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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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BiochimBiophysActa 1991, 1072 (2 – 3):129 – 157. 44.ZhangF,LauSS,MonksTJ: ThecytoprotectiveeffectofNACagainstROS inducedcytotoxicityisindependentofitsabilitytoenhanceglutathione synthesis. 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