Rev. Bras. Reumatol. vol.57 número3

ww w . r e u m a t o l o g i a . c o m . b r

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Original

article

Unbalanced

expression

of

aryl

hydrocarbon

receptor

in

peripheral

blood

CCR6

+

CD4

+

and

CD4

+

CD25

+

T

cells

of

rheumatoid

arthritis

夽

Lin

Cheng

_,

_Long

_Qian

,

Yue

Tan

,

Guo-Sheng

Wang

,

Xiao-Mei

Li

,

Xiang-Pei

Li

,

Chao-Yin

Luo

a_{AnhuiMedicalUniversity,TheSecondAffiliatedHospital,DepartmentofRheumatologyandImmunology,Hefei,China}

b_{AnhuiMedicalUniversity,AffiliatedAnhuiProvincialHospital,DepartmentofRheumatologyandImmunology,Hefei,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24August2015 Accepted17April2016 Availableonline22July2016

Keywords:

Rheumatoidarthritis Arylhydrocarbonreceptor CD4+_CD25+_Tcells

CCR6+_CD4+_Tcells

a

b

s

t

r

a

c

t

Objective:Thegoalofthisstudywastoanalyzetheroleofarylhydrocarbonreceptorin peripheralbloodCCR6+_CD4+_andCD4+_CD25+_{Tcellsofpatientswithrheumatoidarthritis.} Methods:FlowcytometrywasappliedtodeterminetheproportionofAhRpositivecellsin CCR6+_CD4+_T,CD4+_CD25+_{Tandperipheralbloodperipheralmononuclearcellsfromeach}

subject.AhRmRNAandCYP1A1mRNArelativeexpressionlevelsweretestedbyreal-time PCR.

Results:ThepercentageofAhRpositivecellsinperipheralbloodmononuclearcellswas higherinRAgroupthanthatinhealthycases[(35.23±10.71)%vs.(18.83±7.32)%,p<0.01]. TheexpressionlevelsofAhRandCYP1A1werebothincreasedinpatientswithRAwhile comparedtocontrols[(3.71±1.63)vs. (2.00±1.27),p=0.002; (2.62±2.08) vs. (0.62±0.29), p<0.01,respectively].InRApatients,thepercentageofAhRpositivecellsinCD4+_CD25+_T

cellswassignificantlylowerthanthatfromcontrols[17.90(6.10±80.10)%vs.(52.49±19.18)%, p<0.01];Inhealthycontrols,thepercentageofAhRpositivecellsinCD4+_CD25+_Tcellswas

significantlyhigherthanthatinCCR6+_CD4+_{Tcells,andwasalsosignificantlyhigherthan}

thatinPBMCs[(52.49±19.18)%vs.(23.18±5.62)%vs.(18.06±7.80)%,X2_{=24.03,p<0.01];in}

RApatients,thepercentageofAhRpositivecellsinCCR6+_CD4+_{T cellswassignificantly}

increasedthanthatinCD4+_CD25+_{TcellsandPBMCs[(46.02±14.68)%vs.17.90(6.10±80.10)%} vs.(34.22±10.33)%,X2_{=38.29,p<0.01];Nevertheless,nostatisticallysignificantrelationship}

wasfoundbetweenclinicaldataandAhRpositivecellsinCCR6+_CD4+_TandCD4+_CD25+_T

cells.

Conclusion:AhRmayparticipateinthepathologicalprogressofRAbycontrollingthe differ-entiationofTh17andTregcellsinperipheralblood.

©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

夽

StudyoriginatedatDepartmentofRheumatologyandImmunology,TheSecondAffiliatedHospital,AnhuiMedicalUniversity,Hefei, China.

∗ _{Correspondingauthor.}

E-mail:longqian0551@hotmail.com(L.Qian). http://dx.doi.org/10.1016/j.rbre.2016.07.002

