Decreased levels of interleukin 27 in the serum of vitiligo patients,

Anais

Brasileiros

de

Dermatologia

www.anaisdedermatologia.org.br

INVESTIGATION

Decreased

levels

of

interleukin

27

in

the

serum

of

vitiligo

patients

夽,夽夽

Saeed

Malek

Hosseini

_,

_Naser

_Gholijani

_,

_Nooshafarin

_Chenari

_,

Kurosh

Kalantar

a_{DepartmentofImmunology,SchoolofMedicine,ShirazUniversityofMedicalSciences,Shiraz,Iran}

b_{AutoimmuneDiseasesResearchCenter,ShirazUniversityofMedicalSciences,Shiraz,Iran}

Received2June2019;accepted14February2020 Availableonline16June2020

KEYWORDS Cytokines; Inflammation; Vitiligo

Abstract

Background: Vitiligoisacommonskindisorderinwhichmelanocytesaredestroyedby

auto-reactiveimmuneresponses.Thelossofmelanocytesresultsintheappearanceofdepigmented areasindifferentpartsofthebody.Cytokineshaveremarkablerolesinthepathogenesisof vitiligo,suchasIL-1,IL-6,andTNF-␣;interleukin27(IL-27)isanewmemberofthe IL-6/IL-12family,mainlyreleasedbyactivatedantigen-presentingcells.IL-27hasbeensuggestedto functionasapro-inflammatoryaswellasananti-inflammatorycytokine.Alteredconcentrations ofIL-27havebeenshowninvariousauto-immunediseasessuchasmultiplesclerosis,rheumatoid arthritis,andpsoriasis.Nostudieshavebeenconductedtodeterminetheexpressionofthis cytokineinvitiligopatients.

Objective: TheobjectiveofthisstudywastodeterminetheserumconcentrationofIL-27in

vitiligopatientsandcompareitwithnormalindividuals.

Methods: TheserumconcentrationofIL-27in79vitiligopatientswasevaluatedincomparison

to45healthycontrolsusingELISAassay.

Results: ResultsshoweddecreasedconcentrationofIL-27invitiligopatientsascomparedwith

healthysubjects (p=0.026). Furthermore,no correlationbetween IL-27concentrations and diseaseparameterssuch asvitiligoseverityandtheextensionofthedepigmented areawas observed.

Studylimitation: Alargersamplesizewouldbemorerecommendedforthisstudy.

夽 _{Howtocitethisarticle:HosseiniSM,GholijaniN,ChenariN,KalantarK. Decreasedlevelsofinterleukin27intheserumofvitiligo}

patients.AnBrasDermatol.2020;95:570---4.

夽夽_{StudyconductedattheShirazUniversityofMedicalSciences,Shiraz,Iran.}

∗_{Correspondingauthor.}

E-mail:[email protected](K.Kalantar).

https://doi.org/10.1016/j.abd.2020.02.005

0365-0596/©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC

Conclusion: ThereductionintheserumlevelsofIL-27invitiligopatientscomparedtonormal subjects suggestedthe possibleanti-inflammatoryroleofthiscytokine invitiligo.Thus, IL-27maybeconsideredasanewtargetforthemanipulationoftheimmunesysteminvitiligo patients.

©2020SociedadeBrasileira deDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Introduction

Vitiligoisacommonacquireddepigmentationdisorder cha-racterizedbywhitepatcheswhichhavebeendistributedin severalpartsofthebodyandgreatlyaffectsthequalityof lifeandmentalhealthofthepatients.1,2_{Theprevalenceof}

vitiligoindifferentsocietieswaspublishedinarangeof0.5% to1%.3_{Accordingtoarecentmeta-analysis,theprevalence}

ofvitiligoamongdifferentcommunitieswas0.2%,andfrom hospital-basedstudies,itwas1.8%.4_{Vitiligoemergeswitha}

notablepatternofmaculardepigmentation,withavariable appearanceinshapeor size.Thisdiseaseis classifiedinto twosubgroups,generalizedandlocalized,inbothofthem insufficiencyofmelanocytefunctioncanbeobserved.5

