhighintreatedpatientvisitscomparedtothoseofthelowSTAT1subset. Conclusions: Amongthebiomarkersanalyzed,onlyCCL2andCXCL10showedsignificantlyreducedlevelsin treatedcomparedtountreatedSLEpatients.STAT1,CCL2,andCXCL10arepotentiallyusefulindicatorsof therapeuticactioninSLEpatients.FurtherworkisneededtodeterminewhetherhighSTAT1levelsconvey resistancetotherapiescommonlyusedtotreatSLEandwhetherSTAT1inhibitorsmayhavetherapeuticimplication forthesepatients. *Correspondence: echan@ufl.edu1DepartmentofOralBiology,UniversityofFlorida,1395CenterDrive, Gainesville,FL32610-0424,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Dominguez-Gutierrezetal.;licenseeBioMedCentralLtd.Thisisanopenaccessarticledistributedunderthetermsof theCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23 http://arthritis-research.com/content/16/1/R23

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IntroductionSystemiclupuserythematosus(SLE)isasystemicautoimmunerheumaticdiseaseaffectingmultiplesystemsand organsinthebody.SeveralgeneticandenvironmentalfactorshavebeenimplicatedinSLEetiopathogenesis.Even thoughtypeIinterferon(IFN-I:IFN andIFN )wasidentified30yearsagotobeelevatedinSLEpatientserum,it isonlyinrecentyearsthatitsincreasedexpressionhas beenrediscoveredandpostulatedtoplayakeyrolein diseasepathogenesisinthemajorityofpatients[1-4]. InadditiontoIFN-I,STAT1(signaltransducersand activatorsoftranscription1),aninterferon-induciblegene, isinvolvedintypeI,II,andIIIIFNsignalingandisreportedtobeupregulatedinSLE[5].BesidesSTAT1, interferon-regulatedchemokinesalsoplayaroleinSLE pathogenesis[6].C-Cmotifchemokineligand2(CCL2) andC-X-Cmotifchemokine10(CXCL10)havebeenimplicatedinSLEasgoodindicatorsofpotentialflares[7]. TheroleofCCL2indiseasessuchaspsoriasis,rheumatoid arthritis,andmultiplesclerosishasincitedadditional interestonitsroleinSLE[8].BothCCL2andCXCL10 dependupontheJak/STATpathwayactivationforinductionbyinterferon[9-11]andthesetwochemokineswere identifiedasoneofthe12upregulatedproteinsinSLE[6]. TheroleofmicroRNAs(miRNAs)hasalsobeenimplicatedinautoimmunity[12,13].miR-146awasreported tobeunderexpressedinperipheralbloodmononuclear cellsofChineseSLEpatients[14].ThefunctionofmiR146aisnowknowntoregulateinnateimmuneresponse andendotoxintolerance[15-18].miR-146ahasalsobeen reportedtobeoverexpressedinSjgren ’ ssyndrome[19], psoriasis[20,21],andrheumatoidarthritis[22-24]. Inanaccompanyingmanuscript,wedescribedhighand lowSTAT1populationsinSLEpatients[25].Inthelow STAT1population,levelsofSTAT1correlatedwellwith IFNscore;however,inthehighSTAT1populationthey didnot.Moreimportantly,highSTAT1patientsdisplayed elevatedexpressionofCCL2andCXCL10,butnosignificantdifferenceswereobservedforIFNscoreandtumor necrosisfactoralpha(TNF )betweenhighandlow STAT1.Finally,whentheslopeofthelinearregression representingtherateofchangeofCCL2orCXCL10per unitofchangeofIFNscorewasanalyzed,theslopes ofCCL2/IFNscoreandCXCL10/IFNscoreweresignificantlygreaterinthehighSTAT1patientscomparedto thelowSTAT1patientsindicatingthatSTAT1potentiallyenhancedCCL2andCXCL10responsetoIFN-I [25]. ThecurrenttherapiesforSLEprimarilyaimtosuppress theinflammationandautoimmuneresponse.Commonly usedtherapiesincludeprednisone(PDN),mycophenolate mofetil(MMF),andhydroxychloroquine(HCQ).PDNis asyntheticglucocorticoidthatsuppressesinflammation byinhibitingnuclearfactorkappaB(NFB).Itinhibits monocyteandneutrophilinflammatoryfunctionsaswell asBandTcellresponses[26].Syntheticglucocorticoid, suchasdexamethasoneandPDNcaninhibitphosphorylationofSTAT1andpotentiallyblocksIFNinductionby suppressingINFreceptor(IFNAR)signaling[27];however, ithasbeenshownthatdexamethasonealsoupregulates STAT1transcription[27].ThisinhibitionofSTAT1functionwhileincreasingitstranscriptionappearstobecounterintuitivebutmayrepresentacaseofcelladaptingto compensateforthelossoffunctionalSTAT1.Increasesin STAT1levelsmayleadtoundesiredconsequences[28]. MMFisacytotoxicdrugcommonlyusedtopreventorgan rejectionaftertransplantationandalsototreatautoimmunediseasessuchasSLE.MMFisareversibleinhibitorofinosinemonophosphatedehydrogenasethat blocksthe denovo synthesisofguanosinenucleotides[29]. ThelatterisrequiredforgrowthandproliferationofT