Expressão

não

equilibrada

do

receptor

de

hidrocarboneto

arílico

nos

linfócitos

T

CCR6+

CD4+

e

CD4+

CD25+

do

sangue

periférico

na

artrite

reumatoide

Palavras-chave: Artritereumatoide

Receptoresdehidrocarboneto arílico

LinfócitosTCD4+CD25+ LinfócitosTCCR6+CD4+

r

e

s

u

m

o

Objetivo:Analisaropapeldoreceptordehidrocarbonetoarílico(AhR)noslinfócitosTCCR6+ CD4+eCD4+CD25+nosangueperiféricodepacientescomartritereumatoide(AR). Métodos: Foiaplicadacitometriadefluxoparadeterminaraproporc¸ãodecélulasAhR posi-tivasemlinfócitosCCR6+CD4+eCD4+CD25+dosangueperiféricoecélulasmononucleares periféricasdecadaindivíduo.Osníveisdeexpressãorelativadeácidoribonucleico men-sageiro(doinglêsribonucleicacid,RNAm,)deAhReRNAmdeenzimadeprimeiroestágio essencialparaoAhR(CYP1A1)foramtestadosporreac¸ãoemcadeiadepolimerase(doinglês polymerasechainreaction,PCR,)emtemporeal.

Resultados: ApercentagemdecélulasAhRpositivasnascélulasmononuclearesdosangue periféricofoimaiornogrupocomARdoquenosindivíduossaudáveis[(35,23±10,71)%vs. (18,83±7,32)%,(p<0,01)].OsníveisdeexpressãodeAhReCYP1A1estavamaumentados empacientescomARquandocomparadoscomoscontroles[(3,71±1,63)vs.(2,00±1,27), p=0,002;(2,62±2,08)vs.(0,62±0,29),p<0,01,respectivamente].EmpacientescomAR,a percentagemdecélulasAhRpositivasnoslinfócitosTCD4+CD25+foisignificativamente inferioràdos controles[17,90(6,10±80,10)]%vs.(52,49±19,18)%,p<0,01]; emcontroles saudáveis,apercentagemdecélulasAhRpositivasnoslinfócitosTCD4+CD25+foi significa-tivamentemaiselevadadoquenoslinfócitosTCCR6+CD4+etambémfoisignificativamente maiordoquenascélulasmononuclearesdosangueperiférico(doinglêsperipheralblood mononuclearcells,PBMC,)[(52,49±19,18)%vs.(23,18±5,62)%vs.(18,06±7,80)%,X2_=24,03,

p<0,01];empacientescomAR,apercentagemdecélulasAHRpositivasnoslinfócitosT CCR6+CD4+erasignificativamentemaioremcomparac¸ãocomoslinfócitosTCD4+CD25+ ePBMC(46,02±14,68)%vs.[17,90(6,10±80.10)]%vs.(34,22±10,33)%,X2_{=38,29,p<0,01];no} entanto,nãofoiencontradacorrelac¸ãoestatisticamentesignificativaentreosdadosclínicos ecélulasAhRpositivasemlinfócitosTCCR6+CD4+eCD4+CD25+.

Conclusão:OAhrpodeparticipardoprogressopatológicodaARaocontrolaradiferenciac¸ão delinfócitosTh17eTregnosangueperiférico.

©2016ElsevierEditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCC BY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Rheumatoid arthritis (RA) is a systemic chronic inflam-matory disease that affects about 1% of the population.1

The aryl hydrocarbon receptor (AhR), consisted of 806 amino acids, is a transcription factor that belongs to the bHLH (basic Helix-Loop-Helix)-PAS (Per-ARNT-Sim) family.2

AhR combines with chaperone proteins in the cytoplasm and maintains an inactive formin the absence of its lig-andssuchas2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)and 6-formylindolo[3,2-b]carbazole (FICZ). Once activated, AhR dissociatesfromtheproteinsandtransferstothenucleusto inducetheexpressionofdownstreamgenes.

Thelper(Th)17cellsandregulatoryTcells(Treg)aregroups oftheCD4+_{Tcellsfamily.Interleukin(IL)17–producingTh17} cells predominantly express CC chemokine receptor (CCR) 6which isa crucialmarkerfor Th17 cells inautoimmune diseases3_{; previous study has suggested that rheumatoid}

arthritisinanimalmodelscould beimprovedwhentreated withanti-CCR6monoclonalantibody,3 _{whichmayindicate}