The main causes of vitiligo are complex and maybe stem fromgeneticdisorders,which canfeaturepolygenic andmultifactorialinheritance.6_{The maincauseisnotyet}

clear;however,someevidencehasshownthatexchangein immunologicalprogramscontributedtothiskindofaction.7

Vitiligoisassumedtoresultfromautoimmunereactionsthat gradually destroy melanocytes. Despite the detection of self-reactingauto-antibodiesinvitiligopatients,increasing evidencesupportsimpaired cellularimmunityasthemain causeofthisdisfiguringdisease.8 _{Cytokineshaveapivotal}

role as mediators of cellular and humoral immune reac-tions.The imbalance betweenpro- andanti-inflammatory cytokinesfavoringthedominance of aTh1/Th17response rather than a Th2/Treg response has been proposed as a possible underlying mechanismof vitiligo. However,there isconsiderableevidenceinsupportoftheroleofCD8type 1 T cells in the destructionof melanocytes.9,10 _Increased

expressionofpro-inflammatorycytokinesinvitiligopatients ---includingIL-1,IL-6,andTNF-␣hasbeenshowninseveral studies.11---13

IL-27, a rather new member of the IL-6/IL-12 fam-ily, is mainly released by activated antigen-presenting cells, including dendritic cells (DCs), monocytes, and macrophages.14,15 _{It is composed of two subunits,}

EBV-induced 3 (EBI3), an IL-12p40 homolog,and p28,an IL-6 p35homolog.TheIL-27receptor(IL-27R)isaheterodimer composed of an IL-27R specific alpha chain (WSX-1) and agp130 subunit.16 _{The IL-27R isexpressed byseveral cell}

types including but not limited to T, B, and NK cells, DCs, macrophages, keratinocytes, CD8 type1 T-cells, and endothelialcells.17---19_{IL-27Rsignalingresultsinthe}

activa-tionofJAK-STATandp38MAPKpathways.20,21 _IL-27boosts

thedifferentiation ofTh1andTr1cells throughactivation ofSTAT1/3whileinhibitingthedifferentiationofregulatory T-cellsandTh2cells.21---26_{BesidesTh1differentiation,IL-27}

signalingthroughSTAT-1hasalsobeendemonstratedto acti-vateNKcells,whichhavebeenconsideredtohavearolein thepathogenesisofvitiligo.27

Although previous studies identified the inflammatory roleofIL-27,thelatestevidencesuggestsan immunomodu-latoryeffectofthiscytokine.28_{IL-27inducesTr1andinhibits}

Th2andTh17responses,therefore limitingtheseverityof autoimmunediseasesbythesuppressionofTh17cells.29---31

It has also immune-regulatory functions due to the up-regulationofPD-L1,IDO,andIL-10.32_{Thus,ithassuggested}

thatthiscytokinemaybeconsideredasapossible therapeu-ticagentforsomeinflammatoryorautoimmunediseases.

Regarding the complex pro-inflammatory and anti-inflammatory nature of IL-27, several studies have inves-tigated the local or systemic concentrations of IL-27 in differentdiseases.AlterationintheconcentrationofIL-27 anditscorrelationwithautoimmuneparametershavealso beenreportedinseveralTh1/Th17-mediatedinflammatory disorders, such as multiple sclerosis, systemic lupus ery-thematosus,inflammatory bowel disease,and rheumatoid arthritis.33---36

TheconcentrationsofIL-27invitiligopatientshavenot yetbeeninvestigated.Thepresentexaminationintendedto evaluatetheserumlevelsofIL-27invitiligopatients.