andBcells,astheylackthescavengerpathwayandare unabletocompensatefortheinhibitionof denovo synthesisofguanosine.InhibitionofTandBcellgrowthblocks autoimmuneresponseandleadstodecreaseinautoantibodyproductionandT-cell-mediatedtissuedamage.The antimalarialdrugHCQfunctionsbyincreasingthepHof endosomalvesicles.Thisdisruptsantigenprocessingand inhibitingtoll-likereceptor(TLR)3,7,8,and9activity [29-31];furthermore,HCQcaninhibitmacrophageproductionofinterleukin-1andinterleukin-6[29].Since TLR7/9havebeenimplicatedinincitingIFN-Iproduction duetorecognitionofselfRNA/DNA,theblockadeof theseTLRscouldbeattenuatingIFN-Iproductionand antigenprocessingforpresentationofTcellsbyantigenpresentingcellssuchasdendriticcells. Inthisstudy,weanalyzedifferencesintheexpression ofvariousbiomarkers,includingSTAT1,ADAR,CCL2, CXCL10,andmiR-146a,inSLEpatientstreatedwith PDN,MMF,andHCQversusuntreatedandhealthy donors.MethodsHealthydonorsandSLEpatientsPatientinformationisasdescribedintheaccompanying manuscript[25].Inbrief,wholebloodwascollectedfrom atotalof103SLEpatientsand65healthydonorsenrolled intheUniversityofFloridaCenterforAutoimmune Diseasesregistryfrom2008to2011.Healthydonorswere selectedbasedonnohistoryofautoimmunedisease, whileallSLEpatientssatisfiedtheAmericanCollegeof Rheumatologycriteria[32].Therewereatotalof180SLE visitswithsequentialsamplescollectedin60SLEpatients [25].Healthydonorsonlyvisitedthecliniconce;therefore, theyrepresentasinglevisit.Amongthetotalof180visits, SLEpatientswereactivein49visitsaccordingtotheSLE diseaseactivityindex(SLEDAI)score>4.Allhumanblood sampleswereobtainedfromenrolledindividualswiththeDominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page2of15 http://arthritis-research.com/content/16/1/R23

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approvalofinstitutionalreviewboardattheUniversityof Florida.Thisstudymeetsandisincompliancewithall ethicalstandardsinmedicineandinformedconsentwas obtainedfromallpatientsaccordingtotheDeclarationof Helsinki.DatacollectionRNAsampleswereisolatedfromperipheralbloodleukocytesforeachpatientvisitandanalyzedforgeneexpressionusingTaqManreal-timePCRassaysasdescribedin theaccompanyingmanuscript[25].Anti-double-stranded DNA(dsDNA)levels,C3andC4complementlevels,IFN score,andSLEDAIscorewereobtainedasdescribed[25]. C3andC4below90and15mg/dl,respectively,areconsideredsubnormallevels.DataanalysisTaqManreal-timePCRassayswereusedtomeasuregene expression.ThecopynumberofmiR-146awasnormalizedtototalloadedRNAwhereasmRNAlevelswere normalizedto18SRNA.CopynumberofmiR-146awas determinedusingastandardcurvewithsyntheticmiR146a(IntegratedDNATechnologiesInc.,Coralville,IA, USA)[33].RelativeexpressionofmRNAwasdetermined bytheCTmethod[34].SLEpatientswereprimarily treatedwithPDN,MMF,and/orHCQ.Correlationsofall therapiesduringeachpatientvisitwereanalyzedwith levelsofdifferentSLEbiomarkers.NopatientinourSLE cohortwastreatedwithbelimumab,aB-cell-activating factor(BAFF)inhibitorapprovedbytheFDAforSLE[35]. AnalyseswereperformedusingSASversion9.2andJMP Genomicsversion5(SAS,Cary,NC,USA).Wilcoxon/ Kruskal-Wallistestwasusedtoevaluatestatisticalsignificancebetweengroups.Fisher ’ sexacttestwasusedto examinethecontingencybetweenSLEDAIandtherapy. NormaldistributionofIFNscore,CCL2,andCXCL10as wellasthebimodaldistributionofSTAT1inSLEpatients andhealthydonor(HD)visitswasidentifiedasdescribed intheaccompanyingmanuscript[25].SpearmanRho( ) coefficientwasusedtodeterminemonotonicassociations inthestudy.Coefficientofdetermination(r2)wasusedto determinelinearcorrelations.Significancebetweenslopes wasevaluatedbyanalysisofcovariance(ANCOVA). P valueslessthan0.05wereconsideredsignificant.The GeneralizedEstimatingEquation(GEE)modelforrepeated measureswasusedtoaccountforpossiblewithinsubject effectsfrompatientswithmultiplevisits[36].ResultsComparisoninthelevelsofvariousbiomarkersinSLE patientvisitswithorwithouttreatmentChangesinC3,C4,andanti-dsDNAantibodylevelsin SLEpatientvisits,andmRNAexpressionlevelsofvariousbiomarkersinperipheralbloodleucocyteswere examinedforpossibleeffectsoftherapy(Figure1).As expected,SLEDAI(Figure1A)andanti-dsDNAautoantibody(Figure1D)levelsweresignificantlylowerin