s41419-023-05607-4 by Biogenomics

Schwanncell-derivedexosomescontainingMFG-E8modify macrophage/microglialpolarizationforattenuating inﬂammationviatheSOCS3/STAT3pathwayafterspinalcord injury

JieRen1,4,BinZhu1,4,GuangjinGu1,4,WencanZhang2,JunjinLi1,HongdaWang1,MinWang3,XiaomengSong1 ✉,ZhijianWei1,2 ✉ and ShiqingFeng 1,2 ✉

©TheAuthor(s)2023

Macrophage/microgliapolarizationactsasanimportantpartinregulatinginflammatoryresponsesinspinalcordinjury(SCI). However,theregulationofinflammationofSchwanncell-derivedexosomes(SCDEs)forSCIrepairisstillunclear.Therefore,we intendto findouttheeffectofSCDEsonregulatingtheinflammationrelatedtomacrophagepolarizationduringtherecoveryof SCI.Firstly,thethesisdemonstratedthatSCDEscouldattenuatetheLPS-inflammationinBMDMsbysuppressingM1polarization andstimulatingM2polarization.Similarly,SCDEsimprovedfunctionalrecoveryoffemaleWistarratsoftheSCIcontusionmodel accordingtoBBB(Basso,Beattie,andBresnahan)score,electrophysiologicalassay,andthegaitanalysissystemofCatWalkXT. Moreover,MFG-E8wasverifiedasthemaincomponentofSCDEstoimprovetheinflammatoryresponsebyproteomicsequencing andlentiviraltransfection.Improvementoftheinflammatorymicroenvironmentalsoinhibitedneuronalapoptosis.Theknockoutof MFG-E8inSCscanreversetheanti-inflammatoryeffectsofSCDEstreatment.TheSOCS3/STAT3signalingpathwaywasidentifiedto participateinupregulatingM2polarizationinducedbyMFG-E8.Inconclusion,our findingswillenrichthemechanismofSCDEsin repairingSCIandprovidepotentialapplicationsandnewinsightsfortheclinicaltranslationofSCDEstreatmentforSCI.

CellDeathandDisease (2023)14:70;https://doi.org/10.1038/s41419-023-05607-4

INTRODUCTION

Spinalcordinjury(SCI)isadevastatingdiseasethatcancause severedysfunctionofmotor,sensory,andautonomicnerves[1]. Numerousfactorsareinvolvedinthespreadofsecondary damage,includingtrauma-inducedischemia,apoptosis,excitotoxicity,andinflammation[2].Sincemostofthesesecondary injurymechanismscanbeinitiatedandregulatedbytheimmune system,itisobviousthattherapies-influencedinflammatory cascadesarelikelytobenefitthefunctionalrecoveryofSCI[3]. TheCNSmacrophagesmainlyincludemacrophages(bone marrow-derivedmacrophages,BMDM)andmicroglia(CNSresidentmacrophages),whichtogetherconstitutethemaincellsof theinflammatoryresponseafterSCI[4].

Duetothedifferentin fl ammatoryresponsesignalsinthe localmicroenvironmentafterSCI,CNSmacrophagesundergo morphologicalandfunctionalchanges,suchastheproin fl ammatorymacrophages(M1)oranti-in fl ammatorymacrophages(M2)[ 5 , 6 ].Increasingevidenceholdstheviewthatthe

pro-in fl ammatorymacrophages(M1)canincreasetheexpressionofdestructivein fl ammatorycytokines,includingIL-1 β and TNF- α ,aggravatethedamageofhostcellsandhindertherepair oftissues.Onthecontrary,M 2macrophagesplayanantiin fl ammatoryandtissue-repairingrolebyremovingnecrotic tissuedebrisandreleasingprotect ivefactors,includingIL-4and IL-10[ 7 , 8 ].Therefore,thetargetedinterventionofmacrophagescanbeappliedforSCIbyb lockingtherecruitmentand proliferationofmacrophages,inhibitingtheactivationpathway oftheM1phenotype,andpromotingthetransformationtoM2 macrophages[ 9 , 10 ].