Methods

Studypopulation

Seventy-ninevitiligopatientsincluding32(41%)malesand 47(59%)females,and45ageandsex-matchedhealthy con-trolswithnosignofautoimmunediseaseswereenrolledin thestudy.The studypopulation included 72patients with generalizedvitiligoandsevencaseswithlocalizedvitiligo. Participantsinthisstudyassentedtojoinaccordingtothe ethicscommitteeofthemedicalschoolofShirazUniversity ofMedicalSciences(IR.sums.med.rec.1397.400).

Samplecollection

Fromall study populations,5mL of peripheral blood was collected.Aftercentrifugationat3000rpmfor10min,the serawereseparatedandkeptat−70◦Cuntilused.

MeasurementofserumIL-27

Serum concentrations of IL-27 were distinguished by enzyme-linked immunosorbent assay (ELISA) in vitiligo patients and healthy subjects. IL-27 concentrations were quantified using an ELISA kit (DY2526-05, R&D Systems ---UnitedStates)manufacturers’instructions. Thesensitivity oftestswas12.8pg/mL.Briefly,100␮Lofcaptureantibody wascoatedineachwellofa96-wellmicroplateforovernight at room temperature (RT). After washing and blocking,

100␮Lserumsamplesofpatientsandcontrolswereadded for 2h, at RT. Plates were washed again and 100␮L of the detection antibody and then 100␮L of the working dilution of streptavidin horseradishperoxidase wasadded for20min.Thensubstratesolution(100␮L)wasaddedfor 20min;finally,thereactionwasstoppedbyaddingstop solu-tiontoeachwell.Theopticaldensity ofsampleswasread usingamicroplatereaderat450nm.Thelevelsofcytokine wereextrapolatedfromtherelatedstandardcurve.

Statisticalanalysis

All data were analyzed by SPSS v. 16 software (SPSS Inc. --- Chicago, IL, United States) and according to the normalitytest (Kolmogorov-Smirnov test); non-parametric (Mann---WhitneyUand Kruskal---Wallis)testswere used;

p-values<0.05 were considered as significant. Graphs were constructed using Graph Pad Prism (v. 6 --- La Jolla, CA, UnitedStates).Alldataarepresentedasmean±standard errorofthemean(SEM),unlessotherwisespecified.

Results

Demographicanalysis

An overview of the demographic and clinical features of vitiligo patients and healthy subjects participated in the studyisprovidedinTable1.

MeasurementofserumIL-27

TheserumconcentrationsofIL-27in79patientswith gen-eralized(72cases)or localized(sevencases)vitiligowere observedusingtheELISAtechnique.Forty-fiveageand sex-matchedhealthysubjectswereusedasthecontrolgroup. As presented in Figure 1, the results showed that there wasasignificantdifferenceinserumconcentrationsofIL-27 betweenvitiligopatients(5267.8±399pg/mL)andhealthy subjects(7097.7±1502pg/mL)(p=0.0262).

CorrelationsofIL-27withdemographicinformation andclinicalmanifestations

ThecorrelationsbetweenserumconcentrationsofIL-27with gender, the severity of the disease, two types of (local-ized/generalized) vitiligo, segmental/nonsegmental forms ofthedisease,andresponsetotreatmentinpatientswith vitiligowereexamined.Therewasnosignificantcorrelation

Table1 Demographic analysis of populationincluded in thisstudy

Studypopulation Patients Controls

Number 79 45 Age 36.37±14.7 35.06±11.5 Sex Male 32(41%) 18(40%) Female 47(59%) 27(60%) 60000 30000 20000 15000 10000 5000 0.0262 0 IL-27 (pg/ml) Control _Patients

Figure 1 Mean serum concentration of IL-27 (pg/mL) in vitiligopatientsandcontrolgroup.

between IL-27levelsand theseparametersinthepresent study(Table2).

