Control of cytokine mRNA degradation by the histone deacetylase inhibitor ITF2357 in rheumatoid arthritis fibroblast-like synoviocytes : beyond transcriptional regulation

Angiolillietal.ArthritisResearch&Therapy(2018)20:148 https://doi.org/10.1186/s13075-018-1638-4

RESEARCHA RTICLE OpenA c c e s s

ControlofcytokinemRNAdegradationbyt hehistonedeacetylaseinhibitorITF2357inrhe umatoid a r t h r it is f i br ob la s t -

li k e synoviocytes:beyondtranscriptionalre gulation

ChiaraAngiolilli^1,2*,

PawelA.Kabala^1,2†,AleksanderM.Grabiec^2,3†,MarziaRossato^1,4,WiS.Lai⁵,GianlucaFossati⁶, PaoloMa sc ag ni⁶,C h ri s t i a n S t e i n k ü h l e r⁶,P e rr y J . Bl a c ks he ar⁵,K ri s A . R e e d qu i s t^1,2,D o mi n i q ue

L . B a e t e n^2†and

TimothyR.D.J.Radstake^1†

Abstract

Background:Histonedeacetylaseinhibitors(HDACi)suppresscytokineproductioninimmuneandstromalcellsofp atientswithrheumatoidarthritis(RA).Here,weinvestigatedtheeffectsoftheHDACigivinostat(ITF2357)onthetranscription alandpost-transcriptionalregulationofinflammatorymarkersinRAfibroblast-likesynoviocytes(FLS).

Methods:TheeffectsofITF2357ontheexpressionandmessengerRNA(mRNA)stabilityofIL-1β- induciblegenesinFLSwereanalyzedusingarray-

basedqPCRandLuminex.Theexpressionofprimaryandmaturecytokinetranscripts,themRNAlevelsoftristetraprolin(TTP,orZFP3 6)andotherAU-richelementbindingproteins(ARE-BP)andthecytokineprofileoffibroblastsderivedfromZFP36^+/+and

ZFP36^−/

−mice

wasmeasuredbyqPCR.ARE-BPsilencingwasperformedbysmallinterferingRNA(siRNA)- mediatedknockdown,andTTPpost-translationalmodifications

werea n a l y z edb y i m m u n o b lotting.

Results:I T F 2 3 5 7 r e d u c e d t h e e x p ressiono f 8 5 % o f t hea n a l y z e d I L - 1 β-

induciblet r a n s cripts,i n c l u d i n g c y t o k i n e s (IL6,IL8),chemokines(CXCL2,CXCL5,CXCL6,CXCL10),matrix- degradingenzymes(MMP1,ADAMTS1)andother

inflammatorymediators.AnalysesofmRNAstabilitydemonstratedthatITF2357acceleratesIL6,IL8,PTGS2andCXCL2 mRNAdegradation,aphenomenonassociatedwiththeenhancedtranscriptionofTTP,butnototherARE-

BP,andthealteredpost-

translationalstatusofTTPprotein.TTPknockdownpotentiatedcytokineproductioninRAFLSandmurinefibroblasts,w hichinthelattercasewasinsensitivetoinhibitionbyITF2357treatment.

(Continuedonnextpage)

*Correspondence:c.angiolilli@umcutrecht.nl

†PawlKabalaandAleksanderGrabieccontributedequallytothiswork.

†DominiqueBaetenandTimothyRadstakecontributedequallytothiswork.

1LaboratoryofTranslationalImmunologyandDepartmentofRheumatologya ndClinicalImmunology,UniversityMedicalCenterUtrecht,Utrecht,TheNetherland s2

AmsterdamRheumatologyandImmunologyCenter,DepartmentofClinicalImmunol ogyandRheumatologyandDepartmentofExperimental

Immunology,AcademicMedicalCenter/UniversityofAmsterdam, Amsterdam,TheNetherlands

Fulllistofauthorinformationisavailableattheendofthearticle

©TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0Internatio nalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricted use,distribution,andreproductionina nymedium,providedyougiveappropriatecredittotheoriginalaut hor(s)andthesource,providealinktotheCreativeC ommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecom mons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.

Angiolillietal.ArthritisResearch&Therapy(2018)20:148 Page2of13

(Continuedfrompreviouspage)

Conclusions:OurstudyidentifiesthatregulationofcytokinemRNAstabilityisapredominantmechanis munderlyingITF2357anti-

inflammatoryproperties,occurringviaregulationofTTP.TheseresultshighlightthetherapeuticpotentialofITF23 57inthetreatmentofRA.

Keywords:Rheumatoidarthritis,Fibroblast-

likesynoviocytes,Inflammation,mRNAstability,Tristetraprolin,Histonedeacetylasei n h i b i t o r , I T F 2 3 5 7

Background

Rheumatoidarthritis(RA)isac hronicimmune-

mediatedinflammatorydisease,characterizedbytheexcessi veacti-

vationo f t hei mmunes ystema ndt heu ncontrolledp ro- ductionofcytokinesandotherinflammatorymediatorsinsyn ovialj oints.Cytokiness ucha s t umorn e crosisf actor(TNF) a ndinterleukin( IL)-

1βp roducedbymacrophagesandlymphocytesinfiltratingthe synovialtissueleadtotheabnormalactivationoffibroblast- likesynoviocytes(FLS),whichinturncausesboneandc artila gedeterioration[1].Regulationofi nflammatoryc ytokineso c cursa t m ultiplelevelsandresultsfromtheintricatemodulatio nofepigen-

eticr egulatorym e c hanisms,a ctivationo f i ntracellularsign alingp athways,c ontrolo f m essengerR NA( mRNA)stabilit yandproteintranslation.ThecorrectregulationofmRNAdec ayiscriticalforimmunehomeostasis,asital-

lowscellstoquicklyadjusttheexpressionofinflammatorymed iators,t heoverproductionofw hichc oulda dverselyaffectth eorganism[2].ConditionsthatinterferewithstabilityofmRN Aa rea s sociatedw ithd iversed iseases,includingchronicinf lammationandcancer[3].

Adenosineuridine(AU)-

richelements(AREs)representoneofthelargestandmostimp ortantgroupsofcis-

actingmRNAstabilitydeterminants.AREsallowtherecruitm entoftrans-actingAREbindingproteins(ARE-

BP),whichinturnmediatem RNAd egradation[4].S e v eralh umanARE-

BPhavebeenidentified,suchastristetraprolin(TTP,orZFP36 ),T T P f amilym embersB R F1( ZFP36L1)a ndBRF2(ZFP 36L2),AU-richbindingfactor-1(AUF1,orHNRNPD),KH- typesplicingregulatoryprotein(KHSRP),andHua ntigenR ( HuR,o r ELAVL1).ThemajorityofARE-

BPp romotet her e cruitmentofA RE-

containingmRNAstotheexosomesforeventualdegradation, althoughsome,suchasHuRandHufamilymembers,actasm RNAstabilizingfactors[5].

