2011-A marine carotenoid,fucoxanthin,induces regulatory=FX=消炎

Biosci.Biotechnol.Biochem.,75(10),2066–2069,2011

Note

AMarineCarotenoid,Fucoxanthin,InducesRegulatoryTCellsandInhibitsTh17CellDi erentiationinVitro

TadaomiKAWASHIMA

ResearchandDevelopmentDivision,KikkomanCorporation,399Noda,Noda,Chiba278-0037,Japan

ReceivedJune14,2011;AcceptedJuly16,2011;OnlinePublication,October7,2011[doi:10.1271/bbb.110459]

Fucoxanthinisanon-provitaminAcarotenoidcon-tainedinbrownseaweeds.Wefoundthatitsuppressedinterleukin-17secretionfromCD4þTcellsunderIL-17-producingT(Th17)celldevelopmentconditions.ByevaluatingTcelldi erentiationinvitro,fucoxanthinanditsmetabolitefucoxanthinolinhibitedTcelldi er-entiationintoTh17cells.Thissuggeststhatfucoxanthincanimprovein ammatorydiseasesduetoTh17cells.Keywords:

fucoxanthin;interleukin-17;interleukin-17-producingT(Th17)cell;regulatoryTcell

Fucoxanthinisanon-provitaminAcarotenoidinbrownseaweeds,Undariapinnati da.Therearemanyreportsonthebene ciale ectsoffucoxanthinonhumanhealthincludinganti-tumorandanti-obesitye ects.1–3)Recently,aprotectivee ectoftopicalfucoxanthintreatmentagainstskinphotodamagewasreported.4)Asforimmunologicalfunctions,ithasbeenfoundthatintravenousinjectionoffucoxanthinprotectstheeyesfromlipopolysaccharide-inducedin ammationinrats.5)Wehypothesizedthatnon-provitaminAcarotenoids,includingfucoxanthin,cana ectTcelldi erentiationduetoachemicalstructuresimilartovitaminA,whichismetabolizedintoretinoicacid(RA)andinducesforkheadboxP3þ(Foxp3þ)regulatoryT(Treg)cells,resultingininhibitionofinterleukin17-producingT(Th17)celldevelopmentinvitroandinvivo.6–8)

Foxp3þTregcellsareinducedinthepresenceoftransforminggrowthfactor(TGF)- andRAinvitro,whileTh17celldevelopmentisinducedinthepresenceofIL-6andTGF- .6,7)ElevatedlevelsofIL-17areassociatedwiththepathogenesisofmanyin ammatorydiseases,includingautoimmunediseases,in ammatoryboweldiseases,allergicasthma,andatopicdermati-tis.9–12)Therefore,Th17celldi erentiationorIL-17secretionisatargetoftherapeuticdrugsandfoodstobeusedagainstthesein ammatorydiseases.Clinicale ectsofsyntheticretinoidAm80,anagonistofRAreceptors,onautoimmunediseaseshasbeenfoundinmice,13,14)andacommensalbacteriumintheintestineofinfants,Bi dobacteriuminfantis,hasbeenreportedtosuppressIL-17productionexvivousingadextransodiumsulfate-treatedcolonasamodelofin ammatoryboweldiseases.15)Recently,afunctionallydistinct

subsetofintegrin chainCD103-expressingdendriticcells(DCs)wasidenti edinmurinemesentericlymphnodes.15)CD103þDCspromotethedevelopmentofFoxp3þTregcellresponsesdependingonTGF- andthedietarymetaboliteRA.16)Therearemanyreportsontheimmunologicale ectsofvitaminAanditsmetab-olite,RA,asdescribedabove,whilenoreportontheinhibitorye ectofnon-provitaminAcarotenoidsagainstTh17celldevelopmenthasappeared.HenceweevaluatedtheimpactofthesecarotenoidsonTh17celldi erentiationinvitro.