Discussion

The main reason for vitiligo is ill defined. Human and experimental studies have recently providedconsiderable evidence regarding the pattern of autoimmunity in the pathogenesis of vitiligo. Bothhumoral andcellular immu-nity have shown to be involved in the etiology of the disease.37---39 _{Theaccumulatedevidencesuggestsaprimary}

roleforcell-mediatedmechanisms,includingTh1/Th17and Tc1 cells in the pathogenesis of vitiligo.40 _Various

inflam-matorycytokines(IL-1,IL-6,TNF-␣,IL-6,andIL-17)havea keyrolein skin depigmentation,while thelevel of TGF-␤ indicatedreversiblestate.6,41

IL-27 has been suggested to function as a pro-inflammatory as well as an anti-inflammatory cytokine. Altered concentrations of IL-27 have been shown in sev-eralautoimmune andskin disorders.Despiteearly studies identified pro-inflammatory role of IL-27,latest evidence suggeststhatitsuppressesarangeofimmunecell prolifera-tionandcytokineproduction.28_{Thesecytokineapplications}

are related to its pro- or anti-inflammatory activity. The dualroleofIL-27isrelatedtothedifferenttissuesinvolved, theunderlyingmechanism,orthekindandstageof autoim-mune diseases.29 _{Previousstudies revealedthat theblood}

concentrationofIL-27increasedinpemphigusandpsoriatic patients.TheyshowedthatIL-27levelsstronglycorrelated withtheIgGauto-antibodytitersinpemphigus,andalso dis-easeonsetandseverityofpsoriasis.42,43 _{Theexpression of}

IL-27inlesionaleczematousskin hasbeenreportedtoo.43

Thisevidencesupportsapro-inflammatoryfunctionofIL-27. In spite of skin disorders in which IL-27 plasma concen-trations have increased, the serum level of this cytokine is reduced in some auto-immune diseases such as Vogt-Koyanagi-Harada syndrome (VKH), Behc¸et’s disease (BD), andSLE.Wangandhiscolleaguesdescribedthatthe expres-sion of IL-27 p28mRNA by peripheral blood mononuclear cells (PBMCs) and serum concentration of IL-27 in the sera and supernatantsof culturedPBMCs were noticeably reduced in patients with activeBD and VKH. In addition, Gaber et al. have observed that the IL-27 level in SLE patientsismarkedlylowerthaninhealthycontrols.44---46

Table2 ComparisonofserumconcentrationsofIL-27basedondemographicandclinicalmanifestationsinvitiligopatients

n Mean(pg/mL) p-Value

Gender Male 32 4623.8±544.2 0.681

Female 47 4961.5±564.5

Total 79

Severity(spreadoflesions) Bodyarea<20% 49 4793.9_±486.6 0.071

Bodyarea20%---50% 24 4132.7±435.1

Bodyarea>50% 6 7844.7_±2928.6

Total 79

Type:localizedorgeneralized Generalized 72 4925.9±434.3 0.421

Localized 7 3784.3±541.5

Total 79

Type:segmentalornon-segmental Segmental 8 3668.8_±483.0 0.335

Non-segmental 71 4955.0±439.5

Total 79

Responsetotreatment Yes 57 4492.8±380.9 0.154

No 21 5797.1±1083.9

Total 78

Based onthoseexplanations and theresults of several studies about the altered expression of IL-27 in autoim-munity and skin disorders, it was hypothesized that the concentrationofIL-27invitiligopatientsmightbealtered. Consistent with previous studies in SLE and BD patients, thepresentstudy showedthat theconcentrationsofIL-27 serumlevels werereduced in vitiligo patients, which can bejustifiedthroughsomeprobablemechanisms.First,IL-27 actsagainstTh17developmentinadirectmannerthrough modulatingDCsandinducesIL-10productionbynaïveCD4+ T-cells.45 _{Th17 contributes to the pathogenesis of vitiligo}

by IL-17production. IL-27inhibits thedifferentiation and generationof Th17 cells via IL-6andtransforming growth factor-␤ (TGF-␤) suppression, which is dependent on the intracellularsignalingmoleculeSTAT1.44

Conclusion

It can beconcluded that IL-27has an immunomodulatory roleinvitiligo.Accordingtotheobservationsofthepresent study,therewasnoassociationbetweenIL-27serumlevel anddemographicinformation,severityofthedisease,types of vitiligo, and response to treatment in patients with vitiligo.Further studieswithmorepatientsof alltypesof vitiligoareneededtorevealthepossiblealterationsinthe concentration of IL-27 in those patients and their corre-lation withdisease characteristics, including severity and theextensionoftheaffectedskinarea.Inconclusion,IL-27 hastworoles; itshouldbeconsidered asanewtargetfor themanipulationoftheimmunesysteminvarious immune-mediateddisorders.