Thee x pressiono f s everalA R E-

BPw asf o u n d t o b e dysregulatedi n RA,a n d t h eirs i l e n cings h o wnt o a f f ectkeyr e g ulatorym echanismsi n a r t h r i t i s p a t h o g enesis,bothi n v i t r o a n d i n v i v o [6–

10].T o d a t e , TTPi s t h e ARE-

BPt h at h asb e en b es t c h a r a c terizedan d associatedwithR Ad evelopmenta ndd i s e asep r o g r e s s i o n [11,1 2].TTPe xp re ssionisalter edin patientswit h syno viumaffectedb y RA[11]a n d TTP-

deficientm i c e d i s p l a y a severeinflammatoryphenotyp ethatincludessynovialpannusformationanderosiveart hritis[10].Remarkably,

Angiolillietal.ArthritisResearch&Therapy(2018)20:148 Page3of13 overexpressiono f e n d o g e n o u s TTPo r m u t a t i o n s a t T

TPphosphorylationsi t esp r o t ectm i ce i n e x p e r im ental modelsofarthritis[11,13].

GrowinginterestinthemodulationofARE-

BPinRApathologyhasthuspromotedthesearchfornovelinhib itorycompoundsthatcanreversetheaberrantexpressionandfu nctionofARE-BP.Inhibitorsofthemitogen-

activatedproteinkinase(MAPK)p38,acriticalregulatorofthep hos-phorylationstatusandactivityofmultipleARE-

BP,havebeenextensivelyusedtodampenuncontrolledprodu ctionofpro-

inflammatorycytokinesresultingfromdysregulatedmRNAde cay[14].However,p38inhibitorsarecurrentlynotapprovedfor RAtreatmentduetomolecule-

relatedadverseevents,suchascutaneoustoxicity,andlimitedcli n-

icalefficacy[15,16].HDACirepresentanovelclassofsmallm oleculedrugsthathaveshownpromisingresultsinvitroandinvi voinmodelsofRAandimmune-

relateddiseases[17,18]andhavedemonstratedinitialclinical efficacyinthetreatmentofsystemic-

onsetjuvenileidiopathicarthritis[19].Althoughtheprimaryme chanismofactionofHDACiisproposedtorelyontheregulation ofchromatinopeningandtranscription,studieshavereportedth atHDACicanimpaircytokinemRNAexpressiondespitefavor ingtheirtranscriptionalactivation[20,21].Wepreviouslyrepo rtedthatpan-

specificHDACiITF2357(Givinostat)andtrichos- tatinA (TSA)preventedIL-

6productioninRAFLSandmacrophagesbypromotingaccele rateddegradationofIL6mRNA[22].

Int hiss tudy,weaimedtod issectthetranscriptionalandp ost-

transcriptionalr egulationo f cytokinem RNAexpressionb y I TF2357,a ndt o i dentifyw hether,a ndthroughw hichmec hanisms,H DACic anr estoret hebal-anceinm RNA- stabilitymechanismst hata red eregulatedinRA.

Method s

PatientmaterialandFLSisolation

FLSwerederivedfromsynovialtissuespecimensobtain edf r o m p a t i e ntsw i t h RAb y n eedlea r t h r o s c o p y, aspreviouslydescribed[23],culturedinmediumsupple -

mentedw i t h 1 0%f etalb o v i n e s e r u m ( F B S,I n v i t roge n),andu s e d b e t weenp assages4 a n d 1 0 . A l l p a t i e n tsfulfilledthec r i t e r i a f o r the cl assificationo f RAan d ha dactivediseaseincludingclinicalarthritisofthejointfro mwhichthesynovialbiopsieswereobtained[24].

FLStreatmentandstimulation

FLSwe re c u l t u r e d o v e r nightin D u lbecco’sM odi fi edE agleM e d i u m ( D M EM,L i f e T echnologies)c o n t a i n i n g 1%FBSp riort o incubationw ithc ytokines.C ellsw ere pre-incubatedfor3 0 minw itheither250nM pan-

HDACinhibitorITF2357(Italfarmaco)or5μMp38inhibitor(

SB202190,S igma)a nds timulatedw ith1 n g/mlI L- 1β(R&DS ystems).I nformationa boutt hes pecificityo f t he HDACiispublished[25].

RNAextractionandgeneexpressionprofiling

RNeasyMicroKit(Qiagen)wasusedforRNAextraction.Qua ntityandp urityofRNAwa sa s sessedu singaN ano-

drops pectrophotometer( NanodropT e chnologies).R NA wasr e verse-transcribedu singa First-Strandc omplemen- taryD NA(cDNA)s ynthesisk it(ThermoS c ientific)a ndqua ntitative(q)PCRwasperformedusingSybrSelectPCRMaster M ix( AppliedB iosystems).F orq P CRarraya na-

lysis,RNAwasreverse-transcribedusinganRT^{2H T}First StrandK it( Q iagen),c DNAw a s m ixedw ithS ybrG reenqP CRM a sterM ix( Qiagen)a nde xpressiono f 8 3 g enesinvol vedinFLSactivationwasanalyzedusingRT^2Profilercustomiz edq P CRarrays.q P CRr eactionsw erep erformed

ona S tepOnePlusR eal-

TimeP C R S ystem( AppliedBiosystems)andrelativemRN AexpressionwascalculatedusingS tepOneS oftwareV .2.1(

AppliedB iosystems).Sequencesoftheprimersusedarelistedi nAdditionalfile4.Theratiobetweenthegeneofinterestandth eexpressionofhumanGAPDHormurineACTBhousekeepi nggenes,ort hee xpressiono f f iveh ousekeepingg enes(B2M ,HPRT1,RPL13A,GAPDHandACTB)wascalculatedforq PCRandqPCRarrays,respectively.

Proteinextractionandimmunoblotting

FLSw e r e l y s e d i n Laemmli’sb u f f erandp r oteinc o n t e n t wasq u antifiedw i t h a BCAP r o teinAssayK i t ( P i e r ce).

