SENIEURstatusoftheoriginatingcelldonor negatescertain ID: 519206
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RESEARCHOpenAccess SENIEURstatusoftheoriginatingcelldonor negatescertain anti-immunosenescence effects ofebselenandN-acetylcysteineinhumanTcell clonecultures ShivaMarthandan 1,2* ,RobinFreeburn 3 ,SusanneSteinbrecht 2 4 andYvonneBarnett 5 Abstract Background: DamagetoTcellsoftheimmunesystembyreactiveoxygenspeciesmayresultinalteredcell functionorcelldeathandtherebypotentiallyimpactupontheefficacyofasubsequentimmuneresponse.Here, weassesstheimpactoftheantioxidantsEbselenandN-acetylcysteineonarangeofbiologicalmarkersinhuman TcellsderivedfromaSENIEURstatusdonor.Inaddition,theimpactoftheseantioxidantsondifferentMAPkinase pathwaysinTcellsfromdonorsofdifferentageswasalsoexamined. Methods: Tcellcloneswerederivedfromhealthy26,45andSENIEURstatus80yearoldpeopleandtheimpactof titratedconcentrationsofEbselenorN-acetylcysteineontheirproliferationandinvitrolifespan,GSH:GSSGratioas wellaslevelsofoxidativeDNAdamageandonMAPkinasesignalingpathwayswasexamined. Results: InthisinvestigationneitherEbselennorN-acetylcysteinesupplementationhadanyimpactonthe biologicalendpointsexaminedintheTcellsderivedfromtheSENIEURstatus80yearolddonor.Thisisincontrast totheanti-immunosenescenteffectsoftheseantioxidantsonTcellsfromdonorsof26or45yearsofage.The ageandthatEbselenorN-acetylcysteinecoulddecreaseactivation(phosphorylation)inTcellsfrom26or45year olddonors,butnotfromtheSENIEURstatus80yearolddonor. Conclusions: TheresultsofthisinvestigationdemonstratethatthebiologicalphenotypeofSENIEURstatusderived humanTcellsnegatestheanti-immunosenescenceeffectsofEbselenandalsoN-acetylcysteine.Theresults highlighttheimportanceofpre-antioxidantinterventionevaluationtodeterminerisk-benefit. Keywords: Immunosenescence,Ebselen,NAC,Proliferativecapacity,Lifespan,SENIEUR,DNAdamage,GSH:GSSGratio, MAPkinases,JNK,p38,ERK,Totalglutathione Introduction Tcellsneedtoundergorapidclonalexpansionupon antigenicstimulationtoproduceanimmuneresponse. AnyfactorthatinterfereswiththeabilityofTcellsto clonallyexpandmayimpactontheeffectivenessofan immuneresponsewiththepotentialtorenderitsub- optimal. DamagetoTcellsfromreactiveoxygenspecies(ROS), frombothextrinsicandintrinsic(includingsitesofin- flammation)sourcesmayresultinalteredTcellfunction tems,e.g.antioxidantsandDNArepairsystems,tohelp defendagainsttheharmfuleffectsofROS[3].Nonethe- lessthesedefencesystemsarenotperfect,andcanbe- comeoverwhelmed.Inadditionwehaveestablishedthat DNArepaircapacitydeclineswithage invivo [4]andin CD4 + Tcellclones(TCCs)cultured invitro [5,6].This lackofoptimalperformanceatalltimesbythedefence systemsmayresultinanaccumulationofDNAdamage *Correspondence: smarthandan@fli-leibniz.de 1 JenaCentreforSystemsBiologyofAgeing-JenAge,Jena,Germany 2 LeibnizInstituteforAgeResearch,FritzLipmannInstitute,Beutenbergstrasse 11,D-07745Jena,Germany Fulllistofauthorinformationisavailableattheendofthearticle IMMUNITY & AGEING ©2014Marthandanetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated. Marthandan etal.Immunity&Ageing 2014, 11 :17 http://www.immunityageing.com/content/11/1/17 tocriticallevelswithinTcells,resultingincellcyclearrestorevenapoptosis[7],withthepotentialtoimpactadverselyontheTcellmediatedimmuneresponse.PreviousworkfromourgrouphasprovidedevidenceofanincreaseinthelevelofROS-inducedDNAdamagewithageinCD4TCCsculturedat20%O[3,8-10]andanincreaseinDNAdamageandmutationwithageinhumanlymphocytes[11].Amorerecentstudydemon-stratedanti-immunosenescenceeffectsoftwoantioxi-dants,2-phenyl-1,2-benzisoselenazol-3(2H)-one(Ebselen;[12])orN-acetylcysteine(NAC;[13])onCD4TCCsderivedfromhealthy26yearoldand45yearolddonors[10].InthispaperwenowdetailtheimpactofeachofthesetwoantioxidantsonCD4TCCsderivedfromahealthy80yearolddonor(conformingtotheSENIEURprotocolforhealthilyagedindividuals;[14]).TheSENIEURprotocolhelpstoensuretherigorousselectionofhealthilyagedindividuals.Evidencefromtheliteraturesuggestsanage-associatedcompromiseofTcellfunction[15].Anin-verserelationshipbetweenreplicativecapacityanddonorageofTCCshaspreviouslybeenreported[16].However,thereareexceptionswhereastraightforwardrelationshipbetweenageandTcellfunctionbreaksdown.Tcellsfromveryhealthyelderlydonors,includingthoseselectedusingtheSENIEURprotocol[14]isoneexception.Inthesecasesindividualshavebeenshowntobeabletoraiseaneffectiveimmuneresponse,contributedtobyadequateTcellfunc-tion[17-19].Wewereinterestedtoexaminewhethertheanti-immunosenescenteffectsofEbselenorNAC,whichwehavepreviouslyreported,inTCCsfromdonorsof26or45yearsofage[10]werealsopresentwhenTCCsfromaSENIEURselectedhealthyageddonorweretested.Ebselenisalipidsolubleseleno-organiccompoundhavingglutathioneperoxidaselikeactivitywhichenablesthemtoscavengehydroxylradicalsandperoxidesusingglutathione(GSH)asasubstrate[20].Furthermore,Ebselenhasthecapabilitytoinhibitthereleaseofapop-toticfactorcytochromec[21].TheantioxidantpotentialofEbselenhasbeenpreviouslydemonstratedinanum-berofothercelllines;HepGcells[20],humanHL-60[22]andPC-12cells[23].Theirabilitytoscavengeintra-cellularROSresultinginreductionofhydroxylradicalformationmayhavecontributedtotheantioxidantpo-tentialinTCCsderivedfromhealthy26and45yearolddonorsdisplayedbytheirimpactoncertainmarkersofTcellintegrityandfunction[10].IntermsofNAC,thepresenceofacetylatedformoftheaminoacidL-cysteineandsulfhydrylgroupsenablesthemtoactasaprecursorofGSHsynthesisandneutralisefreeradicalsrespectively[24].GlutamateandcysteinesharethesametransporterinthebodyandelevationinlevelsofextracellularglutamatecompetitivelyinhibitcysteinetransportresultingindepletionofintracellularGSHsyn-thesis.TheabilityofNACtoraiseGSHlevelsduetoitscapacitytodonatecysteineaminoacidmayalsosupple-mentitsantioxidantpotential[25].PreviousstudieshaverevealedtheROSscavengingpotentialofNACinHeLacells[26]andHepGcells[20].Althoughtheycancausedamagewithinlivingsystems,ROSactassignals/mediatorsinavarietyofcellularpro-cessesincluding;cellfunction,proliferation,differenti-ation,celldamageanddeath.ROSactasintracellularsignallingmoleculeswithinTcells[27],andtheycanmediatetheireffectsviaseveralsignallingmoleculessuchascalcium,proteintyrosinekinases(PTKs),proteintyrosinephosphatases(PTPs),serine/threoninekinasesandphospholipases.ROShavebeenrevealedtocontrolcellproliferationinducedbylectinandhaveanestab-lishedroleinproteintyrosinephosphorylationandacti-vationofJNK1[28].Mitogenactivatedprotein(MAP)kinases,aprominentfamilyofproteinkinases,operatethroughseveralpathwaysincluding,extracellularsignalregulatedkinases(ERK),c-JunN-terminalkinase(JNK)andp38kinase.Thesepathwaysareinvolvedinproli-feration,differentiationandapoptosis[23,29,30].Inthenovelstudydescribedinthispaper,theimpactofEbselenorNACondifferentMAPkinasepathwaysinhumanCD4TCCsderivedfromhealthy26,45and80yearolddonorshasalsobeeninvestigatedinanattempttoun-derstandthecontributoryfactorstoanyalterationsinthebiologicalendpointsmeasuredinthesupplementedTCCs.CultureofTCCsanddeterminationoftheirproliferativecapacityandlifespanClone399-37wasderivedfromahealthy80yearolddonor(conformingtotheSENIEURprotocolforhealth-ilyagedindividuals;[14]),Clones400-23and385-7werederivedfromahealthy26and45yeardonorrespec-tively.Threeindependentlyderived([31]generalrefe-renceforderivingtheTCCs)humanCD4TCCsofeachofthethreedonorswereseparatelymaintainedinculturein24wellplates(5wells,2mlmediumperwell)containingserum-freemedium,X-Vivo10(BioWhittaker)atconcentrationsof2-4×10cellsperwell,alongwith2×10gamma-irradiated(80Gy)RJK853cellsperwell(EBV-transformedB-lymphoblastoidcelllinewithcom-pletehprtdeletion),asfeedercells.Theclonesweremain-tainedat37°Cunderconditionsof5%COand95%airatmosphereandsupplementedwith400U/mlrecombin-antIL-2(Chiron,UK)ondays1and4ofthe7dayscycle.AviablecellcountwasperformedonharvestedcellsusingaNeubauerCountingChamber,andanewculturecyclewassetupwithfreshmediumandRJK853feedercellsonday7[3,8].Theproliferativecapacityandlifespanwerede-terminedsimilartotheprotocoldescribedpreviouslyMarthandanetal.Immunity&AgeingPage2of10http://www.immunityageing.com/content/11/1/17 [3,8,10].TheTCCsusedinthisstudywerekindlyprovidedbythegroupofProfessorGrahamPawelec.EbselenorNACsupplementationofTCCsFurthertoourpreviousstudy[10]weexaminedtheim-pactoftitratedconcentrationsofEbselen(0,10,30,60,M)orNAC(0,1.25,5,7.5,10mM)inthreepooled399-37(80yearold)TCCs,385-7(45yearold)TCCsand400-23(26yearold)TCCsrespectively.n=3ineachcase.DeterminationoflevelsofoxidativedamagetoDNAinTCCsderivedfromahealthy80yearolddonorThelevelsandtypesofDNAdamageinTCCs,sup-plementedwithorwithoutantioxidants,atvarioustimepointsthroughouttheirlifespanwereassessedusingamodifiedalkalinecometassay[3,8,10].QuantitativedeterminationofGSH:GSSGratioandtotalglutathionelevelinTCCsderivedfromahealthy80yearolddonorAGSH:GSSGratioassaykitwasusedtodeterminethera-tioofreducedglutathione(GSH)tooxidizedglutathione(GSSG),andtotalglutathionelevels[10].AssessingtheimpactofantioxidantsupplementationonMAPkinasesignallingpathwaysinTCCsderivedfromdonorsofallthreeagesusingSDS-polyacrylamidegelelectrophoresis(PAGE)andWesternBlottingEbselenorNACsupplementedandnon-supplementedcloneswereharvestedatdifferentstagesoftheirlifespan(atdifferentPDs).Cellswerewashedin1×PBS(pH7.4).Topreparesamplesforwesternblot,cellswerecountedinaNeubauerCountingChamberandanappropriateamountofcellswerere-suspendedinloadingbufferandincubatedat90°Cfor10minutes.Thesampleswerestoredat-20°CandlaterusedforSDS-PAGE.Loadingbufferconsistsof4%SDS,40%Glycerin,50mMTris/HCL(pH6.8),50mMDithiothreitol(DTT)andbromo-phenolblue.WholecellextractswereelectrophoresedonSDS-PAGEandtransferredtonitrocellulosemem-branes(Protran;SchleicherandSchuell).Themembranewasblockedin5%skimmedmilk/TBS-T(0.5MTrisBase,9%NaCl,0.5%Tween20,pH8.4;Tween20[CarlRoth])andincubatedwithprotein-specificprimaryanti-bodiesfollowedbyhorseradishperoxidaseconjugatedspecies-specificsecondaryantibodies(JacksonImmuno-ResearchLaboratories,Inc.).SignalsweredetectedusingtheECLreagent(GEHealthcare)onimagingfilm(BioMax;Kodak).WesternBlotforAnti--Actinwasperformedastheloadingcontrol.QuantificationofphosphorylatedandtotalMAPkinaseproteinexpressionlevelsinTCCsamplesderivedfromdonorsforallthethreeagesForimmunodetection,primaryantibodieswereusedatthefollowingdilutions:PhosphoJNK(1:50;9251),Phos-phop38(1:100;9211),Phosphop44/p42[ERK1/2](1:500;9101),Phosphoc-Jun(1:50;2361),SAPK/JNK(1:50;9252),p38(1:100;9212),p44/p42[ERK1/2](1:600;4695)andAnti--Actin(1:10,000;A5316).Allanti-bodiesexceptAnti--ActinwerepurchasedfromCellSignalingTechnology,Boston,USA.Anti--ActinwaspurchasedfromSigma-Aldrich.Secondaryantibodies,conjugatedtohorseradishperoxidase(Dako)wereusedat1:10,000dilutionandblotsdevelopedusingECLdetectionsystemandradiographicfilm(GEHealthcare,Germany).Afterthefilmdevelopment,quantificationofthesignalin-tensitiesofthebandsintheWesternblotswascarriedoutusingtheMetamorphsoftware[32].ThesignalintensitiesofthebandsrepresentingthelevelsofphosphorylatedortotalproteinswerenormalizedtothereferencebandofAnti--Actin.StatisticalanalysisofthesamplesTheresultsweretestedforsignificanceusingpairedtwo-sampletype2Studentst-testsassumingequalva-riances;pvaluesarepresentedasappropriate.EffectsofEbselenandNAConintracellularredoxstatus(GSH:GSSGratio)andtotalglutathionelevelsinhumaninvitroderivedfromahealthy80yearolddonorTCCsamplesweretakenfromtheculturesatvarioustimepointsduringtheirlifespanandtheeffectof30Ebselenor7.5mMNAConintracellularredoxstatus(GSH:GSSGratio)andtotalglutathionelevelswithintheTcellsweredetermined.Figure1AandBshowthere-sultsoftheeffectof30MEbselenor7.5mMNAContheGSH:GSSGratio.SupplementationofTCCsfromayounginvitrowith30MEbselen(Figure1A)or7.5mMNAC(Figure1B)hadnoimpactontheGSH:GSSGratioatanyofthetimepointsexaminedwhencomparedtonon-supplementedclones.Inbothcases(antioxidantsupplementedandnon-supplemented),theGSH:GSSGratiosignificantlyde-creasedwithincreasedtimeinculture(Additionalfile1:TableS1AandB).AsimilarscenariowasobservedinTCCsderivedfromahealthy26and45yearolddonorinourpreviousstudy[10].Furthermore,theGSH:GSSGra-tiowassignificantlylowerinTCCsderivedfromahealthy80yearolddonor(supplementedandnon-supplemented)comparedtotheGSH:GSSGratioinTCCsderivedfromeitherofthehealthyyoungageddonors(26and45yearold;Tables1and2;[10]).Marthandanetal.Immunity&AgeingPage3of10http://www.immunityageing.com/content/11/1/17 EitherconcentrationofEbselen(30 M)orNAC(7.5 mM)investigatedinthisstudyhadanyimpactontotal glutathionelevels,atanyofthethreetimepointsin TCCsderivedfromahealthy80yearolddonorcom- paredtonon-supplementedclones,aswasthecasefor GSH:GSSGratio.However,thelevelsoftotalglutathione weresignificantlylowerinthe399-37TCCscompared tolevelsinTCCsfrombothyoungerageddonors(Data notshown). TheimpactofEbselenandNACon invitro proliferative capacityandlifespanofhumanTCCsderivedfroma healthy80yearolddonor TheTCCsusedinthisstudyunderwentapoptosisatthe endoftheirlifespanaftercompletingafinitenumberof PDs.Thisisinlinewithpreviousreports[3,33]. TheeffectofdifferentconcentrationsofEbselen(0,10, 30 M)orNAC(0,1.25,5,7.5mM)ontheproliferative capacityand invitro lifespanofTCCswasinvestigatedby supplementingthemwithoneoftheeitherantioxidants untiltheendoftheirlifespan.Theresultspresentedin Table3indicatethatEbselen(30 M)andNAC(7.5mM) supplementationofTCCsderivedfromahealthy80year olddonorresultedinaslightdecreaseintheaverage numberofPDaccomplishedperweek,thoughnotsta- tisticallysignificant.Neitheroftheantioxidantshadany significantimpactonthecumulativelevelofPDsachieved beforetheendoftheirlifespanintheTCCsderivedfrom ahealthy80yearolddonor,incontrasttothesignificantly enhancedPDsinantioxidantsupplementedTCCsderived fromthehealthyyoungerdonors([10];Table3).However, 30 MEbselenor7.5mMNACsupplementedTCCswere abletosurviveincultureforanadditionalweekandthree weeksrespectivelycomparedtonon-supplementedTCCs. Otherconcentrationsinvestigatedinthestudy,10 M Ebselenand1.25or5mMNACdidnotrevealanimpact oneitherproliferativecapacityorlifespaninTCCsderived fromahealthy80yearolddonor.Higherconcentrations ofEbselen(60-100 M)orNAC(10mM)usedinthis investigationcompletelyinhibitedthegrowthofTCCsde- rivedfromahealthy80yearolddonorwithinaweekof culture(Datanotshown).Asimilarscenariowasobserved Figure1 Impactof30 MEbselenor7.5mMNACsupplementationonGSH:GSSGratioinTCCsderivedfromahealthy80yearold donor.(A&B) Theimpactof30 MEbselen (A) or7.5mMNAC (B) onGSH:GSSGratiointhreepooledTCCsderivedfromahealthy80yearold donor.Thebarsindicatethemean±S.D. Table1GSH:GSSGratioofTCCs+/-Ebselen Timesinculture(Weeks)30 Mebselen400-23(26yrol)385-7(45yrold)399-37(80yrold) 2-42.3±1.744.3±2.437.7±0.4* 2+44.0±1.741.9±1.236.7±0.9* 5-44.2±2.842.0±1.830.8±2.0* 5+47.3±2.446.8±5.131.3±1.5* 9/10-36.1±1.633.4±4.125.5±0.7* 9/10+37.2±1.436.3±2.926.6±0.8* GSH:GSSGratiosinthreepooledCD4 + TCCs(+/-30 MEbselen)derivedfromahealthy80yearolddonoraresignificantlylowerthanineitherofthethree pooledTCCs,eachderivedfromahealthy26ora45yearolddonor(publisheddatafrom[10]). *SignificantlylowerGSH:GSSGratioin399-37clones(80yearold)comparedtoeither400-23(26yearold)or385-7(45yearold)clones. Marthandan etal.Immunity&Ageing 2014, 11 :17 Page4of10 http://www.immunityageing.com/content/11/1/17 