treated(Tx)thanuntreated(UTX)patients,whileC3 (Figure1B)andC4(Figure1C)weresignificantlyhigher inTxthanUTXpatients.Overall,anti-dsDNAautoantibody,IFNscores,adenosinedeaminaseactingon RNA(ADAR),STAT1,CCL2,andCXCL10,weresignificantlylowerinHDthaneitherUTXorTxSLEpatient visits(Figure1D-I).However,therewerenosignificant differencesamongthegroupsformiR-146a(Figure1J) andTNF (Figure1L)expression.pri-miR-146ashowed significantlyhigherlevelonlyinUTXcomparedtoHD. BimodaldistributionofSTAT1inSLEpatientandHD visitswasidentifiedasdescribedintheaccompanying manuscript[25].Tofurtherelucidatetheinfluenceofhigh andlowSTAT1populations,UTXandHDfromFigure1 werefurtherexaminedbycomparingthehigh(blue)and low(red)STAT1groups(SeeAdditionalfile1:FigureS1). Asexpected,regardlessofSTAT1levels,UTXwassignificantlyhigherinanti-dsDNA,IFNscore,ADAR,CCL2, andCXCL10thanHD(SeeAdditionalfile1:FigureS1AC,E,F)whiletherewasnodifferenceinSTAT1,miR-146a, pri-miR-146a,andTNF (SeeAdditionalfile1:Figure S1D,G-I).HighSTAT1HDdisplayedhigherlevelsof STAT1,CCL2,andCXCL10(SeeAdditionalfile1:Figure S1D-F)thanlowSTAT1HD;however,fortheremaining biomarkers,therewerenosignificantdifferences.Levelsof variousbiomarkersinUTXpatientvisitswerenotsignificantlydifferentbySTAT1levelswiththeexceptionof STAT1(SeeAdditionalfile1:FigureS1).Duetothelack ofsignificantdifferenceinlevelsofbiomarkersbetween highandlowSTAT1UTXpatients,UTXwerenotseparatedinanysubsequentanalysis. Next,variousbiomarkerlevelsintreatedpatientswith highversuslowSTAT1visitswerecomparedwithUTX andHD.Overalltwoveryimportantoutcomesbecame apparent.First,thelackofsignificantdifferencebetween UTXandhighSTAT1forSLEDAI,IFNscore,ADAR, CCL2,andCXCL10(SeeAdditionalfile1:FigureS2A-F, H,I)potentiallyindicatingthatthepathologyofhigh STAT1TxpatientsresembledthatofUTXpatients.Second,highSTAT1Txpatientvisitsdisplayedsignificantly higherCCL2andCXCL10(SeeAdditionalfile1:Figure S2H,I)thanthelowSTATgroup,whichmightbeindicatorsofincreasedpathologicalactivity.miR-146aalso showedthesametrend,however,highSTAT1Txpatients havehigherlevelsofmiR-146athanUTX(SeeAdditional file1:FigureS2J).Interestingly,pri-miR-146aappearedto haveanoppositetrend(SeeAdditionalfile1:FigureS2K).ComparisonofindividualtherapiesSincemanypatientswereonmorethanonemedication, wewantedtocomparebiomarkersinpatientswithanDominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page3of15 http://arthritis-research.com/content/16/1/R23

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Figure1 ComparisoninthelevelsofvariousclinicalparametersandbiomarkersinSLEpatientvisitswithorwithouttreatment. (A) Diseaseactivity, (B-C) complementlevels, (D) anti-dsDNAantibodylevels, (E) IFNscore, (F) ADAR, (G) STAT1, (H) CCL2, (I) CXCL10, (J) miR-146a, (K) pri-miR-146a,and (L) .TNF intreated(Tx)anduntreated(UTX)SLEpatientvisitsaswellashealthydonors(HD).Dataarepresentedasboxplot.All groupswerecomparedamongeachotherandonlysignificant P valuesareshownindicatingeachspecificcomparison.Averagetrendlinesforhigh STAT1(blue)andlowSTAT1(red)patientvisitsubsetsarealsoshownforcomparison.DetailcomparisonbetweenhighandlowSTAT1subsetsare showninAdditionalfile1:FigureS2.ADAR,adenosinedeaminaseactingonRNA;CCL2,C-Cmotifchemokineligand2;CXCL10,C-X-Cmotifchemokine10;dsDNA,double-strandedDNA;IFN,interferon;SLE,systemiclupuserythematosus;STAT1,signaltransducerandactivatoroftranscription 1; TNF ,tumornecrosisfactoralpha. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page4of15 http://arthritis-research.com/content/16/1/R23

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individualdrug.AsforPDN(Figure2),byexcludingpatientsnotreceivingPDNfromtheTxgroup,therewas nostatisticalsignificantdifferencebetweenPDNTxand UTXwithSLEDAI,C3,andC4(Figure2A-C).However, SLEpatientsreceivingPDNweremorefrequentlyinactive ( P =0.0071;likelihoodratio:7.44)thanactivebySLEDAI score.Theremainingbiomarkers(Figure2D-L)showed similarsignificanttrendsasseenintheTxpopulation (Figure1D-L),whichmightindicatethattheoverallresultswerefromacombinatoryeffectofthetherapyand/ oralltherapyhadsimilareffectsonthesebiomarkers.To appreciatetheseresults,HCQandMMFwerealsoanalyzedinthesamemanner(Figures3and4).SLEDAI,C3, andC4weresignificantlydifferentbetweenHCQpatients