Financial

support

This study was supported by Shiraz University of Medical Sciences(grantNo.17684).

Authors’

contributions

Saeed Malek Hosseini: Drafting and editing of the manuscript.

NaserGholijani:Collection,analysis,andinterpretation ofdata;participationinthestudydesign.

NooshafarinChenari:Collection,analysis,and interpre-tationofdata;participationinthestudydesign.

Nooshafarin Chenari: Drafting and editing of the manuscript.

Conflicts

of

interest

Nonedeclared.

References

1.NordlundJJ.Vitiligo:areviewofsomefactslesserknownabout depigmentation.IndianJDermatol.2011;56:180---9.

2.ParsadD,DograS,KanwarAJ.Qualityoflifeinpatientswith vitiligo.HealthQualLifeOutcomes.2003;1:58.

3.LeeH,LeeMH,LeeDY,KangHY,KimKH,ChoiGS,etal. Preva-lenceofvitiligoandassociatedcomorbiditiesinKorea.Yonsei MedJ.2015;56:719---25.

4.ZhangY,CaiY,ShiM,JiangS,CuiS,WuY,etal.Theprevalence ofvitiligo:ameta-analysis.PLOSONE.2016;11:e0163806.

5.BonifaceK,SeneschalJ,PicardoM,TaïebA.Vitiligo:focuson clinicalaspects, immunopathogenesis, and therapy. ClinRev AllergyImmunol.2018;54:52---67.

6.Sandoval-Cruz M, García-Carrasco M, Sánchez-Porras R, Mendoza-Pinto C, Jiménez-Hernández M, Munguía-Realpozo P, et al. Immunopathogenesis of vitiligo. Autoimmun Rev. 2011;10:762---5.

7.Laddha NC, Dwivedi M, MansuriMS, Gani AR, AnsarullahM, RamachandranAV,etal.Vitiligo:interplaybetweenoxidative stressandimmunesystem.ExpDermatol.2013;22:245---50.

8.vandenBoornJG,KonijnenbergD,DellemijnTA,vanderVeen JP, BosJD,MeliefCJ, etal. Autoimmunedestructionofskin

melanocytes by perilesional T cells from vitiligopatients. J InvestDermatol.2009;129:2220---32.

9.WuJ,ZhouM,WanY,XuA.CD8+Tcellsfromvitiligoperilesional marginsinduceautologousmelanocyteapoptosis.MolMedRep. 2013;7:237---41.

10.OggGS,RodDunbarP,RomeroP,ChenJL,CerundoloV.High frequencyofskin-homingmelanocyte-specificcytotoxicT lym-phocytesinautoimmunevitiligo.JExpMed.1998;188:1203---8.

11.SinghS,SinghU,PandeySS.SerumconcentrationofIL-6, IL-2, TNF-_␣, and IFN_␥ in vitiligo patients. Indian J Dermatol. 2012;57:12---4.

12.SushamaS,DixitN,GautamRK,AroraP,KhuranaA,Anubhuti A.Cytokineprofile(IL-2,IL-6,IL-17,IL-22,andTNF-alpha)in vitiligo---newinsight intopathogenesisofdisease. JCosmet Dermatol.2019;18:337---41.

13.SinghM,MansuriMS,MansiA.ParasrampuriaandRasheedunnisa Begum.Interleukin1-␣:amodulatorofmelanocytehomeostasis invitiligo.2016;5:2.