inourpreviousstudywhenTCCsderivedfromahealthy26and45yearolddonorwassupplementedwithhighconcentrationsofeitherEbselen(60-100M)orNAC(10mM),[10].Furthermore,asexplainedinourpreviousstudy[10],mechanismsbehindthepro-apoptoticeffectofhighconcentrationsofantioxidantshavealsobeendemonstratedinothermodelsystems[34-36].TheimpactofebselenorNAConlevelsofoxidativeDNAdamageinhumanTCCsasafunctionofinvitroAliquotsofTCCsamplesweretakenfromcultureatva-rioustimepointsandtheeffectof30MofEbselenor7.5mMofNAConthelevelsofoxidativeDNAdamagewithintheTcellsweredetermined.Incontrol(non-sup-plemented)samples,levelsofoxidativedamagetoDNAincreasedasafunctionofage,asmeasuredbythemodi-fiedendonucleaseIII(EndoIII)andformamidopyrimidineDNAglycosylase(FPG)cometassays,inlinewithprevi-ouslypublishedfindings[3,9].TheresultspresentedinFigure2AandBrevealthatoxi-dativeDNAdamagelevelsincreasedasafunctionoftimeinculture,inbothsupplementedandnon-supplementedclones.Neitherdoseofantioxidantsincluding30MforEbselenand7.5mMforNAChadanyimpactonthelevelsofoxidativeDNAdamageinTCCsduringtheirspaninculture.Figure2summarisesthedataobtainedfollowingMEbselen(A)or7.5mMNAC(B)supplementation.ThelevelsofoxidativeDNAdamagesignificantlyin-creasedwithtimeincultureinbothsupplementedandnon-supplementedTCCsderivedfromahealthy80yearolddonor(Table4).ThiswasalsothecaseinTCCsderivedfromahealthy26or45yearolddonor[10].AcomparisonofthelevelsofoxidativeDNAdamageinthe399-37clonesampleswiththosefromtheyoungerdonorsrevealedthatbasallevelsofoxidativeDNAdamage Table2GSH:GSSGratioofTCCs+/-NACTimeinculture(Weeks)7.5mMNAC400-23(26yrold)385-7(45yrold)399-37(80yrold)2-46.3±2.544.2±0.836.7±1.0*2+51.2±4.546.7±3.335.7±0.9*5-37.9±0.841.0±0.529.4±1.2*5+42.5±2.546.1±0.330.7±1.8*9/10-35.5±4.033.2±3.124.2±0.2*9/10+35.8±2.336.6±2.624.1±0.2*GSH:GSSGratiosinthreepooledCD4TCCs(+/-7.5mMNAC)derivedfromahealthy80yearolddonoraresignificantlylowerthanthelevelsineitherofthethreepooledTCCs,eachderivedfromahealthy26ora45yearolddonor(publisheddatafrom[10]).*SignificantlylowerGSH:GSSGratioin399-37clones(80yearold)comparedtoeither400-23(26yearold)or385-7(45yearold)clones. Tables3ProliferativecapacityandlifespanofTCCsonantioxidantsupplementationClone(Ageofthedonor)InitialPDConcentrationAveragePDperweekCumulativePDachievedattheendoflifespaninculture400-23(26yearolddonor)34.5Control0.7±0.144.7±0.3*MEbselen1.2±0.256.2±0.4*385-7(45yearolddonor)31.0Control1.0±0.144.4±0.3*MEbselen1.4±0.450.8±0.4*399-37(80yearolddonor)31.1Control1.2±0.245.0±0.4MEbselen1.2±0.144.6±0.3Clone(Ageofthedonor)InitialPDConcentrationAveragePDperweekCumulativePdachievedattheendoflifespaninculture400-23(26yearolddonor)34.5Control0.7±0.144.7±0.3*7.5mMNAC1.2±0.256.1±0.3*385-7(45yearolddonor)31.0Control1.0±0.144.4±0.3*7.5mMNAC1.4±0.455.8±0.4*399-37(80yearolddonor)31.1Control1.1±0.244.2±0.57.5mMNAC0.9±0.242.3±0.4EffectofEbselenorNACsupplementationontheproliferativecapacityandlifespanofCD4TCCsderivedfromhealthy26,45or80yearolddonors.Dataof26and45yearolddonorsarepublisheddatafrom[].*SignificantlyhighercumulativePDsinsupplemented(+)clonescomparedtocontrols(-).n=3pooledTCCsforeachagegroup.Marthandanetal.Immunity&AgeingPage5of10http://www.immunityageing.com/content/11/1/17 weresignificantlyhigher(+/-supplementation)afterall sampledtimepoints(Additionalfile1:TableS2AandB). TheimpactofebselenorNACondifferentMAPkinase pathwaysinhumanTCCsderivedfromdonorsof differentages TheimpactofEbselenorNACsupplementationon MAPkinasephosphorylationstatusandtotalprotein levelswasdeterminedinTCCsamplesfromhealthyyoung (26yearold),middleaged(45yearold)andelderly(80year old)donors. Figure3ArevealsthatERKwassimilarlyphosphorylated inEbselensupplementedorcontrolTCCs,irrespectiveof TCC invitro age(PD).Incontrast,JNK,p38andc-Jun phosphorylationlevelswereabsent(orlow)inyoungcells (Y)butgreatlyenhancedinlatePDcells(O)fromalldo- nors.30 MEbselendidnotsignificantlyaltertheincrease ofp38phosphorylationinlatePDTCCs.Therewasasig- nificantreductioninJNKandc-Junphosphorylationin youngandmiddle-ageddonorTCCsonEbselensupple- mentation.However,Ebselendidnotresultinareduction inJNKorc-JunphosphorylationinTCCsderivedfroma Figure2 Effectof30 MEbselenor7.5mMNACsupplementationonthelevelsofoxidativeDNAdamageinTCCsderivedfroma healthy80yearolddonor.