andUTX(Figure3A-C);however,onlySLEDAIandC4 weresignificantlydifferentbetweenMMFandUTX patientvisits(Figure4A-C).TheresultsforSLEDAI wereconsistentwithSLEpatientvisitstreatedwith HCQ( P =0.0002;likelihoodratio:13.9)orwithMMF ( P <0.0001;likelihoodratio:16.1)weremorelikelytobe ininactivestates.TheremainingbiomarkersforHCQ (Figure3D-L)andMMF(Figure4D-L)resembledthosein theentireTxpopulation(Figure1D-L). AfterestablishingthebasicroleofhighandlowSTAT1, theircorrelationwasfurtherexploredforeachtherapy. BeginningwithPDN,TNF wassignificantlydecreasedin thelowSTAT1PDNpatientvisitsrelativetoUTXand HD;however,highSTAT1PDNpatientvisitswerenot significantlydifferent(SeeAdditionalfile1:FigureS3L). ThistrendwasnotobservedforeitherHCQorMMF patients(SeeAdditionalfile1:FigureS4L,S5L).Highand lowSTAT1patientsunderPDNtherapy(SeeAdditional file1:FigureS3A-C)didnotdisplayanysignificantdifferencesforSLEDAI,C3,andC4,whichresembledthe earlierresults(Figure2A-C).ThisdifferedforHCQand MMFwherelowSTAT1patientvisitsweresignificantly lowerthanUTXpatientvisitsforSLEDAI,andhigherin C3andC4(SeeAdditionalfile1:FigureS4A-C,5A-C).In PDN,HCQ,andMMFpatientvisits,CCL2andCXCL10 wassignificantlyelevatedinthehighSTAT1population comparedtothelowSTAT1,butsignificantlydifferent fromUTX(SeeAdditionalfile1:FiguresS3H,I;S4H,I; S5H,I).Thisresembledwhatwasobservedearlierinhigh/ lowSTAT1Txpatients(SeeAdditionalfile1:FigureS2H,I) suggestingthathighSTAT1patientsmightmaintainhigh levelsofCCL2andCXCL10regardlessofthetherapy used. TherelationshipbetweenmiR-146aandpri-miR-146was particularlyrevealingwhentheanalysestookintoaccount thedifferenceinhighSTAT1versuslowSTAT1status. WhilemiR-146adidnotshowanysignificantdifferencein PDN,HCQ,andMMFpatientvisits(Figures2J,3Jand4J), highversuslowSTAT1Txpatientvisits(SeeAdditional file1:FigureS2J)aswellaspatientstreatedwithPDN, HCQ,andMMF(SeeAdditionalfile1:FigureS3J,S4J,S5J) revealedthathighSTAT1patientvisitsweresignificantly higherinmiR-146athanlowSTAT1patientvisits,UTX, andHD.Incontrast,pri-miR-146alevelsweresignificantly lowerinhighSTAT1patientvisitsthaninlowSTAT1patientvisits,UTX,andHDforhigh/lowSTAT1Txpatient visits(SeeAdditionalfile1:FigureS2K)aswellaspatients treatedwithPDN,HCQ,andMMF(SeeAdditionalfile1: FiguresS3K,S4K,S5K).Thereversetrendseenbetween pri-miR-146aandmiR-146awasprobablyduetodifferencesinconversionfromprimarytomaturemiRNAor potentialdifferencesintheirintrinsicstability. Therapydosagecouldvarybasedondiseasemanifestationandseverity.Toexaminetheeffectsoftherapy dosage,thePDN,MMF,andHCQtreatedpatientswere separatedbydosage(Figure5).Asdosageincreasedso didthelevelsofthebiomarkersthataresupposedto correlatewithdiseaseactivity.Thismightbeattributed tothewaytherapywasadministered.Asthediseaseactivityofpatientsbecamehigher,prescriptionofhigher dosesoftherapymightbeexpected.Essentially,therapy dosagemightactasamarkerofdiseaseactivity.Interestingly,thehighSTAT1patientvisits(blue)appearedto showhigherlevelsofSTAT1,CCL2andCXCL10than inlowSTAT1patientvisitsastherapydoseincreased (Figure5,Additionalfile1:FigureS6).AssociationbetweenCCL2,IFNscore,andtherapyTheaccumulatedevidencesofarappearedthatpatients withhighlevelsofSTAT1weremaintaininghighCCL2 andCXCL10expressionevenduringtherapy;wetested howSTAT1levelsaffectedtheassociationofCCL2and CXCL10withIFNscore.SinceCCL2andCXCL10are knowntobeinducedbyinterferon,thiswouldsuggesta positivecovariationwhereCCL2andCXCL10increaseas IFNscoreincreases.TheslopeofCCL2/IFNscoreand CXCL10/IFNscorethusrepresentstheassociationbetweenCCL2andCXCL10withIFNscore.Bycomparing theslopebetweengroups,theeffectsoftherapyontheassociationofCCL2andCXCL10withIFNscorecouldbe examined.Forexample,whentheslopeofCCL2/IFN scorewasgreaterforUTXthanthatofaparticulartherapy,itsuggestedthatthedecreasedassociationinCCL2/ IFNscoreforthetreatedpatientswasaresultofthatparticulartherapyorduetootherconditionsofthepatients. WhentheassociationofCCL2withIFNscorewasplottedasshowninFigure6A,threeitemswerenoted.First, bothUTXandTxweremonotonicandincreasedasobservedfromtheSpearmanrhocoefficient( ).Second, bothUTXandTxdisplayedalinearcomponentas describedbythecoefficientofdetermination(r2)and UTXhadagreaterlinearitythanTx.Third,UTXhada significantlygreaterslopeforCCL2/IFNscorethanTx ( P =0.0002,blackversusgreenline)potentiallyindicatingDominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page5of15 http://arthritis-research.com/content/16/1/R23