Equivalentamountsoftotalprot einlysatewerethenmi xedwithloadingbufferandboiledat95°Cfor5min.Protei nsw e r e r e solvedb y e l ectrophoresiso n e i t her4–

12%B i s-

TrisS D S N u PAGEg el s ( I n v i t r o g e n ) f o r 1 h a t constant 2 0 0 V,o r o n 1 0 % S D S-

PAGEg e l s f o r 5 h a t constant7 0 V f o r b e t t e r s e p a r a t i o n o f i m m unoreactivebandsrangi ngbetw een2 6 a nd 5 5 kDa.G els we retrans-

ferredt o p o l y v i nylidened i f l u o r i d e ( PVDF)m e m branes(Bio-

RadLaboratories),m e mbranesw e reb l o c kedi n Tris- bufferedsaline(pH8.0)containing0.05%Tween-20(Bio- Rad)a n d 4 % m i l k ( B i o -

Rad),w asheda n d p r o b e d overnightat 4°C with an t ib o diesrecognizingTTP(C ell Signaling),histone3(H3) (CellSignaling)ortubulin(Sigma-

Aldrich).A fterw ashing,m e m b r anesw e r e i n c u - batedwithhorseradishperoxidase(HRP)-

conjugatedswinea n t i - r abbito r g o a t a n t i -

m o u s e i m m unoglobulinsecondarya n t i b ody( D a k o ) , a

n d p r o t einv i s u alizationwasperformedusingaChemiDoc MPsystem(Bio-Rad).

Luminexa s s a y

RAFLSwereleftunstimulatedorweretreatedwith250n M I T F 2 3 5 7 f o r 3 0 m i n p r i o r t o s t i m u l ationw i t h

1ng/mlIL-

1βf o r 24h.SupernatantswereharvestedandIL- 8, m a t r i x m e t al loproteinase( M M P ) - 3,C XCL- 10,CXCL-5a n d C XCL-

6p r o t e ins ecretiond e t e r m inedb y Luminex(BioRa d)accordingtothemanufacturer’sinstructionsatthecore faci lity o ftheAcademicMedicalCenter(AMC).

AnalysisofmRNAstability

FLSw ereleftu nstimulatedo rw eretreatedw ith2 50nM IT F2357for30minpriortos timulationwith1ng/mlIL- 1β.A fter2 h o f s timulationc ulturem ediumw as discarded, cellswerewashedandfreshmediumcontaining

10μg/mlactinomycinD(ActD)(Sigma-

Aldrich)wasadded.Cellswerethenharvestedat0,2and5hf ollowingtheadditionofActD,R NAwasisolated,andthera tesofmRNAdegradationinthepresenceorabsenceofHDA Ci

assessedusingacustomizedRT^2Profiler™PCRArrayset (SABiosciences)asdescribedabove.Transcriptsdisplaying atleast1 .5-foldc hangei n t her ateofd egradation,c om- paredtoIL-1β-stimulatedcontrolswereanalyzed.

Lambdaphosphatasetreatmen t

FLSwerelysedin1×NEBuffer(NewEnglandBiolabs)sup plementedwith1mMMnCl2.Celllysatewasincubatedonic efor20 min,spundown,andsupernatantwascollectedandi ncubatedwith10,000U/mllambda(λ)phosphatase(NewE nglandBiolabs)at30°Cfor30min.Proteinlysatew a s a dde dt o loadingb uffera ndb oileda t 95° C f or5 m in,a ndf urthe rp rocessedf orimmunoblot-tingasdescribedabove.

siRNAtransfection

RAFLSweretransfectedusingDharmaFECT1( ThermoScie ntific).Thedaybeforetransfection,cellswereincubatedwithD MEMcontaining10%FBSwhichwasthenreplacedwithOPTI -MEMserum-

reducedmedium.AUF1,BRF1,BRF2,KHSRP,HuRandTT PspecificsmallinterferingRNA(siRNA)

(20nM)andcontrolnon-targetingsiRNA(20nM), ( ThermoS cientific)weremixedwithDharma-

FECT1andincubatedfor20minatroomtemperaturepriortotr ansfection;24h aftertransfection,mediumwasreplacedwith DMEMcontaining10%FBSandthiswasleftforanother24h.

TTPwild-typeandknockoutMEF

Mouseembryonicfibroblasts(MEF)werederivedfromlitter mateE14.5Zfp36(TTP)+/+andZfp36(TTP)−/

−embryos,aspreviouslydescribed[10].Zfp36−/

−miceweregeneratedbyinsertinga targetingvectorcontainin ga neomycinresistancegene(neo)intheTTPprotein- codingregion,whichgeneratedmultiplestopcodonsand

precludedsynthesisofthefunctionalprotein.MEFsweremainta inedinmediumcontaining10%FBS,100U/mlpenicillin,100μg /mlstreptomycin,and2mML-glutamine.Zfp36−/

−cellswereregularlymaintainedforonepassageinfeedingmedi umcontaining0.3mg/mloftheselectionantibioticGeneticin(G 418,ThermoFisherScientific).

Statisticala n a l y s is

Dataarepresentedasmean±SEMunlessotherwiseindi- cated.One-

waya nalysiso fv ariance( ANOVA)w a s u sedforanalyzings etsofdatarequiringmultiplecomparisons.Wilcoxonmatche dpairstestandtheratiottestwasusedforallotherpairedcomp arisons.DatawereanalyzedusingGraphPadsoftware7withp values<0.05consideredstatisticallysignificant.

Results

ITF2357rapidlysuppressestheexpressionofIL-1β- inducedinflammato rygenes

Weandothershaveshownthatthepan-HDACiITF2357 isapotentsuppressorofgenesregulatinginflammatoryac- tivation,adhesion,angiogenesis,cellsurvivalandextracellu- larmatrixdegradation[25–

27].Specifically,byscreeningabroadsubsetofgenesrelevantto diseasepathologyinRA,wefoundthattreatmentwithITF2357r educedtheexpres-sionofthemajorityofgenesresponsivetoIL- 1βstimula-

tioninRAFLS[26].Togainmoreinsightintotemporalchangesi ngeneexpressioninthepresenceoftheHDACi,weanalyzedthek ineticsofmRNAregulationof83selectedgenesusingcustomize dqPCRarrays(Fig.1aandAdditionalfile1).ITF2357reducedt heexpressionof85%oftheanalyzedtranscripts,regardlessofth ekineticsofgeneinductionafterIL-

1βstimulation.AsearliershownforIL6[22],thereductionobser vedincytokinemRNAaccu-

mulationafterITF2357treatmentcorrespondedtochangesatthe proteinlevel(Fig.1b).