Exosomesareendoplasmic-derivedsmallvesiclescontaining diversebiologicallyactivemolecules,mainlyincludingnucleic acidsandproteins,whichplayacrucialroleinintercellular communication[11, 12].Beabletocrosstheblood–brainbarrierin theCNS,exosomescanmediatecommunicationbetweenneurons andglialcells,promoteneuronalgrowth,andmodulateinﬂammationresponses[13, 14].Sunetal.reportedthatexosomes

1NationalSpinalCordInjuryInternationalCooperationBase,TianjinKeyLaboratoryofSpineandSpinalCordInjury,DepartmentofOrthopedics,TianjinMedicalUniversity GeneralHospital,Tianjin,China. 2DepartmentofOrthopedics,QiluHospitalofShandongUniversity,ShandongUniversityCentreforOrthopedics,AdvancedMedicalResearch Institute,ShandongUniversity,Jinan,Shandong,China. 3TianjinKeyLaboratoryofLungCancerMetastasisandTumorMicroenvironment,TianjinLungCancerInstitute,Tianjin MedicalUniversityGeneralHospital,Tianjin,China. 4Theseauthorscontributedequally:JieRen,BinZhu,GuangjinGu. ✉email:songxiaomeng@tmu.edu.cn; weizhijian2002@126.com;sqfeng@tmu.edu.cn EditedbyHans-UweSimon

Received:20September2022Revised:17January2023Accepted:19January2023

Fig.1 IdentiﬁcationanduptakeofSCDEs.A SchematicdiagramofBMDMsandSCDEs. B MorphologyofSCDEsunderTEM.Scalebars: 100nm. C ThediameterdistributionofSCDEs. D WesternblotanalysisofthepositiveexpressionofAlix,CD9,andCD63inSCDEs. E RepresentativeimagesofS100(red).NucleiwerelabeledwithDAPI(blue).Scalebars:20µm. F RepresentativeimagesofF4/80(red)and CD11b(green).NucleiwerelabeledwithDAPI(blue).Scalebars:20µm. G RepresentativeimagesofPKH26(red)-labeledSCDEsabsorbedby BMDMs.NucleiwerelabeledwithDAPI(blue).Scalebars:20µm.

derivedfromhumanumbilicalcordmesenchymalstemcellscould reducelocalinﬂammationandpromotenerverepair,thereby improvingfunctionalrecoveryafterSCI[15].

Inrecentyears,Schwanncells (SCs)havebeenshowntoplay apositiveroleinnerverepairbypromotingaxonaldedifferentiationandproliferationandremovingmyelinsheathsand axonaldebris[ 16 , 17 ].Agrowingnumberofstudieshavefound thatSchwanncell-derivedexosomes(SCDEs)canpromote axonalregenerationandcarryproteinscloselyinvolvedin suppressingin ﬂ ammation[ 18 , 19 ].ItisindicatedthatSCDEs havebroadapplicationprospectsforSCI.Ourpreviousstudies havefoundthatSCDEscan,ontheonehand,promoteaxonal

Fig.2 TheregulationofSCDEsonM1/M2polarizationinLPS-stimulatedBMDMs.A RepresentativeimagesofiNOS(red)andF4/80(green). NucleiwerelabeledwithDAPI(blue).Scalebar:20 μm. B RepresentativewesternblotsshowingthereductionofiNOS. C Theimageanalysis resultswerepresentedastherelativemeanintensityofthe fluorescenceofiNOS(n = 5). D QuantitativeanalysisoftheiNOS/GAPDHratio (n = 3). E RepresentativeimagesofCD206(red)andF4/80(green).NucleiwerelabeledwithDAPI(blue)ineachgroup(n = 5).Scalebar:20 μm. F RepresentativewesternblotsshowingtheincreaseofCD206. G Theimageanalysisresultswerepresentedastherelativemeanintensityof the fluorescenceofCD206(n = 5). H QuantitativeanalysisoftheCD206/GAPDHratio(n = 3). I–J FlowcytometryassaydetectedCD86 + M1 phenotype,indicatingthatSCDEstreatmentsuppressedtheM1polarizationinLPS-stimulatedBMDMs(n = 3). K, L Flowcytometryassay detectedCD206 + M2phenotype,showingthatSCDEstreatmentpromotedtheM2polarizationinLPS-stimulatedBMDMs(n = 3).Datawere presentedasmean±SEM.ResultswereanalyzedbyOne-wayANOVA.Significance:*P <0.05,**P <0.01,***P <0.001.