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Figure2 ComparisonofthelevelsofvariousbiomarkersintheSLEpatientvisitswithprednisone(PDN)therapyversusuntreated. DatawereanalyzedasinFigure1exceptonlypatientsreceivingPDNinthetreatedpatientpopulationwereincluded. (A) Diseaseactivity, (B-C) complementlevels, (D) anti-dsDNAantibodylevels, (E) IFNscore, (F) ADAR, (G) STAT1, (H) CCL2, (I) CXCL10, (J) miR-146a, (K) pri-miR-146a, and (L) TNF intreated(Tx)anduntreated(UTX)SLEpatientvisitsaswellashealthydonors(HD).Dataarepresentedasboxplot.Allgroupswere comparedamongeachotherandonlysignificant P valuesareshownindicatingeachspecificcomparison.AveragetrendlinesforhighSTAT1 (blue)andlowSTAT1(red)patientvisitsubsetsarealsoshownforcomparison.DetailcomparisonbetweenhighandlowSTAT1subsetsare showninAdditionalfile1:FigureS3.ADAR,adenosinedeaminaseactingonRNA;CCL2,C-Cmotifchemokineligand2;CXCL10,C-X-Cmotif chemokine10;dsDNA,double-strandedDNA;IFN,interferon;SLE,systemiclupuserythematosus;STAT1,signaltransducerandactivatorof transcription1;TNF ,tumornecrosisfactoralpha. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page6of15 http://arthritis-research.com/content/16/1/R23

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Figure3 ComparisonofthelevelsofvariousbiomarkersintheSLEpatientsvisitswithhydroxychloroquine(HCQ)therapyversus untreated. DatawereanalyzedasinFigure1exceptonlypatientsreceivingHCQinthetreatedpatientpopulationwereincluded. (A) Diseaseactivity, (B-C) complementlevels, (D) anti-dsDNAantibodylevels, (E) IFNscore, (F) ADAR, (G) STAT1, (H) CCL2, (I) CXCL10, (J) miR-146a, (K) pri-miR-146a,and (L) TNF intreated(Tx)anduntreated(UTX)SLEpatientvisitsaswellashealthydonors(HD).Dataarepresentedasboxplot.Allgroupswerecompared amongeachotherandonlysignificant P valuesareshownindicatingeachspec ificcomparison.Averagetrendlines forhighSTAT1(blue)andlowSTAT1 (red)patientvisitsubsetsarealsoshownforcomparison.Detailcompar isonbetweenhighandlowSTAT1subsetsareshowninAdditionalfile1:Figure S4. ADAR,adenosinedeaminaseactingonRNA;CCL2,C-Cmotifchemokineligand 2;CXCL10,C-X-Cmotifchemokine10;dsDNA,double-strandedDNA;IFN, interferon;SLE,systemiclupuserythematosus;STAT1,s ignaltransducerandactivatoroftranscription1;TNF ,tumornecrosisfactoralpha. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page7of15 http://arthritis-research.com/content/16/1/R23

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Figure4 ComparisonofthelevelsofvariousbiomarkersintheSLEpatientsvisitswithmycophenolatemofetil(MMF)therapyversus untreated. DatawereanalyzedasinFigure1exceptonlypatientsreceivingMMFinthetreatedpatientpopulationwereincluded. (A) Disease activity, (B-C) complementlevels, (D) anti-dsDNAantibodylevels, (E) IFNScore, (F) ADAR, (G) STAT1, (H) CCL2, (I) CXCL10, (J) miR-146a, (K) pri-miR-146a, and (L) TNF intreated(Tx)anduntreated(UTX)SLEpatientvisitsaswellashealth ydonors(HD).Dataarepresentedas boxplot.Allgroupswerecompared amongeachotherandonlysignificant P valuesareshownindicatingeachspecificcomparison. AveragetrendlinesforhighSTAT1(blue)andlowSTAT1 (red)patientvisitsubsetsarealsoshownforcomparison.Detailcompar isonbetweenhighandlowSTAT1subsetsareshowninAdditionalfile1:Figure S5. ADAR,adenosinedeaminaseactingonRNA;CCL2,C-Cmotifchemokineligand 2;CXCL10,C-X-Cmotifchemokine10;dsDNA,double-strandedDNA;IFN, interferon;SLE,systemiclupuserythematosus;STAT1,s ignaltransducerandactivatoroftranscription1;TNF ,tumornecrosisfactoralpha. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page8of15 http://arthritis-research.com/content/16/1/R23