Toassessthee ectofnon-provitaminAcarotenoidsastothesuppressionofIL-17production,CD4þTcellswerecollectedfromspleensofC57BL/6mice(JapanCrea,Tokyo)usingCD4microbeads(MiltenyiBiotec,BergischGladbach,Germany)andMACS(MiltenyiBiotec).Anti-CD3andanti-CD28monoclonalantibod-iesforTcellactivationandproliferationwerepurchasedfromeBioscience(SanDiego,CA).AfterculturingCD4þTcellswithnon-provitaminAcarotenoids(fucoxanthin,astaxanthin,lycopene,andlutein,fromWako,Osaka,Japan)inthepresenceofIL-6(20ng/mL,R&DSystems,Minneapolis,MN)andTGF- (2ng/mL,R&DSystems)for3d,IL-17productionwasmeasuredbyaMouseIL-17ELISASet(eBioscience).All-transRA(ATRA,Sigma,St.Louis,MO)wasaddedaspositivecontrolofsuppressionofTh17celldi er-entiation.ATRAandcarotenoidsolutionswerepreparedat5and10mMindimethylsulfoxide(DMSO).Thenthesesolutionsweredilutedandaddedtoaculturemediumat2or4mM( nalconcentration).Ascontrol,thesameconcentrationofDMSO(0.04%v/v)wasaddedtotheculturemedium.ThisconcentrationofDMSOdidnota ecttheexperimentalmodelastocytotoxicityorcytokineproduction.IL-17secretionwassuppressedonlybytheadditionoffucoxanthininadose-dependentmanner,whiletheothercarotenoidsdidnotsuppressIL-17productionbyCD4þTcells(Fig.1).Toanalyzethegeneexpressionofretinoicacidreceptor-relatedorphannuclearreceptor(ROR) t,atranscriptionfactorexpressedinTh17cells,17)andFoxp3,quantitativeRT-PCRwasperformedusingPrimeScriptRTreagent(Takara,Ohtsu,Japan),SYBRPremixExTaq(Takara),andspeci cprimersasfollows:50-TGTCCTGGGCTACCCTACTG-30and50-GTGCAGGAGTAGGCCACATT-30forROR t,50-

Correspondence:Tel/Fax:+81-4-7123-5529;E-mail:takawashima@mail.kikkoman.co.jp

Abbreviations:IL,interleukin;Th17cell,IL-17-producingTcell;Tregcell,regulatoryTcell;RA,retinoicacid;Foxp3,forkheadboxP3;ROR,retinoicacidreceptor-relatedorphannuclearreceptor;TGF,transforminggrowthfactor;RT-PCR,reversetranscriptase-polymerasechain

reaction

FucoxanthinInhibitsTh17CellDi erentiation2067

ATRA

250IL-17 (pg/mL)

20015010050

0++

1++0

Sample (µM)0IL-6/TGF-β-αCD3/αCD28+

Fucoxanthin

250IL-17 (pg/mL)

20015010050

0-+

0++

2++

4++

IL-17 (pg/mL)25020015010050

0-+

Astaxanthin

Sample (µM)IL-6/TGF-βαCD3/αCD280

Sample (µM)IL-6/TGF-βαCD3/αCD28

0++2++4++

250IL-17 (pg/mL)

20015010050

0-+

Lycopene

250IL-17 (pg/mL)

20015010050

0-+

Lutein

Sample (µM)IL-6/TGF-βαCD3/αCD28

0++2++4++

Sample (µM)IL-6/TGF-βαCD3/αCD28

0++2++4++

Fig.1.FucoxanthinSuppressedIL-17ProductionbyCD4þTCells.

CD4þTcells(5Â105)fromthespleensofC57BL/6micewereculturedwithall-transretinoicacid(ATRA)orcarotenoidsinthepresenceofanti-CD3monoclonalantibody( CD3,plate-bound,5mg/mL),anti-CD28monoclonalantibody( CD28,soluble,5mg/mL),IL-6(20ng/mL),andTGF- (2ng/mL)for3d.TheIL-17concentrationinculturesupernatantswasdeterminedbyELISA.ThedataareshownasmeanÆSDoftriplicatecellcultures.Theyarerepresentativeofthreeindependentexperiments.Ãp<0:05,ÃÃp<0:01(Student’st-test).

CCCAGGAAAGACAGCAACCTT-30and50-TTCTC-ACAACCAGGCCACTTG-30forFoxp3,and50-GCTA-CAGCTTCACCACCACAG-30and50-GGTCTTTAC-GGATGTCAACGTC-30for -actin.