ReducedexpressionofasubsetofITF2357- regulatedgenesisassociatedwithmRNAdecay Inapreviousstudywedemonstratedthatboththe pan-HDACiI T F 2 357a ndt r i c hostatinA (TSA)a cceler- atethemRNAdecayofIL6i n RAFLSandhealthydonorm a c rophages[22].T o t e s t w h e t h e r t h i s o b s e rva- tioncoul db eext en dedtooth er inflammatorym e d i ators ,wea n a l yzedm R N A stabilityo f t h e g e ness c r e e n e d i n ourm R N A k i n e t i c s e x periment.I n a d d i tiont o I L 6 mRNA,thestabilityofothertranscripts,suchasIL8,CX CL2,PTGS2,ADAMTS1,andBCL2L1,wasreducedafterI T F2357t r e a t m e n t ( A d d i t i onalf i l e2).I n c o n t r ast,oth ergenesincludingMMP1,MMP3,CXCL5,CXCL6,CX CL10,F o x O 1 an d A DAMTS1we re n o t a f f ectedb y IT F2357atthepost-

transcriptionallevel,eventhoughtheirm R N A e x p r e s s i o n w asr e p roduciblyr e g u l a tedb y ITF2357( A d diti onalf i l e s1a n d2).I n dependentq PCR

assaysc o n f i r m e d r e d u c edm R NAs t a bilityo f I L 6 ,I L 8 ,CXCL2andPT GS2transcriptsafter I TF2357tr eat m ent (Fig.2a).

Ino r d e r t o f u r t h erd i s s ectt h e t r a n s criptionala n d post-

transcriptionale f f ectso f I T F 2 3 5 7 o n m R N A expressi on,w e q u a n t i f i e d u n s p l i cedp r i m a r y t r a n s c r i p tsofthegenesthatwereeitheraffected(IL6,IL8andP TGS2)orunaffected(MMP1)byITF2357atthelevelOFm RNAdecay,andcomparedthemwiththeirrespectivematu retranscripts(Fig.2b).Thedifferencebetweenp r i m a ryan d m a t u r e t r a n s cripte x pressiono f IL6,I L 8 a n d P T GS2 a f t e r 4 h t r e a t m entw i t h I T F 2 357wasm o d e s t ; h o w e ver,i t b ecamem o rep r o m i n e n t a t 8 h andmarkedapronou ncedreductionintheexpressionofthematuretranscript. C on versely,M MP1primaryandm a t u r e transcriptratesw e r e s i m i l a r.T h e s e r e s u l tsconfirmedthatgenesfound unaffectedbyITF2357intermso f m R NAd ecay( e . g.M M P1)a r e p r e d o minantlyregulateda t t h e t r a n s c r i p t i on all evel,w h i l e t h o s e d e s t a -

bilizedbyITF2357treatmentarepost- transcriptionallyregulated.

ITF2357leadstoTTPtranscriptionalandpost- translationalchanges

EarlystudiesincancercellsindicatedthatHDACimodu- lateARE-

BPfunction[28,29].Therefore,weinvestigatedthee xpressio nofb othd estabilizing( T T P,AUF1,B RF1,BRF2,KHRSP) andstabilizing(HuR)ARE-

BPaftershorttreatmentw ithI TF2357i n RAF L S.W e f oundt hatTT P wasinducedbyIL-

1βa ndfurtherupregulatedbyITF2357.C onversely,o therA RE-BPw eree ithern otinducedb y I L-

1βo rd idn ote xhibita s pecificr egulationpatternincombinati onwiththeHDACi(Fig.3a).WeconfirmedthatITF2357ledto sustainedTTPmRNAexpressiona t l atert imep oints( Fig.3 b),w hilen otindu-cinganyoftheotherARE-

BP(datanotshown).Wethusinvestigatedwhetherinduction ofTTPmRNAexpressionbyITF2357couldalsoderivefrom theenhancedstabilityoft het ranscript.Surprisingly,I TF235 7r athera cteda s a destabilizingfactorofTTPmRNA(Fig.3c) ,whileenhan-

cingTTPprimarytranscriptatalltimepoints(Fig.3d).

Followinginflammatoryorgrowthfactor-

drivenp38MAPKa ctivation,T T P p roteini s p hosphorylat edondifferenta minoa cidr esidues,b e c omingi nactivated[3 0].WethereforeexaminedwhetherITF2357couldalsoaffect TTPactivitybyalteringitspost-

translationalstatus.After2hIL-

1βstimulation,weobservedincreasedintensityofanimmuno reactiveband(45kDa)attheexpectedmobilityforTTP(Fig.3 e).Abandwithhighermolecularweighto f a pproximately4 7 k Daw a s d etecteda fter4 h , andc onfirmedb y h igherimmun oblotr esolution(Additionalfile3A),suggestingphosphorylat ionoftheprotein[31].ITF2357significantlyreducedtheintens ityofthehigherband(Fig.3eandAdditionalfile3A),similarly

a

b

Fig.1(Seelegendonnextpage.)

(Seefigureonpreviouspage.)

Fig.1ITF2357suppressestheexpressionofIL-1β-responsivegenes.aRheumatoidarthritis(RA)fibroblast-likesynoviocytes(FLS) (n=3)wereeitherleftuntreatedorweretreatedwithITF2357priortoincubationwithIL-

1βfortheindicatedtime.TemporalchangesinmRNAaccumulationofIL-1β-

induciblegenesweremonitoredusingacustomizedqPCRarraysystem.DataarepresentedasfoldchangesinmRNAlevelscompared tounstimulatedcellsinthepresenceorabsenceofITF2357DifferencesinfoldchangesbetweenIL-1βandIL-

1β+ITF2357conditions,foreachtimepoint,wereanalyzedbyratiottest:*p<0.05,**p<0.01.bRAFLSwereeitherleftuntreatedorweretreatedwithITF2357 priortoincubationwithIL-1βfor24h.FLSsupernatantwasharvestedandlevelsofIL-8,matrixmetalloproteinase(MMP)-3,CXCL-10(n=6)CXCL-5andCXCL- 6(n=5)weremeasuredbyLuminex.Proteinconcentrationswerenormalizedto100%ineachexperimentforsamplesnottreatedwithhistonedeacetyla seinhibitorandexpressedasthepercentageofcontrol:*p<0.05,***p<0.001,ratiottest

top38MAPKinhibition(Additionalfile3B).Additionally,pro teinl ysatet reatmentw ithλ-

phosphataser educedt heintensityofTTPbanduponIL- 1βstimulationbutdidnotfurtherr educeTTP s ignali n s amp lest reatedw ithe itherITF2357orp 38inhibitor( Additionalf i le3 C).T ogether,theser esultss uggestt hatI TF2357h a s a d u alr olei n t heregulationo f T T P,b y i nducingmRNAe xpress iona t t hetranscriptionallevel,a ndb y p reventingT T P p hos phoryl-ationandsubsequentinactivation.