(A&B) Theimpactof30 MEbselen (A) or7.5mMNAC (B) onthelevelsofoxidativeDNAdamageinthree pooledTCCsderivedfromahealthy80yearolddonor.Thebarsindicatethemean±S.D. Tables4LevelsofoxidativeDNAdamageinTCCsonantioxidantsupplementation Timeinculture(Weeks) 30 MEbselen(%ofDNAincomettail) -+ AlkEndFpgAlkEndFpg 219.7±0.621.6±0.622.5±0.620.6±0.822.5±0.823.5±0.8 525.6±0.627.6±0.431.7±0.826.3±1.230.1±1.032.0±1.1 1046.9±0.648.8±0.751.2±1.248.8±0.749.8±0.651.8±2.1 ****** Timeinculture(Weeks) 7.5mMNAC(%ofDNAincomettail) -+ AlkEndFpgAlkEndFpg 221.8±0.822.6±0.623.7±0.322.2±0.323.9±0.124.2±0.2 528.2±1.630.8±0.833.0±1.227.7±1.232.9±0.834.1±0.3 10 48.7±0.449.9±0.752.6±1.050.2±0.352.2±1.253.6±0.9 ****** TheincreaseinlevelsofoxidativeDNAdamageinCD4 + TCCderivedfromahealthy80yearolddonorsupplemented+/-30 MEbselenor7.5mMNAC. *SignificantlyhigherlevelsofoxidativeDNAdamageinweek10oncomparisonwiththeearlierweeks(2and5).n=3pooledTCCsforeachagegroup. Marthandan etal.Immunity&Ageing 2014, 11 :17 Page6of10 http://www.immunityageing.com/content/11/1/17 healthy80yearolddonor(80,O,+).Quantificationofthe signalintensitiesofthebandsintheWesternblotswere performedforbothsupplementedandnon-supplemented clones(Additionalfile1:FigureS1A-L). Asimilarpatternofphosphorylationisseeninthe young(earlyPD)TCCswithandwithout7.5mMNAC supplementation,withonlyERKphosphorylatedtoany significantextent(Figure3B).PhosphorylationofJNK, p38andc-Junwasabsent(orlow)inyoungcells(Y)but greatlyenhancedinagedcells(O)fromalldonors. 7.5mMNACsupplementationinhibitedthisphospho- rylationbyatleast80%(Figure3B)inyoung(26year old)andmiddleaged(45yearold)donorTCCswiththe exceptionofp-JNKinthemiddleageddonorTCC wherealowerreductionwasseen(~25%).However,no significantreductioninphosphorylationofJNK,p38, andc-JunwasfoundinTCCsderivedfromahealthy80 yearolddonortreatedwith7.5mMNAC(80,O,+). Quantificationofthesignalintensitiesofthebandsin theWesternblotswereperformedforbothsupple- mentedandnon-supplementedclones(Additionalfile1: FigureS2A-L). ThetotallevelsofJNK,p38andERK(Additionalfile1: FigureS3CandD)werenotsignificantlydifferentfollo- wing30 MEbselenor7.5mMNAC,comparedtonon- supplementedcontrols. Discussion Previousworkfromourgroupdemonstratedtheanti- immunosenescencepotentialofcertainconcentrations ofEbselen(30 M)orNAC(7.5mM)inCD4 + Tcells exvivoandinCD4 + TCCswhensupplementedfroma young invitro age[10].TheROSscavengingpotentialof theseantioxidantsresultedinenhancementoftheGSH: GSSGratio,asignificantdecreaseinlevelsofoxidative DNAdamageandasignificantincreaseinlifespan,and/ orproliferativecapacityofTCCsderivedfromahealthy 26yearoldor45yearolddonor. Figure3 Impactof30 MEbselen(A)or7.5mMNAC(B)onthephosphorylationlevelsofJNK,c-Jun,p38andERK. Theblotsrevealthe effectofantioxidantsupplementationbetweentheyoung(earlyPD)andaged(latePD)TCCsisolatedfromhealthy26,45or80yearolddonors comparedtonon-supplementedcontrols. Marthandan etal.Immunity&Ageing 2014, 11 :17 Page7of10 http://www.immunityageing.com/content/11/1/17 