UnderTh17celldevelopmentconditions,ROR texpressionwassuppressedbyfucoxanthin(Fig.2A).Moreover,inthepresenceofTGF- withoutIL-6,geneexpressionencodingFoxp3wasupregulatedbyfucox-anthinsupplementation(Fig.2B).TheseresultsindicatethatfucoxanthinsuppressesTh17celldevelopmentandinducesFoxp3þTregcelldi erentiationasRA.

NextweevaluatedtheTcelldi erentiationofna ¨veTcells(CD4þCD62LþTcells)intoTh17cells.Na ¨veTcellswerepreparedfromC57BL/6micebynegativelyisolatingCD4þTcellsusingCD4þTcellIsolationKitII(MiltenyiBiotec),followedbythecollectionofCD62Lþcellsusing uoresceinisothiocyanate(FITC)conjugatedCD62Lantibody(eBioscience)andFITC-labeledmicrobeads(MiltenyiBiotec).Na ¨veTcellswereculturedwithfucoxanthininthepresenceofIL-6andTGF- for3d,andthenthecellswerestimulatedwithphorbol-12-myristate-13-acetate(PMA)/ionomycin(100ng/mLand500ng/mLrespectively,Sigma)andGolgiStop(BDBioscience,SanJose,CA)forintracellularcytokineanalysis.Then

thecellswere xedandpermeabilizedwithaFoxp3StainingSet(eBioscience)andstainedwithFITCconjugatedIL-17antibody(eBioscience)andphycoery-thrin(PE)conjugatedFoxp3antibody(eBioscience).InananalysisofTh17(IL-17þ)andTreg(Foxp3þ)cellpopulationsbyFACS(BDBioscience),fucoxanthinsuppressedTh17celldevelopmentandincreasedthenumberofFoxp3þTregcellsinadose-dependentmanner(Fig.3).Othercarotenoids(astaxanthin,lyco-pene,andlutein)didnota ectTcelldi erentiationlikefucoxanthin(datanotshown).Thisresultisinaccord-ancewiththeresultsforthesuppressivee ectonIL-17production(Fig.1).InananalysisofTcelldi er-entiation,fucoxanthinol(Wako),ametaboliteoffucox-anthinbygutmicro ora,18)wasalsotested.ItwasfoundthatfucoxanthinolinhibitedTh17celldevelopmentaswellasfucoxanthindid(Fig.3).Thisindicatesthatoraladministrationoffucoxanthinshouldexertthesamee ectastoTcelldi erentiationafterabsorptionintheintestine.

Wefoundthatfucoxanthinanditsmetabolitefucox-anthinolinthegutinhibitedTh17celldi erentiationandinducedTregcelldevelopment,resultinginthesuppressionofin ammatoryIL-17production.ManystudieshavefoundthatRAreceptoragonistssuchas

2068T.KAWASHIMA

A

RelativeRORγt expression

604020

RelativeRORγt expression

80

ATRA

80

604020

Sample (µM)0IL-6/TGF-β-αCD3/αCD28+

0++10-310-110

+++

+++

Sample (µM)0IL-6/TGF-β-αCD3/αCD28+

0++1++2++4++

B

RelativeFoxp3 expression

ATRA

6420

0++

10-310-110

+++

+++

Relative

Foxp3 expression

8

6420

Sample (µM)0

TGF-β-αCD3/αCD28+

Fucoxanthin

Sample (µM)0

TGF-β-αCD3/αCD28+0++1++2++4++

Fig.2.FucoxanthinSuppressedROR tmRNAExpressionandIncreasedFoxp3mRNAExpressioninCD4þTCells.