ITF2357s u p p r e s sesc y t o k i nep r o d u c t ioni n d e p e n d e ntlyo f AUF1,BRF1,BRF2,KHSRPandHuR

Despitet hep revalente ffecto f I TF2357o n T T P mRNA induction,w e c ouldn ote xcludet hep ossibilityt hatITF235 7m ayimpacto therA RE-BPbyd ifferentm e c ha-

nismsofaction,e.g.bymodifyingtheirpost-

translationalstate.T o i nvestigatew hethera nyo f t heo ther A RE-

BPincludedinourstudywouldberequiredforITF2357effects onIL6,IL8,CXCL2orPTGS2mRNAexpression,weperfor medknockdownofAUF1,BRF1,BRF2,KHSRPandH uRinR AFLS,achieving8 0–

95%silencingefficiency(Fig.4a).Weobservedthatknockdo wnofthesegenesl edt o m inorc hangesinc ytokineg enee xpr ession(Fig.4 b)a ndt hatI TF2357s imilarlys uppressedc ytoki neproductioninbothc ontrola ndA RE-

BPk nockdownconditions.A t rendt owardsd ownregulatio no f I L6a ndPTGS2mRNAexpressionwasnoticeableupo nKH-

typesplicingregulatoryprotein(KHSRP)knockdown,possib lyindicatingalternativemechanismsofregulationbythisARE -

BP,w hichg o beyondd irectc ontrolofmRNAstability[32].O verall,t heser esultsi ndicatet hat,d espitepotentialp ost- translationalm odificationsoccurringonotherA RE- BPa fterI TF2357t reatment,t hesew ouldn otbes ufficientt o mediatec hangesi n t hee xpressiono f t heinflammatorymedi atorsconsideredinourstudy.

TTPs i l e n c i n g c a u s e s p r o -

i nflammatoryr e sponsesi n R A FLSa n d i s r e q u i r e d t o p r e v e n t I T F 2 3 5 7-

dependentI L6suppressioninmurinefibroblasts WenextinvestigatedtheeffectsofTTPsilencingon cytokinee x pressioni n RAF LS.TTPs i R N A-

mediatedknockdownresultedin50%reductionofTTPmRN Aexpression( F i g .5 a,l ef t p an el ) , co n f i r m e d a t t h ep r o

teinlevel(Fig.5a,rightpanel),whichwassufficienttocaus e

increasedc y t o k i n e e x p r e s s i o n o f I L 6 ,I L 8,CXC L2a n d PTGS2(Fig.5b),butnot MMP1(datanotsho wn)bothinunstimulated(IL6,IL8)andIL-1β-

stimulatedconditions(IL6,IL8,PTGS2).Althoughnots tati sticallysignificant(p=0.05),inductionofCXCL2a fterTTPknockdownw asa l s o o b s e rved.I T F 2 357s t i l l m e d i a t edcytokines u p pressioni n t h e p r e s e n ceo r a b s e nceo f TTP(Fig.5 c),p o s s i b l y b ecauseTTPs i l e n cingo n l y r e d ucedthes t e ad y -

st a t e l evelso f TTPm R NA b ut w asu n a b l e t o fullyp r ev enti tsu pr eg ul ationb y I T F2357( F ig.5 c,ri g ht panel).I n l i n e withthishypothesis,r ecentstudiesindicatedthat,eve nwhenminimal,TTPexpressionissufficienttosuppressinfl ammatoryresponses[30].

BecauseTTPproteindomainsareremarkablyconservedw ithinvertebrates,andshareacommonregulatorymechanism [33,3 4],w e madeu seoff ibroblastsd erived

fromwild-type(ZFP36^+/+)andTTPknockout(ZFP36^−/−) mice[35],a nds timulatedt hemw ithI L-1βtomimicex- perimentalconditionsusedinRAFLS.Higherexpressionof IL6,CXCL2andPTGS2mRNAinZFP36^−/

−fibroblastsindicatedt hatt hesec ytokinesa reT T P t argets, a s p revi-

ouslyd escribed[36–38].I n addition,w hileinw ild- typefibroblastsIL6mRNAe xpressionw as s ignificantlyre ducedbyI TF2357,n o s ignificants uppressionw a s observedinZFP36^−/−fibroblasts(Fig.5d).Asimilartrend wasa lsoobservedf orC XCL2,whileP TGS2w a s n otre ducedbyITF2357inwild-

typefibroblasts.Ourfindingsindicatet hatT T P i s a n imp ortantr egulatorofc ytokinemRNAexpressioninRAFLS,a nds uggestt hatthisARE-

BPisr esponsiblef orm ediatingp arto f t heanti- inflammatorypropertiesofITF2357.

Discussio n

InRAa ndi n otheri mmune-mediatedi nflammatoryd is- eases(IMIDs),theexcessiveproductionandaccumulationo fc ytokinesa ndc hemokinesc ontributest o t hep erpetu- ationofchronicinflammationandimmuneresponses[1].Co ntinuouse xposuret o p ro-

inflammatorys timulid rivesRAFLStodevelopanepigeneti callyimprinted,aggressivephenotypea ndi nflammatorym emoryt hatp romotest hedegradationofsynovialjoints[39].

Developmentofimmu-

nomodulatoryepigeneticinhibitorshasemergedin recent years.A mongt hese,H DACic omprisea c lassofs mallanti -inflammatorymoleculesthatshowedpre-clinical

a

b

Fig.2(Seelegendonnextpage.)

(Seefigureonpreviouspage.)

Fig.2ITF2357acceleratesthemRNAdecayofinflammatorymarkers.aRheumatoidarthritis(RA)fibroblast-likesynoviocytes(FLS) (n=6)wereleftuntreatedorweretreatedwithITF2357priortoincubationwithIL-

1βfor2h.TranscriptionwasblockedwithactinomycinD(ActD)andRNAextractedattheindicatedtimepoints.Graphsshowrepresentativegeneswith enhancedmRNAdegradationinthepresenceofITF2357(toppanel),andexamplesofgenesthatwerenotregulatedbyITF2357attheleveloftranscript stability(bottompanel):*p<0.05,Wilcoxonmatchedpairstest.bRAFLS(n=7)wereleftuntreatedorweretreatedwithITF2357priortoincubationwithIL- 1βfor4and8h.Theexpressionofprimarytranscripts(PT)andmaturetranscriptsofIL6,IL8,PTGS2andMMP1wereassessedbyqPCR.Resultsarepresentedas2^{^(-}

ΔCT)of

thetargetsofinterestnormalizedtoGAPDHhousekeepinggene.PercentagesindicatetheaveragesuppressioncausedbythepresenceofITF2357inthe7inde pendentexperiments:*p<0.05,**p<0.01,***p<0.001,one-wayanalysisofvariancewithGreenhouse-

GeissercorrectionfollowedbyFisher’sleastsignificantdifferencetest

potentialf ort het reatmento f RA[18].D espitee xtensiverese archinrecentyears,themodeofactionofthesecom-

poundsremainslargelyunknown.