Fig.3 FunctionrecoveryofratsbySCDEsafterSCIinvivo.A TheFlowchartofanimalexperiments. B, F TheBBBscoresofSham,SCIgroup, andSCIratgrouptreatedwithSCDEs(n = 5). C Representativepawstepimagesandlimbs’ supportingtimingviewofCatWalkgaitanalysis. D The H&EstainingofthebladdershowedalesserthicknessofthebladderintheSCIrattreatedwithSCDEs.Scalebar:500 μm. E, H Analysisofmotor evokedpotential(MEP)wasperformedasanelectrophysiologicalassessmentinbothgroupsatday28post-injuryinbothgroups(n = 5). G Thewall thicknessofthebladderineachgroup,showingthatthebladderfunctionrecoveredfasterintheSCDEstreatmentgroup(n = 5). I Quantitative analysisofcatwalkatday28post-injury,includingregularityindex,printposition,andstancesofthehindlimb(n = 5,RH:righthindlimb,LH:left hindlimb).Datawerepresentedasmean±SEM.ResultswereanalyzedbyOne-wayANOVA.Signiﬁcance:*P <0.05,**P <0.01,***P <0.001.

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protectionbyincreasingauto phagyandreducingapoptosis throughEGFR/Akt/mTORsignal ingpathway,andontheother hand,increaseToll-likerecept or2expressioninastrocytesand reducethedepositionofchondroitinsulfateproteoglycan,

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therebyimprovingfunctionalrecoveryafterSCI[ 20 , 21 ]. Therefore,thepresentstudiesintendtofurther ﬁ gureoutthe effectofSCDEsonregulatingthein ﬂ ammationrelatedtothe macrophagepolarizationintherecoveryafterSCI.

Fig.4 SCDEsimprovedspinalcordrepairafterSCIinvivo.A RepresentativeimagesofimmunofluorescencestainingofED1(red)andGFAP (green)ineachgroupatday3afterSCI(n = 3).Scalebars:1000µm.Thebottomrowofimageswerehigh-resolutionversionsoftheboxed regionsinthetoprowofimages.Scalebars:50µm. B RepresentativeimagesofimmunofluorescencestainingofGFAP(red)andNF200(green)ineachgroupatday14afterSCI(n = 3).Scalebars:1000µm.Therightimageswerehigh-resolutionversionsoftheboxedregions intheleftimages.Scalebars:20µm. C RepresentativeimagesofimmunofluorescencestainingofChAT(red)intheSCIgroupandSCI + SCDEs group(n = 3).Scalebars:20µm. D TheH&EstainingofthespinalcordshowedthereductionofthedamagedareaintheSCIrattreatedwith SCDEs. E QuantitativeanalysisofthenumberofED1 + cells,showingthatbeingtreatedwithSCDEscanreducethenumberofED1 + cellsin therostral,lesion,andcaudal. F Quantitativeanalysisoftherelativemeanintensityofthe fluorescenceofNF200. G Quantitativeanalysisof thenumberofChAT+ cells,showingthatthenumberofChAT+ cellsintheSCIrattreatedwithSCDEswashigher.Datawerepresentedas mean±SEM.ResultswereanalyzedbyStudent ’s t-test.Significance:*P <0.05,**P <0.01.