CCR6expressionplaysaprimaryroleinautoimmunediseases. CCR6+_CD4+_{TcellisthemaincomponentofTh17cells,}3–6_and

meanwhileCCR6isregardedasabiomarkerofhumanTh17 cells.5,6 _TheCD4+_CD25+ _{lineage ofTreg is an} immunosup-pressive cell which playsa vital characterin immune and autoimmuneresponsesandthetranscriptionfactorforkhead boxproteinP3(Foxp3)hasbeenthoughttodeterminetheTreg cells lineage.7 _{Recently,studieshavefoundAhRwashighly}

expressedinTh17andTregcells,8,9 _{whichcouldactivateits}

signalingpathways,controlthedifferentiationofthecells,and furtherinvolveinthepathogenesisofautoimmunediseases inaligand-specificmanner.8,10,11

Patients

and

methods

Studysubjects

Thirty-fivepatientswithRA(meanage49.22±8.94,8males and 27 females) according to the 1987 revised criteria of AmericanRheumatismAssociation12_{wererecruitedfromthe}

DepartmentofRheumatology,TheSecondAffiliated Hospi-talofAnhuiMedicalUniversity.Theinclusioncriteriaofthe study(a) adefinitediagnosis,(b)freeofotherautoimmune diseases,(c)undergoingnoinfectiousdiseases,(d)treatment withsamedrugsforRApatients.Meanwhile,fourteenageand sexmatchedhealthycontrolswere randomlyselectedfrom healthcenter(meanage46.5±7.5,3malesand11females). Theclinicalparametersandthediseaseactivityscorein28 joints(DAS28)werecalculatedindetail.TheEthics Commit-tee of the Hospital approved the study. Informed consent wasobtainedfromthepatientsorfromtheirrelativesifthe patientswereincapableofconsent.

Cellisolation

5-mL venous peripheral blood was stored in anticoagula-tiontubecontainingEDTAastheanticoagulant.PBMCswere separatedusingdensity-gradientcentrifugation(Beijing Solar-bioScienceandTechnologyCompany,Beijing, China).Total PBMCsnumberofeachsamplewasnotlessthan1×106_.

Flowcytometry

Cellsurfacemarkers

5␮LCD25-PEand2␮LCD4-PerCP-Cy5.5werebothaddedto

themarkedflowtubes(a,b),atthesametime5␮LCCR6-PE

and2␮LCD4-PerCP-Cy5.5wereaddedtotheothertwotubes

(c,d).Aftertheliquidinfourtubeswasmixed,weplacedthem inarefrigeratorat4◦_{Cfor20min.Then2-mLbufferwas} pre-paredtowashthemforonce.Theabove-mentionedantibodies werepurchasedfromBecton,DickinsonandCompany(BD)of American.

Fixationandperforation

100-␮LPBMCswasmixedwith500␮Lfixatives(Becton,

Dickin-sonandCompany,American)infourtubestogiveincubation avoidinglightat4◦_{Ctemperatureandafterthat,1-mLbuffer} wastowashthemfortwotimes.

TheindirectmethodofmarkingAhR

Eachtubewasaddedthemixtureof2␮Lfirstantibody(the

antibodyofanti-arylhydrocarbonreceptor,AbcamCompany, England)and2␮Lphosphatebufferedsaline(PBS).1-mLbuffer

wasappliedtowashthemafterputthemixtureatroom tem-peraturetoavoidlightfor1handthen2␮Lsecondantibody

(mouse monoclonal(2A9)secondary antibodytorabbit IgG heavychain(FITC),AbcamCompany,England)wasaddedto thefourtubesandplacedtheminchamberatroom temper-aturetoavoidlightforhalfanhour.2␮Lbufferwasusedto

washthemfortwotimesandatlast200␮LPBSwasaddedto

thetubes.

Table1–Primersusedforthequantitativereal-time RT-PCR.

Genes Primer

AhR

Forwardprimer 5′_{-ATACCGAAGACCGAGCTGAAT-3}′

Reverseprimer 5′_{-CCAGCAGACACCTTAGACGAC-3}′

ˇ-Action

Forwardprimer 5′_{-AGCGAGCATCCCCCAAAGTT-3}′

Reverseprimer 5′_{-GGGCACGAAGGCTCATCATT-3}′

CYP1A1

Forwardprimer 5′_{-CATCCCCCACAGCACAACAAGAGA-3}′

Reverseprimer 5′_{-GCAGCAGGATAGCCAGGAAGAGAA-3}′

AhR,arylhydrocarbonreceptor;CYP1A1,vitalphaseIenzymeof AhR.

Flow-cytometricanalysis

The percentage of CD4+_CD25+ _{and CCR6}+_CD4+ _{T cells in} PBMCs and the proportionof AhRpositive cells inPBMCs, CD4+_CD25+_TandCCR6+_CD4+_{Tcellswerealldetectedby} Cel-lQuestsoftware(FACSaclibur,BDCompany).Thenumberof cellswastestedatleast100,000atatimeandFlowJo7.6.1was appliedtoanalyzethedata.

IsolationandreversetranscriptionoftotalRNA

TotalRNAfromRAperipheralbloodmononuclearcellswas separated usingTrizol reagent(Invitrogen, California,USA). PrimersweredesignedandsynthesizedbyTakara Biotechnol-ogy(Tokyo,Japan).Thespecificprimersequenceswereshown inTable1. ThePrimeScriptRT reagentKit(Takara Biotech-nology,Japan) wasused toperformthe synthesis ofcDNA accordingtothemanufacturer’sinstructions.

Real-timequantitativePCR

ThePCRwasperformedina20␮Lreactionsystem

contain-ing1.6␮Lprimers(0.8␮Lforwardprimerandreverseprimer,

respectively), 2␮L cDNA, 0.4␮L ROX Reference dye II, 6␮L

dH2O,and10␮LSYBRPremixExTaqTMII.Allthesampleswere

amplifiedatthesamesiteonABI7500real-timepolymerase chainreactionsystem(AppliedBiosystems,FosterCity,CA). 2−ct_{wasusedtocomputetheexpressionvalueofAhRand} CYP1A1.

Laboratorymeasurement

ForalltheRApatients,erythrocytesedimentationrate(ESR), Creactiveprotein(CRP)andanti-cycliccitrullinatedpeptide antibody(A-CCP)weremeasured.

Statistical

analysis

60

40

20

0

Control

RA

AhR, %

P<.01

Fig.1–ThepercentageofAHRinperipheralblood mononuclearcellsfromRApatientsandhealthycontrols.

datainthreegroups,andthecomparisonbetweentwogroups inthethreegroupswasanalyzedbyLSD-torMann–WhitneyU test.Spearmancorrelationanalysiswasappliedinourstudy. p<0.05wasthoughttobestatisticallysignificant.

Results

ThepercentagesofAhRinPBMCsfrompatientswithRA andcontrols

AsshowninFig.1,thepercentageofAhRpositivecellswas decreased in healthy controls (18.83±7.32)% than that in patientswithRA(35.23±10.71)%(p<0.01).

TheincreasedmRNAlevelsofAhRandCYP1A1inPBMCs fromRApatients

AsshowninFig.2,AhRandCYP1A1mRNAexpressionlevels werebothincreasedinpatientswithRAwhencomparedwith controls[(3.71±1.63)vs.(2.00±1.27),p=0.002;(2.62±2.08)vs. (0.62±0.29),p<0.01,respectively].

ThepercentageofAhRinCCR6+_CD4+_TandCD4+_CD25+_T

cellsfromRAcasesandhealthysubjects

As shown in Fig. 3, the percentage of AhR positive cells in CCR6+_CD4+_{T cells was clearly higher in patients} (46.02±14.68)%thanthatinhealthysubjects(23.18±5.62)%

(p<0.01)(Fig.3A).Incontrast,thepercentageofAhRpositive cellsinCD4+_CD25+_{TcellswasdecreasedinRApatients[17.90} (6.10,80.10)]%thanhealthygroup(52.49±19.18)%(Fig.3B). Sig-nificantdifferenceswereexistedbetweentwogroups(p<0.01).

ThecomparisonaboutthepercentageofAhRpositivecells inperipheralbloodCD4+_CD25+_T,CCR6+_CD4+_Tcellsand

PBMCsfromNormalcontrolandRAgroup

In the healthy group, the percentage ofAhR positive cells in CD4+_CD25+_{T cells was significantly higher than that in} CCR6+_CD4+_{Tcells, and wasalso statisticallyhigher than in} PBMCs[(52.49±19.18)%vs.(23.18±5.62)%vs.(18.06±7.80)%, X2_{=24.03, p<0.01] (Fig. 4A). Nevertheless, the percentage} ofAhRpositivecells inCCR6+_CD4+_{Tcells wassignificantly} increasedthanthatinCD4+_CD25+_{Tcells,andwasalso} sig-nificantlyhigherthanthatinPBMCs[(46.02±14.68)%vs.17.90 (6.10,80.10)%vs.(34.22±10.33)%,X2=38.29,p<0.01](Fig.4B).