Herew e s howt hatinRAFLS,p an-HDACiI TF2357 efficientlys uppressesc ytokinep roductionindependentlyof thek ineticso f g eneinductionb y I L-1β.A s w e p revi- ouslyfoundthatIL-

6,akeycytokinecontributingtoRApathobiology,wassuppr essedbyITF2357viaaccelerationofIL6mRNAdecay[22],we aimedtoinvestigatewhetherpost-

transcriptional,r athert hant ranscriptional,r egulatoryevents c ouldb e t hek eyf actore xplainingt heb roadanti-

inflammatoryeffectsof ITF2357.Ofnote,r e centreportsindi catet hatp rolongede xposuret o T NFleadst o ag radualr esha pingoft heF L S t ranscriptome,w hichislargelyd ependento n m RNAs tabilityp rocesses[14],highlightingtheimportanc eofpost-transcriptionalregula-

torymechanismsinm aintainingthec hronicityofinflam- mationinRA.WeextendedouranalysistoadditionalIL-1β- inducedc ytokines(IL8,CXCL2)a ndm ediatorsofinflammat oryresponses,matrixdegradation,andcellsur-

vival(PTGS2,ADAMTS1,andBCL2L1).Weconfirmedth atasubsetofthesegenes,specificallyIL8,CXCL2andPTGS 2,w eres ubjectt o m RNAs tabilityr egulationbyITF2357.

On t hec ontrary,s omet argetsd isplayedsustainedstability(

MMP1–3,CXCL10,CXCL5–

6),whileothersrapidlydecayedovertimebutwerenotfurtherd estabilizedbyITF2357(IRF1,FoxO1).Moreintriguingly,kin eticsplayedanimportantroleinthetranscriptionalorpost- transcriptionalregulationofgenesaffectedbyITF2357.Indeed, whilemostlyaffectedatthetranscriptionallevelaftershorterexp osuretoITF2357,themRNAexpres-

sionofIL6,IL8andPTGS2waspost-

transcriptionallyregulatedatlatertimepoints.Theseresultsindic atethattheinitialanti-

inflammatoryeventsmediatedbyITF2357occurbysuppressing thenascentproductionofcytokinemRNA,whilesubsequentim munesuppressivefunctionsarerelatedtothedestabilizationofthe irtranscripts.

Akeymechanismresponsibleforthepost-

transcriptionalregulationofgeneexpressionisARE-BP- mediatedmRNAdecay[4].Wee valuatedwhetherARE- BPcouldmediatetheeffectsofITF2357oncytokinemRNAstabi lityinRAFLSandfoundthatT TPmRNAexpressionrapidlyinc reasedaftershortexposuretoIL-

1β.AftertreatmentwithITF2357,TTPmRNAwasnotstabilize ddespitebeing

inducedatalltime-

points,implyingthata transcriptionalcomponentisresponsiblef ortheregulationofthisARE-BPbyHDACi.Todate,themRNA- destabilizingTTPhasbeenbestdescribedasaregulatorofinflam matoryprocesses[40].TTPexpressionissignificantlyincrease dinthesynovialjointsinRAcomparedtonon-

inflamedjoints,anditisabundantinmacrophagesandsynovialfi broblasts[41],possiblyindicatingarelevantroleforthisARE- BPinthesecells.Studiesofperipheralbloodmononuclearcellsin RAhavereporteda globalreductionofTTPexpression,compar edtohealthycontrolsandpatientswithosteoarth-ritis(OA) [12,42].Inanimalstudies,knockoutofTTPhasbeenshownass ufficienttothedevelopmentofacomplexinflammatoryp hen otype,c haracterizedb y a uto-

immunityandp olyarthritis[10]Ont hec ontrary,i nductionof T T P expressionisprotectiveincollagen-

inducedarthritis(CIA)[13].

Ino u r s t u dy,w e o b servedt h a t b e s i d e s a f f ectin gt h e transcriptionalr e g u l a tiono f TTP,I T F2357a d d i tionallyreducedt h e a b u n d a n c e o f a h i g h e r m o l ecular- weightformo fTTP.Treat mentwit hp h osphataseco nfirm edthisast h e p h o s phorylatedf o r m o f t h e p r o t ein.Thedest abilizinge f f ectso f I T F2357o n TTPm R N A d ecayfurth ersupportthisfi nding,asdep hosphorylatedandactiveTTP w o u l d a l s o c a u s e i tso w n m R N A t o b e degraded[13].T h e e q u i l i briumb e t weent h e p h o s p h o r y-

latedandthedephosphorylatedpoolsofTTPhasfrequentlyb e enr e p o rtedt o b e a c r i t i c a l f e a t urei n t h e determi nationo f t h e i n f lammatoryr e s p o n se[30].M i c e expr essinga p h o s p h o r ylation-

deficientf o r m o f TTP,i n whichs e r i n e s 5 2 a n d 1 7 8 a r e c o n v e r t edt o a r g i nineresidues,a r e p r o t ectedf r o m C I A,asa c o nsequenceo f increasedfun cti ona lityoft hep rot ein [11,30].Al so ,activationo r d e p l etiono f p h o s phatasest h a t r evertTTPtoi tsd e p h o s p horylatedf o rm , s u c h asP P 2Aa n d D u s p 1 , reduceth e p r o du ct i ono fcy t ok i nessu chasI L 6 ,IL8andTNF,a ndi n c r e aseb r o a d p r o - i n f l a m m atoryg e n e e x pres-

sion,respectively[11,43,44].

Phosphorylationist hemostc ommonp ost- translationalmodificationofTTPandotherARE- BP,butothermodifi-

cationshavebeenreported[45,46].Thus,itremainspossiblet hatITF2357mayenhancetheacetylationlevelsofT T P a nds ubsequentlyr educeitsp hosphorylation.Indirectr egulationo fT T P p hosphorylationbyH DACiis

a

b c d

e

Fig.3Tristetraprolin(TTP)expressionandactivityareinducedbyITF2357.a,bRheumatoidarthritis(RA)fibroblast- likesynoviocytes(FLS)wereleftuntreatedorweretreatedwithITF2357beforeincubationwithIL-

1βforeither2h(a,n=4)orfor4and8h(b,n=6).ThemRNAexpressionoftheAdenosineuridine-richelements(ARE)bindingproteins(ARE-

BP)wasanalyzedbyqPCR.cRAFLS(n=6)weresubjecttomRNAstabilityanalysisasspecifiedinFig.2aandTTPexpressionwasanalyzedbyqPCR:*p<0.05,Wilcoxonmat chedpairstest.dRAFLS(n=4)weretreatedwithITF2357beforeincubationwithIL-

