“Inflamm-aging” influences immune cell survival factors in human bone marrow Luca Pangrazzi.pdf

该【“Inflamm-aging” influences immune cell survival factors in human bone marrow Luca Pangrazzi 】是由【妙玉】上传分享，文档一共【34】页，该文档可以免费在线阅读，需要了解更多关于【“Inflamm-aging” influences immune cell survival factors in human bone marrow Luca Pangrazzi 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..“Inflamm-aging”influencesimmunecellsurvivalfactorsinhumanbonemarrow111233LucaPangrazzi,AndreasMeryk,ErinNaismith,RafalKoziel,JulianLair,MartinKrismer,KlemensTrieb4,BeatrixGrubeck-,InstituteforBiomedicalAgingResearch,UniversityofInnsbruck,Rennweg10,Innsbruck,,InstituteforBiomedicalAgingResearch,Universit?tInnsbruck,Rennweg10,Innsbruck,Austria3DepartmentofOrthopedicSurgery,InnsbruckMedicalUniversity,Anichstrasse35,Innsbruck,Austria4DepartmentofOrthopedicSurgery,HospitalWels-Grieskirchen,Grieskirchnerstrasse42,Wels,AustriaCorrespondingauthor:-LoebensteinHeadInstituteforBiomedicalAgingResearch,UniversityInnsbruck,Rennweg10A-6020Innsbruck,AustriaPhone:0043-512-50750855,Fax:0043-512-50750899E-Mail:Beatrix.******@:21/06/2016;Revised:21/10/2016;Accepted:14/12/2016eptedforpublicationandundergonefullpeerreviewbuthasnotbeenthroughthecopyediting,typesetting,paginationandproofreadingprocess,:..:..Keywords:Bonemarrow,aging,immunosenescence,immunologicalmemory,ROSListofabbreviations:APRIL:aproliferation-inducingligand;BM:bonemarrow;BMMCs:BMmononuclearcells;DC:ell;DHE:Dihydroethidium;hi:high;MFI:meanfluorescenceintensity;NAC:N-acetylcysteine;PBMCs:peripheralbloodmononuclearcells;ROS:reactiveoxygenspecies;SOD1:superoxidedismutase1;WT::..ABSTRACTThebonemarrow(BM)playsakeyroleinthelong-,theimpactofagingontheproductionofsurvivalfactorsforeffector/-15,whichprotectspotentiallyharmful+-CD8CD28senescentTcells,increases,IL--6,whichmaysynergizewithIL-15,,aproliferation-inducingligand(APRIL),aplasmacellsurvivalfactor,-y,-15andIL-6expressionarestimulatedbyIFN-yandcorrelatewithROSlevelsinBMmononuclearcells(BMMCs).BothcytokinesarereducedbyincubationwiththeROSscavengersN-acetylcysteine(NAC)-15andIL-6arealsooverexpressedintheBMofSOD1paredtotheirwild-type(WT),ourresultsdemonstratetheroleofinflammationandoxidativestressinage-relatedchangesofimmunecellsurvivalfactorsintheBM,:..INTRODUCTIONOneofthemostprominentchangesoftheagingimmunesystemistheinvolutionofthethymus,whichisresponsibleforadramaticearlydeclineinthegenerationofnewna?veTcells[1,2].Forthisreason,adaptiveimmunityinoldageismainlysupportedbyaturnoverofanexistingpopulationofantigen-experiencedcells,themaintenanceofwhichiscrucialtofightinfections[3].RecentstudiesperformedinbothmiceandhumansdescribetheBMasapreferredsiteforthesurvivalofeffector/memoryCD4+andCD8+Tcellsandlong-livedplasmacells[4-10].Survivalfactorspromotelong-termmaintenanceofeffector/-livedplasmacellsresideinsurvivalanizedbyreticularstromalcellsexpressingthechemokineCXCL-12(stromalcell-derivedfactor1;SDF1)[11].SurvivalfactorssuchasAPRILandIL-6,mainlyproducedbymyeloidcelltypes,arealsoknowntobeimportantforthemaintenanceoflong-livedplasma+cells[12-16].Effector/memoryCD4TcellshavebeendescribedalsotomigratetoBMnicheswheretheyinteractwithVCAM-1+stromalcellsproducingthesurvivalfactorIL-7[5,+17,18].Maintenanceofeffector/memoryCD8TcellsalsorequiresIL-7[19].ThecytokineIL-15cancontributetothisprocess,anditisparticularlyimportantforthepreservationofhighlydifferentiatedCD8+effectorTcells[20-23].DCs,monocytes(CD14+),CD34+hematopoieticprogenitorcellsandBMstromalcellshavebeendescribedtoproduceIL-15+[24,25].Inparticular,IL-15transpresentedbyDCsandCD14cellsisimportantforeffector/memoryCD8+T-cellgenerationandmaintenance[26-28].:..umulationofhighlydifferentiatedeffectorCD8+Tcells,whichlacktheexpressionofthecostimulatorymoleculeCD28[29-31].IthasbeensuggestedthathighnumbersofCD8+CD28-Tcellsaredetrimentalforelderlypeople,umulationisassociatedwithhighinflammationlevelsandanincreasedriskofage-relateddiseasesandmortality[32,33].SinceIL-15andIL-6havebeenshowntocontributetothegenerationandmaintenanceofCD8+CD28-Tcells,theexpressionofthese+cytokinesshouldbecontrolledinordertoguaranteesufficientBMspaceforCD4helpercellsandplasmacellsinoldage,umulationofexhaustedTcells[34,35].Inthepresentstudy,weanalyzedtheexpressionofeffector/memorycellsurvivalfactorsintheBMofpeopleofdifferentages,-15andIL-6couldbestimulatedwithIFN-+regulationofBMIL-15andIL--relatedimpairmentsinthemaintenanceofimmunologicalmemoryinoldagebychangingtheexpressionpatternofeffector/memorycellsurvival+-:..RESULTSEffector/(Figure1).WefoundthatIL-15andIL-6mRNAincreasewithage,whereastheexpressionofIL-7andAPRILdecrease(Figure1A-D).IL-15andIL-6levelswereverylowandinsomesamplesalmostundetectableinpersonsunder65years,whiletheywerefrequentlyhigherintheagegroup>,someolderdonorshadaveryhighexpressionofbothcytokine’-±(p=)andIL-±(p=)--±(p=)±>paredtotheagegroupof<-relatedchangeswereobservedforthechemokineCXCL-12,withgreatdeviationsatallages(Figure1E).FlowcytometryexperimentsconfirmedtheresultsdescribedformRNAattheproteinlevel(Figure2).IL-15andIL-6levelsincreasedwhileAPRILdecreasedandCXCL--relatedchangesweresmall,-relatedchangesofIL-:..(Figure2E-F).ThesecretionofCXCL-paredtoyoungerdonors(Figure2G).IL-7immunofluorescencecouldnotbeperformedsincenoflowcytometryantibody(Ab)-15norIL-7couldbedetectedinthesupernatantsbyELISA,-termmaintenanceofeffector/memoryTcellsandlong-+Antigen-presentingCD11candCD14cellshavebeenconsideredtobethemaincelltypesproducingAPRILandIL-6andproducingandtrans-presentingIL-15toTcellsintheBM[14,16,25].CD34+myeloidprogenitorcellshavealsobeenfoundtoproduceIL-15[24].Here,weinvestigatedwhichoftheseBMcellsubpopulationswereresponsiblefortheage-relatedchangesofeffector/(SupportingInformationFigure1B-D).IL-15levelshi++increasedinoldageinCD11candCD34cells,butdidnotchangeinCD14cells(Figure3A-C).AsimilarstainingintensityforIL--6wasupregulatedwithageinbothCD11chiandCD14+cells(Figure3D-E)butwasnotexpressed+hi+byCD34cells(Figure3F).Again,+contrast,APRILexpressionnegativelycorrelatedwithageinCD34cellswhiletherewasnocorrelationwithageinCD11chiandCD14+cells(Figure3G-I).Inmostdonors,:..hi++ells,-relateddiseases[36].WeaimedtoassesswhethertheproductionofproinflammatorycytokinesotherthanIL-15andIL--γ(Figure4A)andTNF(Figure4B),althoughthecorrelationbetweenIFN-,theage-relatedincreaseintheexpressionofbothproinflammatorycytokineswasconfirmedinBMMCsupernatantsusingELISA(Figure4C-D).+-CD8CD28TcellsareenrichedintheagedBM(Figure4Eand[34]).Sincethissubpopulationisknowntocontributetoage-relatedinflammation[33,37],weanalyzedthe+-productionofIFN-γ-andTNFwithintheCD8CD28Tcellpopulationinagroupofyounger(<60years)andinagroupofold(>65years)paringitwiththeexpressionofthesamemoleculesinCD8+CD28+andCD4+Tcellsubpopulations(Figure4F-G).:..+-percentagesofCD8CD28TcellsexpressedIFN-γ--relateddifferencesinthepercentageofcytokineexpressingcellswerefoundamongCD8+CD28-andCD4++CD28+TcellsfromtheoldergroupexpressedIFN-γcomparedtotheyoungergroup(p=),contributingtooxidativestress[38,39].InlinewiththeincreasedlevelsofIFN-γandTNFin+-theBMwithageandthehighpercentageofIFN-γ-andTNF-producingCD8CD28BMcells,(Figure4H).,paredtoPBMCsfromthesamedonors(Figure4I).-15andIL-,weinvestigatedwhetherROScorrelatedwitheffector/-15andIL-6,butnotAPRILexpressioncorrelatedwithROSlevelsinBMMCs(Figure5A-C).ToconfirmthatROSalsoplayacausalroleinregulatingtheexpressionofIL-15andIL-6,unstimulatedBMMCswereincubatedwiththeROSscavengersNACandvitaminCatdifferentconcentrations(Figure5D-E).WhileNACorvitaminCalonedidnotchangeIL-15expression,:..significantlydownregulatedIL-paredtountreatedcontrols(Figure5D).IL-6wasbinationofNACandvitaminC(Figure5E).InordertomodelthesituationinthehumanBMinvitro,wetestedhowwellIL-15andIL-6couldbestimulatedinCD11chicellsbyIFN-(Figure3