CorrelationbetweentheAhRpositivecellsinperipheral bloodCCR6+_CD4+_T,CD4+_CD25+_{Tcellsandmajorclinical}

parametersfromRApatients

WeconductedSpearman’scorrelationtoassesstheassociation betweenAhR/CYP1A1mRNAexpressionlevels,AhRpositive cellsinperipheralbloodCCR6+_CD4+_T,CD4+_CD25+_Tcellsand majorclinicalparameters.However,nosignificantcorrelation wasfoundbetweenthem,andthedetailsweresummarized inTable2.

Discussion

RAisasystemicautoimmunediseaseprimarilymanifestas polyarthritis,especiallythesmalljointsofhandsandfeet,that ischaracterizedbyjointdestructionandchronicdisability.13

However,themechanismsabouttheimmunepathwaysinRA havenotbeenidentifiedyet.Forthepastfewyears,awealthy ofdatahasshowedself-toleranceplaysadominantrolein autoimmunityincludingRAasit erroneouslywasactivated in response to its self-antigen. The self-tolerance in RA patientsmayberegulatedbyvariousfactorssuchascytokine, environmentalrisksandlifestylefactors.14 _{Currently,itwas}

believed thataberrantT-cellhomeostasisplaysagreatpart inthepathogenesis.Moreover,Tregcellsdysfunctionand/or Th17 cells dysregulation are thought to contribute to the

10

8

6

4

2

0

10

8

6

4

2

0 P<.05

P<.01

AhR mRNA relative expression

CYP1A1 mRNA relative

expression

RA

Control Control

RA

A

B

100

80

60

40

20

AhR positive cells, % AhR positive cells, %

0

100

80

60

40

20

0

P<.01 P<.01

RA Control

RA Control

A

B

Fig.3–ThepercentagesofAHRpositivecellsinCCR6+_CD4+_T(A)andCD4+_CD25+_{T(B)cellsfromhealthycontrolsandRA}

patients.

100

80

60

40

20

0

100

80

60

40

20

0

AhR positive cells, %

AhR positive cells, %

PBMCs

CCR6+CD4+T CD4+CD25+T

PBMCS

CCR6+CD4+T CD4+CD25+T

P_<.01

P_<.01

P_<.01 _P

<.01

P_>.05

P_<.01

A

B

Fig.4–ThepercentagesofAHRpositivecellsinPBMCs,CCR6+_CD4+_T,CD4+_CD25+_{Tcellsfromnormalcontrol(A)andRA}

group(B).

development of autoimmune disorders. Similarly, accu-mulated evidence indicates AhR plays a critical place in regulatingthedifferentiationofTh17andTregcells, affect-ingthe developmentofanumber ofautoimmunediseases includingRA.11,15–20

AhRislocatedinthecytoplasmandmaintainsaninactive formintheabsenceofitsligandssuchasTCDDandFICZ.Once

activated,AhRcomplexcombineswithAhRnuclear transloca-torprotein(Arnt)tobeheterodimerinthenucleuswherethey bindtospecificDNAsequencesknownasxenobiotic-response elements(XRE),andthisbindingwillproducesomereaction. Duringthisactivatedprocess,avitalphaseIenzymeofAhR (CYP1A1)thatisamostwidelystudiedresponsivegenehada higherexpression.21_{Severalgroupspreviouslyhaveshowna}

Table2–ThecorrelationbetweenpercentagesofAhRpositivecellsinCCR6+_CD4+_T/CD4+_CD25+_Tcells,

AhR/CYP1A1mRNAexpressionfromRApatientsandclinicalparameters.