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Figure5 ComparisonofthelevelsofvariousbiomarkersintheSLEpatientvisitsgivenPDN,MMF,andHCQtherapyathighorlow dosage. DifferencesbetweendosesofPDN (D,G,J,M) ,MMF (B,E,H,K,N) ,andHCQ (C,F,I,L,O) werenotsignificantwiththeexceptionof SLEDAI (A) ,andinfactSLEDAIscoreswerehigherforPDNdoseof20to60mg/daycomparedtothe2to18mg/dayofdose.HCQ, hydroxychloroquine;MMF,mycophenolatemofetil;PDN,prednisone;SLE,systemiclupuserythematosus;SLEDAI,SLEdiseaseactivityindex. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page9of15 http://arthritis-research.com/content/16/1/R23

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Figure6 AssociationbetweenCCL2,IFNscore,andtherapy.(A) TherelationshipofCCL2versusIFNscorepresentedasaslopewasanalyzed inuntreated(UTX,black)andtreatedSLEpatientvisits(Tx,green).SimilaranalyseswerecarriedoutforPDN-treated (C) ,MMF-treated (E) ,and HCQ-treatedpatientvisits (G) aswellasforhighSTAT1(blue)andlowSTAT1(red)forTx (B) ,PDN-treated (D) ,MMF-treated (F) ,andHCQ-treated patientvisits (H) .CCL2,C-Cmotifchemokineligand2;HCQ,hydroxychloroquine;IFN,interferon;MMF,mycophenolatemofetil;PDN,prednisone; SLE,systemiclupuserythematosus;SLEDAI,SLEdiseaseactivityindex;STAT1,signaltransducerandactivatoroftranscription1. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page10of15 http://arthritis-research.com/content/16/1/R23

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thattherapydecreasedCCL2responsivenesstoIFN-I.In Figure6B,TxwassegregatedintohighandlowSTAT1. Similarly,highSTAT1TxandlowSTAT1Txweremonotonic,increasingandlinear.HighSTAT1Txdisplayeda significantlyhigherslopethanlowSTAT1Tx(Figure6B, P <0.0001,blueversusredline)andsignificantlyhigher slopethanTx(Figure6A-B, P <0.0001,blueversusgreen line)indicatingthatCCL2responsivenesstoIFN-Iinhigh STAT1patientswasmoresimilartothatoftheUTX patients.OverallsimilarresultswereobservedforPDN, MMF,andHCQ(Figure6C-H).Thesameanalysiswas performedforCXCL10(Figure7).TheresultsweresimilartothoseofCCL2(Figure6)withtheexceptionfor PDNandMMFinthehighversuslowSTAT1patient visits(Figure7D,F).ForPDN,highSTAT1patientvisits werenotsignificantlydifferentthanlowSTAT1(blueversusredline);inaddition,highSTAT1PDNwassignificantlylowerthanUTX(Figure7C-D, P =0.0005,blue versusblackline)andthismightindicatethatPDNaffectedCXCL10responsetoIFN-1.ForMMF,highSTAT1 patientvisitshadsignificantlyhigherslopethanlow STAT1patientvisits(Figure7F, P =0.038,blueversusred line);however,highSTAT1MMFwasnotsignificantly differentinCXCL10fromMMF-treatedpatientvisits (Figure7E-F,blueversusgreenline).ExpressionofCCL2andCXCL10inhighversuslowSTAT1 patientsubsetswithindividualandcombinedtherapyFinally,allpossibletherapycombinations(MMF,PDN, HCQ,HCQ+MMF,PDN+MMF+HCQ,PDN+MMF, PDN+HCQ,andUTX)werecomparedfortheexpressionofallbiomarkers.Interestingly,whiletherewasno significantdifferencesinIFNscore,STAT1,ADAR,primiR-146a,andmaturemiR-146aobservedbetweenUTX andthevarioustreatments(datanotshown),CCL2and CXCL10displayedsignificanttrends(Figure8).Fornearly everytreatment,CCL2wasdecreasedcomparedtoUTX (Figure8A).OverallsignificantdecreaseinCCL2transcriptsinthosetreatedcomparedtoUTXpatientvisitsindicatedthattherapywasaffectingCCL2transcription; however,thismightnotbetrueforhighSTAT1patient visits(blueline)astheyweresignificantlyhigherinCCL2 thanthelowSTAT1patients(redline)fornearlyevery treatment(Figure8A).ThelowSTAT1patientsappeared toberesponsivetotherapyastheyweresignificantlylower thanUTXandthemajoritywasnotsignificantlydifferent fromHD(SeeAdditionalfile1:FigureS7A).Thiswas reversedinthehighSTAT1patientswhereHDwere significantlylowerthantreatedpatientsandthemajority werenotsignificantlydifferentfromUTXpatients(See Additionalfile1:FigureS7b). TheresultsforCXCL10werenotasconsistentas CCL2.UTXpatientsweresignificantlyhigherinCXCL10 thananytreatedgroups(Figure8B).Boththetreated patientvisits,highSTAT1patientvisits,andthemajority oflowSTAT1patientvisitsweresignificantlylowerthan UTX(Figure8A,Additionalfile1:FigureS8).Whilethe lowSTAT1patientvisitsweresignificantlylowerin CXCL10thanUTX,thehighSTAT1werenotsignificantly differentfromUTX(SeeAdditionalfile1:FigureS8)potentiallyagainsupportingthathighSTAT1levelscontributetomaintainthehighlevelofCXCL10inpatients undertherapy.DiscussionOurstudyfocusedonthedifferenceinthelevelsofSLEbiomarkersandtheirrelationshipwithinterferon,CCL2,and CXCL10inSLEpatientsgivendifferenttherapy.IFN-Iand