Milkfatglobule-epidermalgrowthfactor-factor8(MFG-E8)isa secretedmultifunctionalglycoprotein[22].Duringmultiple pathologicalprocessesintheCNS,MFG-E8isupregulatedin microglia/macrophages,whileastrocytesandneuronscanexpress smallamountsofMFG-E8[23, 24].Recently,MFG-E8hasbeen foundtoplayabeneficialroleintreatingneurodegenerative diseasesandtraumaticbraininjury,inhibitthereleaseofproinflammatorymediators,andmodulateimmuneresponse[25, 26]. Furthermore,MFG-E8promotesthetransitionofthemicroglia phenotypetowardtheM2phenotype[27, 28].Thence,onthe basisoftheresultsofproteomicsequencing,wespeculatethat MFG-E8maybeinvolvedwhenSCDEsplayapartinin inflammatoryregulationandmacrophagepolarization.

ThisstudysystematicallyanalyseswhetherSCDEscanexertan anti-inflammatoryeffect,whichisachievedbyinhibitingM1 polarizationandpromotingM2polarizationofmacrophage/ microglia,thuspromotingthefunctionalrecoveryafterSCI. Meanwhile,MFG-E8hasbeenverifiedtobethekeycomponent ofSCDEsininflammatoryregulation.Theknockout(KO)ofMFG-E8 inSCscanreversetheanti-inflammatoryeffectsandM2 polarizationofmacrophage/microgliacausedbySCDEstreatment. TheSOCS3/STAT3signalingpathwayhasbeenidentifiedto participateinupregulatingtheM2polarizationinducedbyMFGE8.These findingscanenrichthemechanismofSCDEsinrepairing SCIandprovidepotentialapplicationsandnewinsightsforthe clinicaltranslationofSCDEtreatmentforSCI.

RESULTS

CharacterizationsanduptakeofSCDEs

PrimaryBMDMsandSCswereextractedfromadultfemaleWistar rats(Fig. 1A).TEM(TransmissionElectronMicroscopy)wasusedto observethemorphologyofSCDEs,andtheresultsshowedthat mostdisplayedroundorovalshape,withanaveragediameterof 100nm(Fig. 1B,C).Threekindsofmarkerproteinscontainedin exosomesweredetectedbywesternblot:Alix,CD9,andCD63 (Fig. 1D).ToidentifySchwanncells,immunoﬂuorescencestaining wasperformedonS100proteins(Fig. 1E).ToidentifyBMDMs,they werestainedwithF4/80andCD11b(Fig. 1F).Todetectthe phagocytosisofBMDMexosomesinvitro,PKH26-labeledSCDEs wereco-culturedwithBMDMsandthephagocytosisofexosomes wasobservedviaaconfocalmicroscopy(Fig. 1G).

InvitroSCDEspromotedM2polarizationinLPS-stimulated BMDMs ALPS-inducedBMDMsinflammationinvitromodelwas establishedtofurtherverifytheregulationofSCDEsonM1/M2 phenotypes.Immunofluorescencestainingwasperformedonthe macrophagemarkerofF4/80andtheM1phenotypemarkerof iNOS(Fig. 2A).Theresultshowedtherelativemeanintensityof iNOSwassignificantlyreducedintheLPS + SCDEsgroup(Fig. 2C). Meanwhile,itwasconfirmedbywesternblotwiththesignificantly lowexpressionlevelofiNOSintheLPS + SCDEsgroup(Fig. 2B,D). Immunofluorescencestainingwasperformedonthemacrophage markerofF4/80andtheM2phenotypemarkerofCD206(Fig. 2E), showingthattherelativemeanintensityofCD206wasobviously

enhancedintheLPS + SCDEsgroup(Fig. 2G).Meanwhile,itwas confirmedbywesternblotwiththesignificantlyhighexpression levelofCD206intheLPS + SCDEsgroup(Fig. 2F,H).Similarly, flow cytometrydetectedtheM1phenotypeofCD86+ (Fig. 2I,J)and theM2phenotypeofCD206+ (Fig. 2K,L),indicatingthatSCDE treatmentsuppressedM1polarizationandpromotedM2polarizationinLPS-stimulatedBMDMs.