Clinicalparameters ESR CRP Anti-CCP DAS28scores Course

AhRmRNA

r-Value 0.115 0.014 0.003 0.068 −0.107

p-Value 0.511 0.936 0.987 0.698 0.541

CYP1A1mRNA

r-Value 0.007 0.149 −0.035 0.143 0.031

p-Value 0.967 0.392 0.842 0.411 0.860

CCR6+_CD4+_T

r-Value −0.262 −0.273 0.055 −0.187 −0.309

p-Value 0.14 0.124 0.761 0.296 0.08

CD4+_CD25+_T

r-Value −0.129 0.013 0.140 −0.045 −0.081

p-Value 0.475 0.943 0.436 0.804 0.653

stateAhRwashighlyexpressedinubiquitouscellsincluding Th17,Treg,dendriticcells(DCs).8,9,22

Interestingly, the essential factor of the pathogenesis ontraditional view inautoimmune diseases ismainlydue to Th1 and Th2 cells ratio imbalance. However, recent researchers had a tendency to focus more on the bal-ancebetween Th17 and Treg cells belong tothe family of CD4+_{Tcells.Quintanaetal.}17_{showedthatAhRactivationby}

its ligand2,3,7,8-tetrachlorodibenzo-p-dioxininduced func-tionalTregcellsthatsuppressedexperimentalautoimmune encephalomyelitis(EAE), butboostedTh17 cells differentia-tionandfurthermoreincreasedtheriskofEAEiftheligand wasFICZinanimalmodels. Thisstudy furtherilluminated theimportanceofthebalanceonTh17andTregcells.Astudy bydeKleeretal.23_{showedasignificantlyhighernumberof}

CD4+_CD25+_{Tregcellswasfoundinjuvenileidiopathic} arthri-tis(JIA)thanthatinextendedoligoarticularJIA(ext-OAJIA), andsuggestedthehigherfrequenciesofCD4+_CD25+_Tregcells insynovialtissueofpatientswithJIA,thelowerseverityofthe disease.IthasbeenreportedCD4+_CD25+_{Treg-deficiencymight} exacerbatesymptomsinpatientswithRA.24,25_Inthe

mean-time,Nguyenetal.26_{showedthediseaseprogressioncouldnot}

bepreventedbyCD4+_CD25+_{Tregcells,buttheseveritywould} beloweredforitsimmunosuppressivefunction.

Todate,thestudy aboutAhRinperipheralblood aswell asinTh17and TregofRApatientsismuchless.Basedon the mentioned studies and the notion, we conducted the experiment.Consistentwithpreviousfindings,8,10,11,17_firstly,

we found a statistically higher frequency of AhR positive cellsinRApatientswhen comparedwithhealthycontrols. Inaddition,wealsogot AhRand CYP1A1mRNAwere both overexpressedinpatientswithRA.Giventheresult,wemight speculateAhRwasactivatedandplayedasignificantrolein RApatients.Secondly,ourstudyshowednotonlythe percent-ageofAhRinCCR6+_CD4+_{Tcellswas enrichedbut alsothe} percentageinCD4+_CD25+_{TcellswasreducedinRApatients} comparedtohealthysubjects.Moreover,inpresentstudy,the highestfrequencyofAhRpositivecellsinCD4+_CD25+_Twas testedofCCR6+_CD4+_{TandPBMCsinhealthypersons,} how-everitsproportioninCCR6+_CD4+_{Tcellswasincreasedthan} PBMCsandbothofthemwere higherthan inCD4+_CD25+_T cells.Allthesefindingscollectivelysupportedtheviewthat AhRplaysanessentialroleindifferentiationandfunctionof CD4+_CD25+_TregandCCR6+_CD4+_{Th17,controllingthebalance} ofTh17andTregcells,andwhatcouldprovidemore thera-peuticapproachesinRA.However,nosignificantcorrelations betweenAhR/CYP1A1mRNAexpressionlevels,theAhR posi-tivecellsinCD4+_CD25+_{TaswellasCCR6}+_CD4+_{TcellsandCRP,} ESR,A-CCP,DAS28,courseofdiseasewerefound.Itpotentially toldustheycouldnotreflectthediseaseactivityorinsufficient samplesizemightbeoneofthereasons.

Inconclusion,ourfindingsfirstdemonstratean associa-tionbetweenAhRandCD4+_CD25+_Treg,CCR6+_CD4+_Th17cells inperipheralbloodfromRApatients,whichmaybroadenour horizonsintheetiologyofRAandprovidenewinsightsinto thepathogenesisofRA.OurresultssuggestthatAhRmayplay acentralroleincontrollingthebalanceofTh17andTregforits unbalancedexpressioninperipheralbloodandfurther partic-ipateinthepathogenesisofRA.Ofcourse,theseresultsmay beneededtoelucidateinafurtherresearch.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisworkwassupportedbyallthedonorsforfriendly provid-ingthebloodsamples.

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