1βforeither2h,4hor8handtheexpressionlevelsofTTPprimaryandmaturetranscriptwereanalyzedbyqPCR.Graphshowsthe2^{^(-}

ΔCT)r a ti o

oftheprimary/maturetranscriptnormalizedtoGAPDHhousekeepinggene.eRAFLSwereleftuntreatedorweretreatedwithITF2357beforein cubationwithIL-

1βfor2and4h.ProteinlysateswereprocessedforimmunoblottingwithantibodiesrecognizingTTPandtubulin(leftpanel)andsignalintensity(n=8) wassubsequentlyquantifiedbydensitometryanalysis(rightpanel).Bandwithhigherapparentmolecularmasscorrespondstothepost-

translationallymodifiedprotein.d,e*p<0.05,**p<0.01,one-wayanalysisofvariancewithGreenhouse-

GeissercorrectionfollowedbyFisher’sleastsignificantdifferencetest.ActD,actinomycinD;AUF,AU-richbindingfactor;KHSRP,KH- typesplicingregulatoryprotein;HuR,HuantigenR

Page10of13 Angiolillietal.ArthritisResearch&Therapy(2018)20:148

a

b

Fig.4Knockdownofadenosineuridine-

richelementsbindingproteinsdoesnotcausechangesintheexpressionofinflammatorymediators.aRheumatoidarthritis(RA)fibroblast- likesynoviocytes(FLS)(n=3)wereleftuntransfectedorweretransfectedfor72hwith20nMcontrolnon-

targetingsiRNA(siCtrl)or20nMspecificsiRNAtargetingAUF1(siAUF1),BRF1(siBRF1),BRF2(siBRF2),KHSRP(siKHSRP)andHuR(siHuR).Knockdownefficie ncywasverifiedatthemRNAlevelbyqPCR.DataarepresentedasfoldexpressionofmRNAcomparedtosiCtrltransfectedcells:*p<0.05,**p<0.01,ratiottest .bRAFLS(n=3)weretransfectedasinaandfurtherleftuntreatedortreatedwithITF2357,priortostimulationwithIL-

1βfor8h.IL6,IL8,CXCL2andPTGS2expressionweredeterminedbyqPCR.DataarepresentedasfoldexpressionofmRNAcomparedtosiCtrl-treatedIL- 1βstimulatedcells:*p<0.05,**p<0.01,***p<0.001,****p<0.0001,one-wayanalysisofvariancefollowedby

Fisher’sleastsignificantdifferencetest

Page11of13 Angiolillietal.ArthritisResearch&Therapy(2018)20:148

2^(-dCT)×100mRNAexpression(foldchange)mRNAexpression(foldchange) mRNAexpression(foldchange)

a b

TTP

1.2 ** ⁴⁰⁰⁰₃₅₀₀

IL6

Med* 150000

IL8 * ₈₀₀₀ ^CXCL2p=0.05 1600

PTGS2

*

0.8

0.4

0

I L-1 β siCtrls i T T

P TTP

H3

30002500 20001500 1000 2.52.0 1.51.0 0.50.0

IL-1β

**

100000

50000

4 *

3 2 1 0

6000 4000 2000

2.0 ns

1.5 1.0 0.5 0.0

1200 800 400

2.0 ns

1.5 1.0 0.5 0.0

c

1.5

1.0

0.5

0.0

IL6

-40%

**

-43%

** ^2.₀

1.

5

1.

0

0.

5

0.0

IL8-43%

**

-42%

**

2.0

1.

5

1.

0

0.

5

0.

0

CXCL2 -54%

**

-46%

**

2.5

2.

0 1.

5 1.

0 0.

5 0.

0

PTGS2 -46%

**

-39%

*

TTP

8 +486%

6 *

4 +506%

*

2

0

IL-1β IL-1β+ITF2357

siCtrls i T T P siCtrls i T T P siCtrls iTTP

siCtrls i T T P siCtrls i T T P

d

mIL6 ns

ns

0.13 0.10 0.0 7 0.0 4

mCXCL2

ns 9

ns

6

mPTGS2 p=0.06

ZFP36+/

+ZFP36-/

-

0.03

0.00

**

*

0.01 8x10^-4 ns 6x10^-4

ns4x10^-4 2x10^-4 0

3

ns ns

0

p=0.06

++++

-+-+ ++++

-+-+

++++

-+-+

++++

-+-+

++++

-+-+

++++

-+-+

IL- 1βITF23 57

4h 8h 4h 8h 4h 8h 4h

8h

4h

8h

4h 8h

Fig.5Tristetraprolin(TTP)silencinginrheumatoidarthritis(RA)fibroblast-

likesynoviocytes(FLS)andmurinefibroblastsinducescytokineexpression.aRAFLS(n=4)wereleftuntransfectedorweretransfectedwithcontrolnon- targetingsiRNA(siCtrl)orspecificsiRNAtargetingTTP(siTTP).KnockdownefficiencywasverifiedatthemRNAlevelbyqPCR(leftpanel)andbyimmunoblotti ng(rightpanel).Forimmunoblotsamples,cellswerestimulatedwithIL-

1βfor2h.ProteinlysatewasanalyzedwithantibodiesrecognizingTTPorcontrolH3:**p<0.01,ratiot-

test.bRAFLS(n=7)weretransfectedasina,andfurtherleftuntreatedortreatedwithITF2357,priortostimulationwithIL-

1βfor8h.GeneexpressionwasdeterminedbyqPCR.DataarepresentedasfoldchangeinmRNAexpressioncomparedtosiCtrlconditions.cRAFLSwereprocess

Page12of13 Angiolillietal.ArthritisResearch&Therapy(2018)20:148

edasinb,IL6,IL8,CXCL2andPTGS2(n=7)andTTPexpression(n=3)wasdeterminedbyqPCR.DatapresentedasfoldchangeinmRNAexpressioncomparedtosiCtrl-IL- 1βstimulatedconditions.dWild-type(ZFP36^+/+)orTTPknockout(ZFP36^−/−)murinefibroblasts(n=4)wereeitherleft

untreatedortreatedwithITF2357andwerefurtherstimulatedwithIL-1β.GeneexpressionwasdeterminedbyqPCR.Resultsarepresentedas2^{^(-}

ΔCT)×100ofthetargetofinterestnormalizedtoACTBhousekeepinggene.b-d*p<0.05,**p<0.01,one-wayanalysisofvariancewithGreenhouse- GeissercorrectionfollowedbyFisher’sleastsignificantdifferencetest

yetanotherpossibility.Infact,evidencefromtheliteraturesug gestst hatHDAC1,2a nd3 c anb indt o a nda cetylateDusp1[4 7].Onthec ontrary,d irecteffectsofH DACionp38activationa relikelytobeexcluded,aswefoundpan-

HDACitoleavep38phosphorylationunalteredin

IL-1β-

stimulatedFLS[22].Similarly,ITF2357mayaffectmRNAsta bilityindependentlyofthec-JunN-

terminalkinase(JNK)signalingpathway,asFoxO1mRNAst ability,previouslys hownt o bemediatedb y J NKi nhibition[4 8]wasnotaffectedbyITF2357.