InvivoSCDEsimprovedfunctionalrecoveryafterSCI

ToassessthefunctionalrecoveryafterSCI,theBBBscorewas observedbytwotrainedobserversat1daybefore,and1,3,7,14, 21,and28daysafterSCI.TheBBBscoreoftheSCI + SCDEsgroup wassigniﬁcantlyhigher(Fig. 3B,F).Theresultsofgaitanalysis showedthattherearlimbcoordinationoftheSCI + SCDEsgroup wasgreatlyimproved,especiallyintheregularityindex,print position,andstanceofthehindlimb(Fig. 3C,I).Meanwhile,the H&Estainingofthebladdershowedalesserthicknessofthe bladderintheSCIrattreatedwithSCDEs,whichmeantthatthe functionsofthebladderrecoveredfasterintheSCDEstreatment group(Fig. 3D,G).Toassesstheimprovementinnerve conduction,electrophysiologicalassaywasperformedonrats. TheMEPresultsshowedthattheMEPlatencywasshortenedand theamplitudeincreasedintheSCI + SCDEsgroup(Fig. 3E,H). TheseresultssuggestedthatSCDEspromotedthefunctional recoveryafterSCI.

TheroleofSCDEsinimprovingspinalcordrepairafterSCI InordertopreliminarilyexplorethemechanismofSCDEs repairingspinalcordinjury,immunofluorescencestainingofED1 (aspecificmarkerofactivatedmacrophages/microglia)was performed.Theresultsshowedthatthemacrophages/microglia infiltrationwasreducedintheSCI + SCDEsgroup(Fig. 4A,E). ImmunofluorescencestainingofGFAP(red)andNF-200(green)in eachgrouponday14afterSCIwasperformedtoevaluatethe injuredareaofthespinalcordandnervesurvivallevels(Fig. 4B), whichshowedthatafterSCDEstreatment,therecoveryoflesion volumeandtherelativemeanintensityofNF-200werebetter thanthoseoftheSCIgroup(Fig. 4F).Cholineacetyltransferase (ChAT)isthemostspecificindicatorformonitoringthefunctional statusofcholinergicneuronsintheCNS.SCDEssignificantly improvedmotorfunctionafterSCI,soimmunofluorescence stainingofChATwasperformed(Fig. 4C)andtheresultsshowed thattheChAT-positivecellssignificantlyincreasedinnumberafter thetreatmentofSCDEs(Fig. 4G).TheH&Estainingofthespinal cordshowedthatthedamagedareaintheSCIratwasreduced afterbeingtreatedwithSCDEs(Fig. 4D).

SCDEsmodulatedmacrophage/microgliapolarizationtowards M2phenotypeinvivo

PKH26-labeledSCDEswereinjectedviathetailveinandwere uptakenbymacrophage/microglialcellsinvivo(Fig. 5A).SCDEs canreducethenumberofinﬁltratedmacrophages/microglia invivo,butthepolarizationdirectionwasnotclear.Toclarifythe M1polarizationofmacrophages/microglia,immunoﬂuorescence stainingwasperformedonthemacrophage/microgliamarkerof ED1andtheM1phenotypemarkerofiNOS(Fig. 5B),which

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showedthatthepositivecellsofED1andiNOSweresignificantly reduced(Fig. 5D).Also,asverifiedbywesternblot,theexpression levelofiNOSintheSCI + SCDEsgroupwassignificantlylower (Fig. 5F).Ontheotherhand,toclarifytheM2polarization ofmacrophages/microglia,immunofluorescencestainingwas

performedonthemacrophage/microgliamarkerofED1andthe M2phenotypemarkerofCD206(Fig. 5C),whichindicatedthat thepositivecellsofED1andCD206weresigniﬁcantlyreducedin theSCI + SCDEsgroupandthattheproportionofCD206+ cells wasobviouslyincreased(Fig. 5E).Also,asveriﬁedbywesternblot,