A,CD4þTcells(5Â105)fromthespleensofC57BL/6micewereculturedwithall-transretinoicacid(ATRA)orfucoxanthininthepresenceofanti-CD3monoclonalantibody( CD3,plate-bound,5mg/mL),anti-CD28monoclonalantibody( CD28,soluble,5mg/mL),IL-6(20ng/mL),andTGF- (2ng/mL)for3d.ROR tmRNAexpressionwasdeterminedbyquantitativeRT-PCR.Thedataarerepresentativeofthreeindependentexperiments.B,CD4þTcells(5Â105)wereculturedwithATRAorfucoxanthininthepresenceof CD3, CD28,andTGF- for3d.Foxp3mRNAexpressionwasmeasuredbyquantitativeRT-PCR.Thedataarerepresentativeofthreeindependentexperiments.AandB,Thedataareshownasexpressionrelativetocellsstimulatedwith CD3/ CD28only.

αCD3 + αCD28

IL-6 + TGF-β

Control2.1

10.1

Control

ATRA (1 µM)1.2

2.410.638.3

Fucoxanthin(2 µM)7.8

3.8

Fucoxanthin(4 µM)

FITC-IL-17

15.8

Fucoxanthinol

(2 µM)

PE-Foxp3

7.1

23.0

Fucoxanthinol

(4 µM)5.2

18.123.3

Fig.3.FucoxanthinandFucoxanthinolInhibitedTh17CellDevelopmentandInducedFoxp3þRegulatoryTCellDi erentiation.

Na ¨veTcells(5Â105)fromthespleensofC57BL/6micewereculturedwithall-transretinoicacid(ATRA),fucoxanthin,andfucoxanthinolinthepresenceofanti-CD3monoclonalantibody( CD3,plate-bound,5mg/mL),anti-CD28monoclonalantibody( CD28,soluble,5mg/mL),IL-6(20ng/mL),andTGF- (2ng/mL)for3d.Thecellswerecollected,incubatedwithPMA/ionomycinandGolgiStop,and xedandpermeabilized.TheywereanalyzedbyFACSstainingwithFITCconjugatedIL-17antibodyandPEconjugatedFoxp3antibody.Thedataarerepresentativeofthreeindependentexperiments.Valuesrepresentratiosofcellpopulations.

FucoxanthinInhibitsTh17CellDi erentiation2069

ATRAandsyntheticretinoidssuppressTh17celldevelopment,6–8,13,14)andhenceweassumethatfucox-anthinandfucoxanthinolexertactivitysimilartoATRAviaRAreceptors.Althoughfucoxanthinandfucoxan-thinolweree ectiveathigherconcentrationsthanATRA,adi erenceina nityforRAreceptorsmightbeinvolvedinthedi erenceine ectiveconcentrations.Accordingtoapreviousreport,thestructure,especiallytheH6–H7loop,isinvolvedintheinteractionbetweenseveralsyntheticretinoidsandRAreceptors.19)Thusstructuraldi erencesamongthetestedcarotenoidsandtheirdi erenta nitiesforRAreceptorsmightcontributetotheirsuppressivee ectsonTh17celldevelopment.

Inconclusion,ourresultssuggestthatoraladmin-istrationoffucoxanthininhibitsTh17celldevelopment,probablythroughRAreceptors,andworksagainstTh17-dependentin ammatorydiseasessuchasauto-immunediseases,in ammatoryboweldiseases,andasthma.Inregardtothesafetyoforaladministrationoffucoxanthin,ithasbeenreportedthatnoabnormalchangeswereobservedintheliver,kidney,spleen,orgonadaltissuesunderoraladministrationof1,000mg/kgofbodyweightover30d.20)SinceitisexpectedthattheRA-likeactivityoffucoxanthinmighta ectthehomeostasisofvitaminAmetabolism,weintendtoinvestigatetheinvivoe ectsoforaladministrationoffucoxanthinonin ammatorydiseaseswithoutsidee ectsonvitaminAmetabolismusingmousemodelsuchasexperimentalautoimmuneencephalomyelitisorexperimentalcolitis.Wealsointendtoinvestigatethemechanismsofthesedi erentactivitiesamongfucox-anthin,fucoxanthionol,andothernon-provitaminAcarotenoids,focusingonstructuralfeaturesandthea nityforRAreceptors.

Acknowledgments

WethankDr.A.Obata,Dr.N.Kajiyama,Dr.I.Nishimura,andMr.K.Matsushimaforscienti cdiscussion.WealsothankMrs.K.Nagamurafortechnicalassistance.

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