WetestedwhethersinglesilencingofmultipleARE- BPcouldresultinthedifferentialregulationofIL6,IL8,CXCL 2andPTGS2.UnlikeotherARE-

BP,TTPsilencingcausedincreasedexpressionofinflammator ymediatorsandp rovedt o bea c riticalr egulatoryf actorinRA F L S.Additionally,ITF2357reducedIL6andCXCL2product ion

inZFP36^+/+butnotZFP36^−/−murinefibroblasts,overall demonstratingc rucialinvolvementofT T P i n I L6regula- tionbyHDACi.

Conclusions

Recents tudiessuggestedt hatthei nducede xpressiona ndther educedp hosphorylationofT T P couldb e b eneficialindamp eninginflammatoryresponsesinarthritis[11].Ourresultsindi catethatITF2357functionsasanactivatorofTTPfunctionand transcriptioninRAFLS,andprovidenovelu nderstandingo f h owH DACid ictaten oto nlyt hetranscriptional,butalsothep o st-

transcriptionalregulationofinflammatoryg enes.Thesef indi ngsp rovider ationaleforf urthere v aluationofH DACia s a t h erapeutict oolinRAandotherchronicinflammatorydiseases.

Additionalf i l e s

Additionalfile1:FigureS1. Kineticsof mRNAregulati onby ITF2 357.(PDF293kb)

Additionalfile2:FigureS2.EffectsofITF2357onmRNAstability.

(PDF816kb)

Ad d i t i o na l f i l e 3:F ig ur e S 3 . T T Pp ost-

translationalc h a n g e s a f t e r ITF2357treatment.(PDF283kb) Additionalfile4:Sequencesofprimers.(PDF111kb)

Abbreviations

ActD:ActinomycinD;ARE:Adenosineuridine-richelements;ARE- BP:ARE-bindingp r o t e i n s ; A U F 1 : A U - r i c h b i n d i n g f a c t o r - 1;D M E M : D u l b e c c o ’sModifiedEa gleMediu m;F BS :Fe t a l bovinese r um; FLS:Fib rob la st -

like synoviocytes;H D A C i : H i s t o n e d e a c e t y lasein h ib itor;Hu R: H u a n t i g e n R ;

IL:Interleukin;JNK:c-JunN-terminalkinase;kDa:KilotDalton;KHSRP:KH- typesplicingregulatoryprotein;MAPK:Mitogen-activatedproteinkinase;

MMP:Matrixmetalloproteinase;mRNA:messengerRNA;PT: Prim arytranscripts;RA:Rhe umatoida rth rit is;T NF: Tumorne crosisfa ct o r; TSA:TrichostatinA;TTP:Tristetraprolin

Acknowledgements

Thea u t h o r s w o u l d l i k e t o t h a n k D r R L u t t e r a n d t h e L u m i n e x c o r e f a c i l i t y (AcademicMedicalC e n t e r , U n i v e r sityo f A m sterdam)f o r pe rformingthemeasuremento f s o l u b l e p r o t e i n s i n R A F L S s u p e r n a t a n t s.

Funding

AMGrabieciscurrentlysupportedbytheNationalScienceCenter,Poland(POLO NEZfellowshipUMO-2015/19/P/NZ7/03659);thisprojecthas

receivedfundingfromtheEuropeanUnion’sHorizon2020researchand innovationprogramundertheMarieSkłodowska-

Curie(grantagreementnumber665778KA).WSLaia ndPJBlackshearares u pported b ytheIntramuralR esearchP rogramo ftheN IEHS,NIH.Reedquisti ss upportedby theD utchA rthritisAssociation(NR11–1–

403);TRRadstakeissupportedb y ag rantf romt h eEuropeanResearchC o u n c il(ER C);DLBaeteniss upportedbya VICIg rantf r o mt heNetherlandsS c i e n t i f i cOrgani zation(NWO)anda

ConsolidatorgrantfromtheERC.

Availabilityofdataandmaterials

Thedatasetssupportingtheconclusionsofthisarticleareincludedwithint hearticleanditsadditionalfiles.

Authors’contributions

CA,PAKandAMGcontributedtoresearchdesign,performedexperiments,analyze ddataandcontributedtowritingthepaper;WSL,GF,PM,CSandPJBinterpretedda taandcontributedtowritingthemanuscript.MR,KAR,DLBandTRDJRdesigned theresearch,interpreteddataandcontributedtowritingthemanuscript.Allauth orsreadandapprovedthefinalversionofthemanuscript.

Ethicsapprovalandconsenttoparticipate

FLSisolationwasapprovedbythemedicalethicscommittee(METC)o f theAcade micMedicalCenter,Universityo fA msterdam,Amsterdam,theNetherlands(ME TC2013_069).Informedwrittenconsentwasobta ined

frompatientsp r i o rtoi nclusioni nt h es tudy.

Consentforpublication Notapplicable.

Competinginterests

Theauthorsdeclarethattheyhavenocompetinginterests.

Publisher’sNote

SpringerNatureremainsneutralwithregardtojurisdictionalclaimsin publishedmapsandinstitutionalaffiliations.

Authordetails

1LaboratoryofTranslationalImmunologyandDepartmentofRheumatologya ndClinicalImmunology,UniversityMedicalCenterUtrecht,Utrecht,TheNetherland s.²AmsterdamRheumatologyandImmunologyCenter,

DepartmentofClinicalIm m u nologyandRheum atologya nd Departmen tofExperimentalImmunology,AcademicMedicalCenter/UniversityofAmsterd am,Amsterdam,TheNetherlands.³DepartmentofMicrobiology,FacultyofBioch emistry,BiophysicsandBiotechnology,JagiellonianUniversity,Kraków,Poland.⁴Functi onalGenomicsCenter,UniversityofVerona,Verona,Italy.⁵SignalTransductionLaboratory ,NationalInstituteofEnvironmentalHealthSciences,ResearchTrianglePark,NC27 709,USA.⁶ItalfarmacoResearchandDevelopment,CiniselloBalsamo,Italy.

Received:10March2018Accepted:1June2018

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