Frontiers - The Upregulation of Toll-Like Receptor 英文文字文档

Frontiers|TheUpregulationofToll-LikeReceptor3viaAutocrineIFN-βSignalingDrivestheSenescenceofHumanUmbilicalCordBlood-DerivedMesenchymalStemCellsThroughJAK1IntroductionMesenchymalstromalcells(MSCs)aremultipotentstemcellswiththecapacityforself-renewalandthepotentialtodifferentiateintodiversecelltypes,includingosteoblasts,adipocytes,myocytes,neurons,hepatocytes,andchondrocytes().MSCsalsosecretevariouscytokinesandgrowthfactorstoregulatetheirvariouscellularfunctions().MSCsisolatedfromumbilicalcordblood,bonemarrow,andadiposetissuehavebeenextensivelyinvestigatedasabasisforcellulartherapyindiseasesettings().Thehumanumbilicalcordblood-derivedMSCs(UCB-MSCs)havebeenconsideredthestandardchoiceforcell-basedtherapyforvariousdiseasesandforregenerativemedicinebecauseoftheirreadyisolationfromtissues,highproliferativecapacity,littleornoimmunogenicity,andlowtumorigenicity(,).ToacquiresufficientnumbersofcellsforMSC-basedclinicalapplications,thereliableandefficientexpansionofthesecellsinvitrothroughlong-termcultivationisrequired.However,similartoprimarytissuecells,thisexpansioninculturecausescellularsenescenceandaconsequentdeclineincellfunctionwhichpotentiallycompromisestheuseofthesecellsintherapeuticapplications().Cellularsenescencecanbetriggeredbydiversestimuli,includingtelomeredysfunction,oxidativestress,genomicinstability,oncogenicandmetabolicinsults,andepigeneticchanges().Theprogressionofcellstowardreplicativesenescenceisaccompaniedbythegradualexpressionincreaseinthetranscriptionfactorp53,cyclindependentkinaseinhibitors,p21WAF1(p21),andp16INK4a(p16)().Senescenceisparalleledbyanincreaseinspecificmarkersincludingsenescence-associatedgalactosidase(SA--gal),DNAdamagefoci,anenlargedcellsize,cellcyclearrest,andthesenescence-associatedsecretoryphenotype(SASP)().SASPentailsthesecretionofproinflammatorycytokines,chemokines,growthfactors,andextracellularmatrixremodelingproteases(,).GiventhatSASPcomponentssecretedbysenescentMSCsincreasethecomplexityofparacrinecommunicationamongthesecellsandtheirphysiologicalmicroenvironment(),thecellularsenescencephenotypecanbesustainedandamplifiedbytheautocrineorparacrineregulatoryloopofSASP().SASPhasbeenpreviouslyassociatedwithTolllikereceptor(TLR)pathways().TheTLRsarekeymediatorsofinnateandadaptiveimmuneresponses().Furthermore,increasingevidencehasdemonstratedthatTLRsareexpressedinMSCsandmodulatevariousbiologicalfunctionsofthesecellsincludingproliferation,differentiation,migration,andimmunologicalbehavior().TLRsregulatetheimmuneresponsesmediatedbyproinflammatorycytokinesandtypeIinterferons(IFNs)().ThetwotypesofIFN,TypeI(mainlyIFN-andIFN-)andTypeII(IFN-)bindtotheirreceptorsandinitiateacascadeofeventsknownastheJanuskinase(JAK)/signaltransducerandactivatoroftranscription(STAT)signalingpathwaywhichisalsoinvolvedincellularsenescence(,).Theincreasedlevelsofcirculatinginflammatorymarkers,includingIFN-,IFN-,IL-1,IL-6,andTNF-areassociatedwithsenescence().PreviousstudieshavereportedthatIFN-upregulatesp53-dependentsenescenceinnormalhumanfibroblasts()andinducescellularsenescencebymodulatingtheJAK/STAT-inducedpromyelocyticleukemiaprotein(PML)inpapillomavirus-transformedkeratinocytesandtumorcells(,,).Furthermore,TLRfunctionwasfoundtobedysregulatedinthecontextofaging(),implyingtheinvolvementofTLRsinsenescence.TheJAK/STATpathwayplaysanimportantroleinregulatingcytokineproduction()anditsinhibitionsuppressesSASPinpreadipocytesandendothelialcells()andreprogramstheSASPinsenescenttumorcells().JAK1mediatesthebiologicalfunctionsinducedbythreemajorcytokinereceptorsubfamiliesincludingclassIIcytokinereceptors(thereceptorsforIFN-/,IFN-,andIL-10),cytokinereceptorsthatutilizethecreceptorsubunit(thereceptorsforIL-2,IL-4,IL-7,IL-9,andIL-15)andcytokinereceptorsutilizingthegp130subunit(thereceptorsforIL-6,IL-11,LIF,OSM,CNTF,andCT-1)().AknockdownofJAK1inhibitscellproliferation,invasion,andinvivotumorgrowthanddisruptsthePI3K/mTORpathway().JAK1,JAK2,andSTAT3areinvolvedincellgrowth,survival,invasion,andmigrationincolorectalcancercells()andJAKinhibitorsreduceinflammationandalleviatefrailtyinagedmice().Thus,theJAKpathwayisconsideredasacrucialfactorinthesenescentcells,however,theirroleinthesenescenceofMSCsremainslargelyunclear.Inourpresentstudy,weshowforthefirsttimethatTLR3ispredominantlyupregulatedinsenescenthumanUCB-MSCsandthattheactivationofthisreceptorleadstoMSCsenescence.Additionally,weidentifyJAK1asakeyregulatoryfactorlinkedtothecellularsenescenceofMSCstriggeredbyTLR3activationthroughtheuseofclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/Cas9knockout(GeCKO)libraryscreeningofMSCsaftercontinuoustreatmentwithpolyinosinic-polycytidylicacid(polyIC).Finally,ourfindingsindicatethatJAK1activationmediatestheIFN-actiontoincreaseTLR3expression,therebyreinforcingTLR3-mediatedMSCsenescence.ThesefindingsemphasizethedistinctroleofIFN-asacomponentoftheTLR3-dependentSASPinvolvedinTLR3-mediatedMSCsenescence.MaterialsandMethodsCellCultureandReagentsTheisolationofhumanMSCswasapprovedbytheInstitutionalReviewBoardofMEDIPOSTCo.,Ltd.(Seongnam,Korea)andMSCsusedinthisstudyweredonatedbyMEDIPOSTCo.,Ltd.Umbilicalcordbloodwascollectedfromumbilicalveinsafterneonataldeliverywithmaternalinformedconsent.Umbilicalcordblood-derivedMSCswereseparatedaspreviouslydescribed().Thecellswerethenplatedat2103cells/cm2andsplitat70%confluencyevery5days.293TcelllineswereculturedinDulbeccosmodifiedEaglesmedium(DMEM;GEHealthcare,Chicago,IL)containing10%FBSandpenicillin/streptomycin.Allcellsweremaintainedat37Cinahumidified5%CO2atmosphere.PolyICwaspurchasedfromInvivogen(SanDiego,CA)andtofacitinib,ruxolitinib,andGLPG0634wereobtainedfromSelleckchem(Houston,TX).Real-TimeQuantitativePolymeraseChainReaction(qPCR)TotalRNAwasisolatedfromMSCsusingTRIzolReagent(ThermoFisherscientific,Waltham,MA)inaccordancewiththemanufacturersprotocol.TotalRNAaliquots(2g)werethenreversetranscribedusingaTOPscriptcDNAsynthesisKit(Enzynomics,Daejeon,Korea).qPCRwasperformedontheCFXConnectReal-TimePCRDetectionSystem(Bio-rad,Hercules,CA).Theprimersusedaredescribedin.GeneexpressionwasnormalizedtothatofGAPDH,whichwasusedasaninternalcontrol.TherelativequantitiesofmRNAofinterestwerecalculatedusingthecomparativethresholdcycles(2Ct)method.WesternBlottingWesternblottingwasperformedaspreviouslydescribed().Eachmembranewasblockedin0.1%TrisbufferedsalinewithTween20(TBST)containing5%bovineserumalbuminandincubatedwiththeindicatedprimaryantibodiesagainstphospho-p53,p21,phospho-pRb,TLR3,JAK1,phospho-STAT1(CellSignalingTechnology,Danvers,MA),IL-6,p16,EZH2,CyclinD1(Abcam,Cambridge,UK),PCNA(Biosciences,SanJose,CA),BMI1(ActiveMotif,Carlsbad,CA),survivin(Genetex,Irvine,CA),and-actin(Sigma-Aldrich).Themembraneswerethenincubatedwiththecorrespondingperoxidase-conjugatedsecondaryantibodiesfor1hatroomtemperature.TheblotsweredevelopedusingtheAdvanstaWesternBrightECLHRPSubstrateKits(Advansta,SanJose,CA)anddetectedusingaC-DiGitBlotScanner(LI-COR,Lincoln,NE).ThesignalofeachbandwasquantifiedusingImageJdensitometrysoftware(Version1.6,NationalInstitutesofHealth,Bethesda,MD)fordensitometricevaluation.Thetargetedproteinlevelswerenormalizedtothelevelof-actin.EnzymeLinkedImmunosorbentAssay(ELISA)TheconcentrationsofhumanIFN-(VeriKineHumanIFNELISA;PBLAssayScience,Piscataway,NJ),IL-6(HumanIL-6ELISAMAXDeluxe,Biolegend,SanDiego,CA),IL-7(HumanIL-7QuantikineHSELISAKit;RDsystems,Minneapolis,MN),andIL-15(HumanIL-15QuantikineELISAKit;RDsystems)inconditionedmediafromhumanMSCsweremeasuredinaccordancewiththemanufacturersprotocols.Allsampleswereexaminedintriplicateforeachexperiment.SenescenceAssociatedBetaGalactosidaseStaining(SA--GalStaining)HumanMSCs(2104cell/well)wereseededandtreatedwithpolyICortofacitinib.SA--galstainingwasperformedaspreviouslydescribed().5-Bromo-2-Deoxyuridine(BrdU)CellProliferationAssayCellproliferationwasmeasuredusingBrdUcellproliferationELISA(RocheAppliedScience,UpperBavaria,Germany),whichquantifiestheincorporationofBrdU,athymidineanalog,duringDNAsynthesis.Cellproliferationwascalculatedrelativetothepercentageofcontrolcells.GenerationoftheGeneKnockoutMSCsUsingtheCRISPR/Cas9TechniqueOligonucleotidepairsforthetargetsequences(JAK1,TLR3,IFNAR1)wereannealedandthefragmentswerethenclonedintotheBsmBIsiteofplentiCRISPRv2plasmid(Addgene,Watertown,MA,USA).TheOligonucleotidesusedaredescribedin.Lentiviruswasproducedviatheco-transfectionofthepMD2.G(Addgene)envelopeandpsPAX2(Addgene)packagingplasmidsinto293TcellsusingNeonTransfectionSystem(ThermoFisherscientific).Thevirus-containingsupernatantwascollected72haftertransfectionandpassedthrougha0.45mfiltertoeliminatecells.MSCswereinfectedbyvirus-containingsupernatant.At72hafterinfection,viruswasremovedandcellswereselectedwiththe2.5g/mlpuromycin(Sigma-Aldrich).CRISPR/Cas9Knockout(GeCKO)ScreeningGeCKOLibraryLentivirusProductionLentiviruswasproducedviatheco-transfectionof80gofplentiCRISPRplasmidlibrary(Addgene)withthe40gpMD2.G(Addgene)envelopeand60gpsPAX2(Addgene)packagingplasmidsinto4107293TcellsusingtheNeonTransfectionSystem(ThermoFisherscientific).Cellswerepulsedtwicewithavoltageof1,100andawidthof20.Thevirus-containingsupernatantwascollected72haftertransfectionandpassedthrougha0.45umfiltertoeliminatecells.Aliquotswerestoredat80C.CellTransductionUsingtheGeCKOLibraryTointroducetheGeCKOlibraryvirusesintoMSCs,1106cellswereseededandincubatedovernight.Cellsweretheninfectedwith20mlGeCKOlibrarylentivirus-containingsupernatant.At72hafterinfection,theviruswasremoved,andcellswereselectedwith2.5g/mlpuromycin(Sigma-Aldrich)for48h.Puromycin-selectedGeCKO-library-MSCsweremaintainedfor7daysandthenstoredinfreezingmediumat80Cuntiluse.PolyICResistanceScreenGeCKOlibrary-MSCswereculturedwithorwithoutpolyIC(1g/ml).CellswereeitherpassagedorfreshmediacontainingpolyICwasaddedevery2days.TheMSCswerefinallyharvestedat14daysafterpolyICadditionatwhichpointthescreenwasterminated.GenomicDNASequencingGenomicDNAextraction,libraryconstruction,tagging(indexing)ofsamples,deepsequencingusingtheIlluminaMiSeqplatform,anddataprocessingwereperformedbyMacrogenInc.(Seoul,Korea).StatisticalAnalysisAllquantitativeexperimentswereperformedatleastintriplicate.Dataarepresentedasmeansstandarderrorofthemean(SEM)ofatleasttwoindependentexperiments.TheMann-Whitneytest(,)orKruskal-Wallistest(,,,,)wereusedtodeterminesignificance.P0.05wasconsideredstatisticallysignificant.FIGURE1Figure1.Cellularsenescenceleadstotheu