interferonsignaturegeneswerereportedtobeelevatedboth atthemRNAlevelbasedondatafrommicroarrayanalyses andevenattheproteinlevelintheserumofSLEpatients [4,37-39].Notsurprisingly,ourresultsreaffirmtheelevated expressionofADAR,STAT1,CCL2,andCXCL10inSLE patients[25]asreportedintheliterature[1,2,6,7,37,40]. CCL2andCXCL10levelsarelowerintreatedversus untreatedSLEpatients.ThemajorityofSLEpatient visitswerereceivingtherapyatthetimeofsamplecollection.SLEpatientvisitsusingPDN,MMF,andHCQ aswellastherapycombinationsdisplayednosignificant decreaseofIFNscore,STAT1,ADAR,pri-miR-146a, andmaturemiR-146acomparedtountreated.Linear regressionanalysestreatingthepatientvisitsasindependentvariables(inFigures6and7)yieldedessentially thesameconclusionwhencomparedtousingtheGEE modelforrepeatedmeasures(datanotshown). PDNisaglucocorticoidthatsuppressesNFBsignaling [41].ItisunclearhoworevenifPDNsuppressesIFNproduction.Glucocorticoidshav ebeenreportedtosuppress STAT1phosphorylation(pSTAT1)[27],butdepending uponcelltypeandprofile,theycanalsoleadtochangesin thetranscriptionofSTAT1[28,42].STAT1isimportantfor CCL2andCXCL10inductionbyINF[43-45].Furthermore, thedecreaseinpSTAT1couldexplainwhyCCL2and CXCL10decreasedinthelowSTAT1patients.TheincreaseinSTAT1expressionmaybeanattempttocompensatefordecreasedpSTAT1levelsandmaypossiblyexplain theoccurrenceofthehighSTAT1patients.Thismayalso bethereasonforCCL2andCXCL10increaseinhigh STAT1patientsandwhyCCL2andCXCL10arenotassignificantlylowerinSLEpatientsundergoingtherapyinthe highSTAT1patientscomparedtothelowSTAT1patients. Ontheotherhand,CCL2andCXCL10expression levelsinSLEpatientsundergoingtherapyweresignificant lowerthanuntreatedpatients.PDNhasbeenpreviously reportedtodecreaseCCL2andCXCL10expression [46-48].IfPDNreducespSTAT1levels,thismayexplain inpartthedecreaseofCCL2andCXCL10expressiondue totheroleofSTAT1inchemokinesignaling[43-45].InDominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page11of15 http://arthritis-research.com/content/16/1/R23

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Figure7 AssociationbetweenCXCL10,IFNscore,andtherapy. DatawereanalyzedasinFigure6exceptthatCCL2wassubstitutedby CXCL10. (A) TherelationshipofCCL2versusIFNscorepresentedasaslopewasanalyzedinuntreated(UTX,black)andtreatedSLEpatientvisits (Tx,green).SimilaranalyseswerecarriedoutforPDN-treated (C) ,MMF-treated (E) ,andHCQ-treatedpatientvisits (G) aswellasforhighSTAT1 (blue)andlowSTAT1(red)forTx (B) ,PDN-treated (D) ,MMF-treated (F) ,andHCQ-treatedpatientvisits (H) .CCL2,C-Cmotifchemokineligand2; CXCL10,C-X-Cmotifchemokine10;HCQ,hydroxychloroquine;IFN,interferon;MMF,mycophenolatemofetil;PDN,prednisone;SLE,systemiclupus erythematosus;SLEDAI,SLEdiseaseactivityindex;STAT1,signaltransducerandactivatoroftranscription1. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page12of15 http://arthritis-research.com/content/16/1/R23

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highSTAT1SLEpatients,CCL2andCXCL10didnotsignificantlychangefromuntreatedSLEpatients,possiblyindicatingthattheelevatedlevelsofSTAT1arefacilitatinga pathogenicpatternoccurringintheuntreatedpatients.In part,STAT1maybeincreasingtocompensateforinhibitionofSTAT1phosphorylationandmaintainCCL2and CXCL10levelsasintheuntreatedpatients.STAT1has beenassociatedwiththerapyresistanceincancer.STAT1 overexpressionprotectscancersfromDNA-damaging agentsincludingradiationtherapiesandchemotherapies indifferentcancertypes[49].Radioresistantnu61derived fromradiosensitiveSCC61tumorsdisplayed49overexpressedgenes;ofthese49genes,31wereISGsalso includingSTAT1[50].FurthermorewhenSTAT1was overexpressedinSCC61cells,itdisplayedradioresistance [51].Similarly,humanfibroblastsrepeatedlyexposedto IFN-Idisplayedradio-resistance[52].In10cancercell lines,STAT1expressioncorrelatedwithresistanceto doxorubicinandtopoisomerase-IIinhibitors[53].In addition,14ovariancancerlineswereobservedforresistancetoplatinumcompoundswhereSTAT1wasassociatedwithresistancetocisplatinandAMD473[54]. TheseassociationsbetweentherapyresistanceandSTAT1 incancermayexplaintheassociationofSTAT1levels withhigherCCL2andCXCL10andtheapparentlackof therapysensitivityinhighSTAT1patients. Conclusions