Fig.5 TheeffectofSCDEsontheregulationofM1/M2polarizationinvivo.A RepresentativeimagesofPKH26(red)-labeledSCDEs absorbedbymacrophages/microgliainvivo.Scalebars:20µm.Therightimageswerehigh-resolutionversionsoftheboxedregionsintheleft images.Scalebars:2µm. B RepresentativeimmunofluorescencestainingimagesofiNOS(green)andED1(red).NucleiwerelabeledwithDAPI (blue)ineachgroup(n = 3).Scalebar:20 μm. D QuantitativeanalysisoftheED1+ cellsandiNOS+ cells(n = 3). F Representativewesternblots showingthereductionofiNOSinvivo(n = 3). C RepresentativeimmunofluorescencestainingimagesofCD206(green)andED1(red).Nuclei werelabeledwithDAPI(blue)ineachgroup(n = 3).Scalebar:20 μm. E QuantitativeanalysisoftheED1-positivecellsandCD206-positivecells (n = 3). G RepresentativewesternblotsshowingtheincreaseofCD206invivo(n = 3).Datawerepresentedasmean±SEM.Resultswere analyzedbyOne-wayANOVA.Significance:*P <0.05,**P <0.01,***P <0.001.

theexpressionlevelofCD206intheSCI + SCDEsgroupwas signiﬁcantlyhigher(Fig. 5G).

MFG-E8WastheKeyComponentofSCDEsinRegulating InﬂammatoryResponses

Exosomescontaindifferentbiologicallyactivemolecules,includingRNAs,proteins,andlipids.Inordertoexplorethekey componentsinexosomesrelatedtoregulatingtheinflammatory responses,weperformeddifferentialproteomicanalysisbetween SCDEsandSCsandscreenedoutMFG-E8asaproteinthatwas highlyexpressedinSCDEscomparedwithSCs(Fig. 6A).Toverify whetherMFG-E8playsakeyroleinregulatinginflammation,we knockedouttheMFG-E8inSCsvialentiviraltransfection,which wasprovedsuccessfulbyGFP fluorescence(green)(Fig. 6D).Next, weconfirmedthesuccessfulknockoutofMFG-E8inSCsvia westernblot(Fig. 6B,C).Inordertofurtherverifywhetherthe contentofMFG-E8proteininSCDEs(MFG-E8-KO)decreased, SCDEsextractedfromSCs(MFG-E8-KO)wereinjectedintothe ratsviathetailveinandimmunofluorescencestainingwas performedtoobservethechangesofthecontentofMFG-E8. Finally,theimmunofluorescencestainingofMFG-E8showedthat MFG-E8wassignificantlydecreasedintheSCDEs(shMFG-E8-KO) (Fig. 6E,F).

TheknockoutofMFG-E8suppressedtheM2polarization invitroandinvivo

WefurtheranalyzedtheregulatoryeffectofMFG-E8onmacrophage/microgliapolarizationinvitroandinvivo.Concerningthe modelofLPS-inducedinflammationinBMDMs,immunofluorescencestainingwasperformedonthemacrophagemarkerofF4/ 80,theM1phenotypemarkerofiNOS,andtheM2phenotype markerofCD206(Fig. 7A,B).Theresultshowedthattherelative meanintensityofthe fluorescenceofiNOSwassignificantlyhigher andtherelativemeanintensityofthe fluorescenceofCD206was significantlylowerintheLPS + SCDEs(shMFG-E8-KO)group (Fig. 7C,D).Thatistosay,theknockoutofMFG-E8attenuated theM2PolarizationofBMDMsinvitro.Similarly,immunofluorescencestainingwasperformedonthemacrophagemarkerofED1, theM1phenotypemarkerofiNOS,andtheM2phenotypemarker ofCD206invivo.(Fig. 7E,F).TheresultshowedthattheiNOS+ cellswerehigherandtheCD206+ cellswerelowerinthe SCI + SCDEs(shMFG-E8-KO)group(Fig. 7G,H).Thatistosay,the knockoutofMFG-E8attenuatedtheM2Polarizationinvivo.Also, flowcytometrydetectedCD86 + M1phenotype,indicatingthat MFG-E8knockoutpromotedM1polarization(Fig. 7I,J)while inhibitingM2polarization(Fig. 7K,L).