14.PflanzS,TimansJC,CheungJ,RosalesR,KanzlerH,GilbertJ, etal.IL-27,aheterodimericcytokinecomposedofEBI3andp28 protein,inducesproliferationofnaiveCD4+Tcells.Immunity. 2002;16:779---90.

15.MorrowKN,CoopersmithCM,FordML.IL-17,IL-27,andIL-33: Anovelaxislinkedtoimmunologicaldysfunctionduringsepsis. FrontImmunol.2019;10:1982.

16.KasteleinRA,HunterCA,CuaDJ.DiscoveryandbiologyofIL-23 andIL-27:relatedbutfunctionallydistinctregulatorsof inflam-mation.AnnuRevImmunol.2007;25:221---42.

17.Ruckerl D, Hessmann M, Yoshimoto T, Ehlers S, Hölscher C. AlternativelyactivatedmacrophagesexpresstheIL-27receptor alphachainWSX-1.Immunobiology.2006;211:427---36.

18.Hunter CA,Kastelein R. Interleukin-27: balancing protective andpathologicalimmunity.Immunity.2012;37:960---9.

19.Meka RR, Venkatesha SH, DudicsS, AcharyaB, Moudgil KD. IL-27-inducedmodulationofautoimmunityanditstherapeutic potential.AutoimmunRev.2015;14:1131---41.

20.Charlot-RabiegaP,BardelE,DietrichC,KasteleinR,Devergne O. Signalingeventsinvolvedininterleukin27(IL-27)-induced proliferationofhumannaiveCD4+Tcellsand Bcells.JBiol Chem.2011;286:27350---62.

21.GuzzoC,CheMatNF,GeeK.Interleukin-27inducesa STAT1/3-andNF-kappaB-dependentproinflammatorycytokineprofilein humanmonocytes.JBiolChem.2010;285:24404---11.

22.WangH,MengR,LiZ,YangB,LiuY,HuangF,etal.IL-27induces thedifferentiationofTr1-likecellsfromhumannaiveCD4+T cellsviathephosphorylationofSTAT1andSTAT3.ImmunolLett. 2011;136:21---8.

23.ArtisD,VillarinoA, Silverman M,HeW, ThorntonEM, MuS, etal.TheIL-27receptor(WSX-1)isaninhibitorofinnateand adaptiveelementsoftype2immunity.JImmunol.2004;173: 5626---34.

24.Diveu C,McGeachyMJ, Boniface K, Stumhofer JS, SatheM, Joyce-ShaikhB,etal.IL-27blocksRORcexpressiontoinhibit lin-eagecommitmentofTh17cells.JImmunol.2009;182:5748---56.

25.El-behiM,CiricB,YuS,ZhangGX,FitzgeraldDC,RostamiA. Dif-ferentialeffectofIL-27ondevelopingversuscommittedTh17 cells.JImmunol.2009;183:4957---67.

26.SchneiderR,YanevaT,BeauseigleD,El-KhouryL,ArbourN. IL-27increasestheproliferationandeffectorfunctionsofhuman naiveCD8+Tlymphocytesandpromotestheirdevelopmentinto Tc1cells.EurJImmunol.2011;41:47---59.

27.Ziblat A, Domaica CI, Spallanzani RG, Iraolagoitia XL, Rossi LE, Avila DE, et al. IL-27 stimulates humanNK-cell effector functionsand primesNKcells forIL-18 responsiveness.EurJ Immunol.2015;45:192---202.

28.Villarino AV, Huang E, Hunter CA. Understanding the pro-and anti-inflammatory properties of IL-27. J Immunol. 2004;173:715---20.

29.Fabbi M, Carbotti G, Ferrini S. Dual roles of IL-27 in cancer biology and immunotherapy. Mediators Inflamm. 2017;2017:3958069.