IncreasesinCCL2andCXCL10havebeenassociatedwith SLEpatientsenteringastateofflareactivity[6,7].We considerreductionofCCL2andCXCL10asgoodindicatorsofsuccessfultherapy,whileelevationinSTAT1levels mayindicatetherapyresistance.Furtherworkisneededto determinetherolethatSTAT1playsintherapy,butthis studygivesinsighttoapotentiallynewroleforSTAT1in SLE.OurstudyraisesaninterestingquestionwhetherSLE patientswithhighSTAT1statuscanbenefitfromtherapy withspecificSTAT1inhibitors[55]. Additionalfile Additionalfile1:FigureS1. Expressionofdifferentbiomarkersin highversuslowSTAT1populationsinbothSLEandhealthydonors. FigureS2. ComparisonofhighandlowSTAT1subsetsofalltreatedto untreatedSLEpatientvisits. FigureS3. Comparisonofhighandlow STAT1subsetsofPDNtreatedpatientvisitstountreatedpatientvisits. FigureS4. ComparisonofhighandlowSTAT1subsetsofHCQtreated patientvisitstountreatedpatientvisits. FigureS5. Comparisonofhigh andlowSTAT1subsetsofMMFtreatedpatientvisitstountreatedpatient visits. FigureS6. ComparisonofhighandlowSTAT1anddosage subsetsonexpressionlevelsofthevariousbiomarkersintheSLEcohort. FigureS7. SeparateanalysesofhighandlowSTAT1effectsonCCL2 expressioninvariouscombinedtherapies. FigureS8. Separateanalyses ofhighandlowSTAT1effectsonCXCL10expressioninvarious combinedtherapies. Abbreviations ADAR: adenosinedeaminaseactingonRNA;CCL2:C-Cmotifchemokine ligand2;CXCL10:C-X-Cmotifchemokine10;dsDNA:double-strandedDNA; HCQ:hydroxychloroquine;HD:healthydonors;IFNAR:interferonreceptor; IFN-I:typeIinterferon;miRNA:microRNA;MMF:mycophenolatemofetil; PDN:prednisone;pSTAT1:phosphorylationSTAT1;SLE:systemiclupus erythematosus;SLEDAI:SLEdiseaseactivityindex;STAT:signaltransducers andactivatorsoftranscription;TLR:toll-likereceptor;TNF :tumornecrosis factoralpha;Tx:treated;UTX:untreated. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions PRDGcarriedouttheexperiments.PRDG,MSandEKLCdesignedthestudy. PRDG,AC,andMSperformedthestatisticalanalysis.ESS,AC,andWHR enrolledpatientsforthestudy,collectedinformationandmaintainedthe database.PRDG,AC,andEKLCdraftedthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Figure8 TheexpressionofCCL2andCXCL10inhighversuslowSTAT1patientsubsetswithindividualandcombinedtherapy. (A) CCL2levelsinHD(healthydonor),untreated(UTX),andotherpatientvisitsundertreatmentwithdifferentcombinationsofPDN,MMF,andHCQ wereplotted(blackbars).OnlysignificantdifferencescomparingeachtreatmentgrouptoeitherHDorUTXareindicatedasblacklineswith P value shown.DatasegregatingintohighSTAT1(blueline)andlowSTAT1(redline)subsetsarealsoshownandsignificantdifferencesforthesesubsetsare indicatedbygreenasterisks. (B) CXCL10datawereanalyzedsimilarly.CCL2,C-Cmotifchemokineligand2;CXCL10,C-X-Cmotifchemokine10;HCQ, hydroxychloroquine;HD,healthydonor;IFN,interferon;MMF,mycophenolatemofetil;PDN,prednisone;SLE,systemiclupuserythematosus;SLEDAI SLEdiseaseactivityindex;STAT1,signaltransducerandactivatoroftranscription1. Dominguez-Gutierrez etal.ArthritisResearch&Therapy 2014, 16 :R23Page13of15 http://arthritis-research.com/content/16/1/R23

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Acknowledgments SupportedinpartbyagrantfromtheLupusResearchInstituteandthe NationalInstitutesofHealthgrantAl47859.PRDGwassupportedbyNIH traininggrantT90/R90DE007200.WethankallthestaffattheDivisionof Rheumatologyforcollectionofbloodsamplesandclinicalinformation. Authordetails1DepartmentofOralBiology,UniversityofFlorida,1395CenterDrive, Gainesville,FL32610-0424,USA.2DivisionofRheumatologyandClinical Immunology,DepartmentofMedicine,UniversityofOccupationaland EnvironmentalHealth,Japan,1-1Isei-ga-oka,Yahata-nishi-ku,Kitakyushu, Fukuoka807-8555,Japan.3SchoolofHealthSciences,Universityof OccupationalandEnvironmentalHealth,Japan,1-1Isei-ga-oka, Yahata-nishi-ku,Kitakyushu,Fukuoka807-8555,Japan.4Currentaddress: RheumatologyandClinicalImmunology,HumanitasClinicalandResearch Center,ViaA.Manzoni56,20089Rozzano,Italy.5Currentaddress:BIOMETRA Department,UniversityofMilan,ViaFestadelPerdono,7,20122Milan,Italy.6Currentaddress:DepartmentofUrology,UniversityofFlorida,1600SW ArcherRoad,Gainesville,FL32610-0247,USA. 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