Incontrast,inthepresentstudy,supplementationofaTCCderivedfromahealthy80yearolddonor(conform-ingtotheSENIEURprotocol;[14])with30MEbselenor7.5mMNAC,fromayounginvitroage(31.1InitialPD)didnotsignificantlyalterthelifespan,theprolifera-tivecapacity(Table3),levelsofoxidativeDNAdamage(Figure2AandB),intracellularredoxstatus(GSH:GSSGratio;Figure1AandB)orthetotalglutathionelevels.Barnettandcolleagueshavepreviouslypublishedthat20mMCarnosine(anantioxidant)supplementationfromthemidpointoftheirinvitrolifespandidnotalterthelongevityofTCCsderivedfroman80yearolddonor[3].Inthatcase,itwassuggestedthatCarnosinemaynothavebeenabletorevealitsantioxidantpotentialduetothehighbackgroundofbiomoleculedamagethatalreadyexistedintheseTcells,accumulatedduringear-lierstagesoftheirinvitrolifespanunderconditionsof20%Othatmayhavecompromisedarangeofintracel-lularsystems.OnepieceofevidenceinthisregardisthemeasuredincreaseinbasallevelsofoxidativeDNAinTCCsfromahealthy80yearolddonor,comparedwithbasallevelsinTCCsfromhealthy26or45yearolddonors(Additionalfile1:TableS2A,B).Theresultsob-tainedinthiscurrentinvestigationsuggestthatarangeofantioxidantsupplementationsdonothaveanimpactonthebiologicalendpointsmeasuredintheTCCsfromhealthy80yearolddonors.AsimilarscenarioisapplicableintermsoftheGSH:GSSGratio.Intracellularredoxstatus(asreflectedintheGSH:GSSGratio)isanimportantmechanismhavinganinvaluableroleasamediatorinapoptosisinmanycellsystems[37].Previousfindingsrevealthatintracellularreducedglutathione(GSH),amaindeterminantofin-tracellularredoxstatus,isdepletedbeforetheonsetofapoptosis[38].TheGSH:GSSGredoxcoupletmaintainstheredoxenvironmentofthecellandGSHisabundantinthecell[39].TheoxidationofevenasmallamountofGSHresultsintheformationofGSSGtherebyloweringtheGSH:GSSGratiosuggestedtoberesponsibleforseveralhumandiseases[40].However,inthisstudy,theGSH:GSSGratiodidnotsignificantlychangeonantioxi-dantsupplementationcomparedtothenon-treatedcon-trols,andtheratiodecreasedascellsapproachedthelatterstageoftheirinvitrolifespan.AlthoughROSaregenerallythoughtofasharmfulmolecules,theydoplayanimportantroleinTcellsig-nallingevents[41]includingtheMAPkinasepathways.MAPkinaseshaveseveralpathwaysidentifiedincludingERK,JNKandthep38kinasepathways.ERKphospho-rylationhasbeenshowntoactasacellsurvivalfactoragainstoxidativestress,whereasphosphorylationofJNKandp38contributestocelldeathmachinery[23].TcellsignallingeventssuchasproteintyrosinephosphorylationandactivationofJNKaswellascellularproliferationinducedbylectinaresomeofthefewinstancesthatre-quirethepresenceofROS[28].ReducedROSlevelsmightinterferewiththesignallingpathwaysinvolvedinTcellac-tivationandproliferation,forexample,theredox-sensitiveactivationoftranscriptionfactorssuchasnuclearfactorkappalightchainenhancerofactivatedBcells(NF-kB)oractivatorprotein-1(AP-1)[42].Thispaperdescribestheinvestigationoftheeffectoftheantioxidants,EbselenorNAC,onthephosphoryl-ationofp38andJNK(SAPK)inTCCsfromdonorsofdifferentages.JNKactivationprimarilyresultsinapop-tosisbythephosphorylationofc-Jun(serine63),whichisacomponentofthetranscriptionfactorcomplexAP-1thatbindstoaspecificDNAsequenceintheAP-1site[43]resultinginincreaseofDNAbindingandultimatelyapop-tosis.PreviousfindingshaveindicatedinhibitionofHinducedp38MAPkinaseactivation,c-JunphosphorylationandJNKactivationbyEbseleninaconcentration-dependentmanner[23].Furthermore,earlierstudieshaverevealedthatNACdecreasedbothJNKandp38phosphorylationinducedby2,3,5-tris-(glutathion-S-yl)hydroquinone(TGHQ)inhumanepithelialcells[44],sel-eniteinhepatocytes[29]andtaxol(chemotherapeuticagent)inleukaemiccells[30].Theresultsofthepresentstudysuggestthatpro-apoptoticpathwaysbecomeacti-vatedinallTCCsasthecellsreachanoldinvitroagewithactivationofJNK,p38andc-JunacrossalloldTCCsirrespectiveofdonorage(Figure3AandB).TheresultsofourstudyalsoreinforcetheradicalscavengingpotentialofEbselenandNACwithasignificantdecreaseinphospho-rylationofJNKandc-JuninlatePDTCCsinvitroderivedfromahealthy26or45yearolddonoronsupplemen-tationwith30MEbselen(Figure3A)or7.5mMNAC(Figure3B)comparedtonon-supplementedTCCs,al-thoughonlyNACsupplementationwasabletodecreasep38phosphorylationintheselatePDTCCs.However,nei-therantioxidantcouldsignificantlyalterphosphorylationofp38,JNKorc-JuninlatePDTCCsinvitroderivedfromahealthy80yearolddonor(Figure3AandB).OurresultssuggestthatneitherEbselennorNACcouldaltertheactivationofp38,JNKandc-JuninTCCsfromveryhealthyelderlydonorsandthusfailtoimpactuponthetimetoonsetofapoptosis.Thisisanotherpieceofevidencewhichsuggeststhattherearealterationstointra-cellularprocesses,whichhaveaccumulatedduringtheprolongedexistenceofTcellsfromelderlydonors.Incontrasttotheresultsobtainedfromourstudy,othershavepublishedthatNACsupplementationin-creasedERKactivationinhumankidneyproximaltu-buleepithelialcells(HK-2)[44].However,resultsfromthispresentstudyrevealedconsistentactivationofERKinallTCCsirrespectiveofdonororinvitroagewithnosignificantchangeinthelevelsofERKphosphorylationinanyoftheagegroupsuponsupplementationwithMarthandanetal.Immunity&Ageing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either30 MEbselenor7.5mMNAC,comparedto non-supplementedTCCs(Figure3AandB). Theresultsofthisstudyhighlightaheterogeneous potentialofEbselenorNACasanti-immunosenescent interventivestrategiesinhumanTcells.If invivo anti- oxidantsupplementationistobeattemptedthencareful pre-interventionevaluationshouldbeundertakentode- terminerisk-benefit. Additionalfile Additionalfile1:TableS1. DecreaseinGSH:GSSGratiolevelswith timeincultureof80yearoldTCCs. TableS2. (A)Higherlevelsof oxidativeDNAdamagein80yearoldTCCs+/-Ebselen .TableS2. (B)HigherlevelsofoxidativeDNAdamagein80yearoldTCCs+/-NAC. FigureS1. (A-D)EffectofEbselenonMAPkinasepathwaysin26year oldTCCs. FigureS1. (E-H)EffectofEbselenonMAPkinasepathwaysin 45yearoldTCCs. FigureS1. (I-L)EffectofEbselenonMAPkinase pathwaysin80yearoldTCCs. FigureS2. (A-D)EffectofNAConMAP kinasepathwaysin26yearoldTCCs. FigureS2. (E-H)EffectofNACon MAPkinasepathwaysin45yearoldTCCs. FigureS2. (I-L)EffectofNAC onMAPkinasepathwaysin80yearoldTCCs. FigureS3. (C)Impactof 30 MEbselen(C)ontotallevelsofJNK,c-Jun,p38andERK. FigureS3. (D)Impactof7.5mMNAC(D)ontotallevelsofJNK,c-Jun,p38andERK. Abbreviations AP-1: Activatorprotein-1;CD:Clusterofdifferentiation;ERK:Extracellularsignal regulatedkinase;GSH:Reducedglutathione;GSSG:Oxidisedglutathione; HK-2:Humankidneyproximaltubuleepithelialcells;JNK:c-JunN-terminal kinase;ERK:Extracellularsignalregulatedkinases;MAP:Mitogenactivated protein;NAC:N-Acetylcysteine;PD:Populationdoubling;TCCs:Tcellclones; TGHQ:2,3,5-tris-(glutathion-S-yl)hydroquinone;PTKs:Proteintyrosinekinases; PTPs:Proteintyrosinephosphatases;EndoIII:EndonucleaseIII; FPG:FormamidopyrimidineDNAglycosylase. Competinginterest Theauthorsdeclarethattheyhavenocompetinginterest. Authors contributions SMundertookthelaboratoryworkforthisstudyandwrotethemanuscript. SSdidmostofthewesternblots.YBproducedthehypothesisforthis investigationandproofreadthemanuscript.RFgaveextensiveadviceon theMAPkinasestudyandGPsuppliedtheTcellclones.Allauthorsreadthe manuscript,studieditcriticallyforitsintellectualcontentandapprovedthe finaldraft. Acknowledgements WethankProfessorStephanDiekmannandDr.PeterHemmerichfortheir contributionwithregardstothesignallingwork.Theworkdescribedhereis partoftheresearchprogrammeoftheJenaCentreforSystemsBiologyof Ageing-JenAge.WeacknowledgeJenAgefundingbytheGermanMinistry forEducationandResearch(BundesministeriumfürBildungundForschung BMBF;supportcode:0315581).RJK853cellswereagiftfromDr.JaneCole, MRCCellMutationUnit,UniversityofSussex,UK). 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ToxicolSci 2011, 120 (1):87 97. doi:10.1186/s12979-014-0017-5 Citethisarticleas: Marthandan etal. : SENIEURstatusoftheoriginating celldonornegatescertain anti-immunosenescence effectsofebselen andN-acetylcysteineinhumanTcellclonecultures. Immunity&Ageing 2014 11 :17. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Marthandan etal.Immunity&Ageing 2014, 11 :17 Page10of10 http://www.immunityageing.com/content/11/1/17