30.PotC,ApetohL,AwasthiA,KuchrooVK.Kuchroo.Inductionof regulatoryTr1cellsandinhibitionofTH17cellsbyIL-27.Semin Immunol.2011;23:438---45.

31.Stumhofer JS, Hunter CA. Advances in understanding the anti-inflammatory properties of IL-27. Immunol Lett. 2008;117:123---30.

32.AwasthiA,CarrierY,PeronJP,BettelliE,KamanakaM,Flavell RA.Adominantfunctionforinterleukin27ingenerating inter-leukin10-producinganti-inflammatoryT cells.NatImmunol. 2007;8:1380---9.

33.SénécalV,DebloisG,BeauseigleD,SchneiderR,Brandenburg J,NewcombeJ,etal.ProductionofIL-27inmultiple sclero-sislesionsbyastrocytesandmyeloidcells:modulationoflocal immuneresponses.Glia.2016;64:553---69.

34.GaberW,SayedS,RadyHM,MoheyAM.Interleukin-27andits relationtodiseaseparametersinSLEpatients.Egypt Rheuma-tol.2012;34:99---105.

35.LaiX,WangH,CaoJ,LiY,DaiY,XiangY,etal.CirculatingIL-27is elevatedinrheumatoidarthritispatients.Molecules.2016;21, pii:E1565.

36.Schmidt C, Giese T, Ludwig B, Mueller-Molaian I, Marth T, ZeuzemS,etal.Expressionofinterleukin-12-relatedcytokine transcriptsininflammatoryboweldisease:elevated interleukin-23p19 and interleukin-27p28 in Crohn’s disease but not in ulcerativecolitis.InflammBowelDis.2005;11:16---23.

37.Ongenae K, Van Geel N, Naeyaert JM. Evidence for an autoimmune pathogenesis of vitiligo. Pigment Cell Res. 2003;16:90---100.

38.HannSK,ShinHK,ParkSH,ReynoldsSR,BystrynJC.Detection ofantibodiestomelanocytesinvitiligobywestern immunoblot-ting.YonseiMedJ.1996;37:365---70.

39.Alikhan A, Felsten LM, Daly M, Petronic-Rosic V. Vitiligo: a comprehensive overview. Part I. Introduction, epidemiology, qualityoflife, diagnosis,differentialdiagnosis,associations, histopathology, etiology,and work-up.J AmAcad Dermatol. 2011;65:473---91.

40.Kalaiselvi R, Rajappa M, Chandrasekhar L, Thappa DM, MunisamyP.ImmunophenotypeofcirculatoryT-helpercellsin patientswithnon-segmentalvitiligo.PostepyDermatolAlergol. 2019;36:449---54.

41.Gholijani N, Yazdani MR, Dastgheib L. Predominant role of innatepro-inflammatorycytokinesinvitiligodisease.Arch Der-matolRes.2020;312:123---31.

42.HennericiT,PollmannR,SchmidtT,SeipeltM,TackenbergB, MöbsC,etal.Increasedfrequencyoftfollicularhelpercells andelevatedinterleukin-27plasmalevelsinpatientswith pem-phigus.PLOSONE.2016;11:e0148919.

43.ShibataS,TadaY,KandaN,NashiroK,KamataM,KarakawaM, etal.PossiblerolesofIL-27inthepathogenesisofpsoriasis.J InvestDermatol.2010;130:1034---9.

44.GaberW,SayedS,RadyHM,MoheyAM.Interleukin-27andits relationtodiseaseparametersinSLEpatients.2012;34:99---105.

45.WangC,TianY,LeiB,XiaoX,YeZ,LiF,etal.Decreased IL-27expressioninassociationwithanincreasedTh17response in Vogt-Koyanagi-Harada disease. Invest Ophthalmol Vis Sci. 2012;53:4668---75.

46.WangC,TianY,YeZ,KijlstraA,ZhouY,YangP,etal.Decreased interleukin 27expression isassociated withactiveuveitis in Behc¸et’sdisease.ArthritisResTher.2014;16:R117.