ImprovedInﬂammatoryMicroenvironmentInhibited NeuronalApoptosisinVitro

ToexplorewhetherSCDEsaffectneuronalapoptosisafterthe inﬂammatorymicroenvironmentisimproved,PC12cellswerecoculturedwithBMDMs(Fig. 8A).Afterbeingco-culturedfor24h, PC12cellswereisolatedforapoptosisassay.Theresultsofthe westernblotshowedthatSCDEsreducedtheexpressionof CleavedCaspase3andincreasedtheexpressionofBcl-2inPC12 cells,andthattheknockoutofMFG-E8reversedtheanti-apoptosis effect(Fig. 8B,E).Similarly, ﬂowcytometryalsodemonstratedthe

sameresult(Fig. 8C,F).TheTUNELstainingshowedthatSCDEs reducedTUNEL + cells,whileMFG-E8knockoutincreased TUNEL + cellsinPC12cells(Fig. 8D,G).

M2PolarizationInducedbyMFG-E8KnockoutinSCsWas UpregulatedviaSOCS3/STAT3SignalingPathway Inorderto ﬁndoutthedetailedmolecularmechanismofMFG-E8 inregulatingtheM2polarizationofmacrophages/microglia, westernblotwasperformed.ItwasfoundthattheSOCS3/ STAT3signalingpathwayplayedanimportantpartinupregulating M2polarizationinducedbyMFG-E8knockdown(Fig. 9).TheMFGE8inSCDEsreducedtheexpressionofiNOSandincreasedthe expressionofCD206,indicatingthatMFG-E8suppressedM1 polarizationandpromotedM2polarization,whileMFG-E8knockoutreversedtheeffectsofSCDEsinvitro(Fig. 9A,B).Similarresults wereveriﬁedinvivo(Fig. 9C,D).Meanwhile,itwasfoundthat MFG-E8couldreducetheexpressionofphosphorylatedSTAT3 whentheexpressionofSOCS3wasincreased,whileMFG-E8 knockoutcouldreduceSOCS3andincreasethephosphorylated STAT3invivo(Fig. 9C,E).

DISCUSSION

SCIisadevastatingdiseasethatcancauseseveredysfunctionof motor,sensory,andautonomicnerves,thusimposingahuge economicburdenandexertingagreataffectonthequalityoflife [1, 2].Therapies-influencedinflammatorycascadesarelikelyto benefitthefunctionalrecoveryafterSCI.Ourstudydemonstrates thatSCDEscanattenuatetheinflammatoryresponsebyregulatingmacrophage/microgliapolarization,reducingneuronalapoptosis,andpromotingthefunctionalrecoveryafterSCI.Moreover, MFG-E8isthekeycomponentofSCDEsinimprovinginflammation.KOofMFG-E8canpartlyreversetheanti-inflammatory effectsandpromoteM2polarizationofmacrophage/microgliavia theSOCS3/STAT3signalingpathway(Fig. 10).Theseresultscan enrichtheanti-inflammatorymechanismofSCDEstreatmentin repairingSCIandprovideanewpotentialandtherapeuticoption forrepairingSCI.

Exosomeshavebeenverifiedtopromoteneuronalrepairand growthandmodulateinflammationresponsesinmanystudies [29].Wehaverecentlypublishedthe findingthatSCDEscan promotethefunctionalrecoveryafterSCIbyincreasingautophagy anddecreasingapoptosis,inducingaxonalprotection,decreasing thedepositionofchondroitinsulfateproteoglycan(CSPG)and theirdetailedmolecularmechanism.Thatistosay,SCDEs transplantationhasapotentialapplicationprospectinthe treatmentofSCI[20, 21].Inourstudy,itwasevidentthatthe histologicallevelsinratsafterspinalcordinjurywererecovered aftertailveininjectionofSCDEs,especiallythebladdertissue function.AfterSCI,long-termdysuriaandurineretentionwilllead tothehypertrophyofthebladderwallwithahighratioof collagentomuscle[30, 31].Itwasfoundthatthethicknessofthe bladderwallafterSCIwascorrelatedwiththerecoveryofbladder function,andthatthemechanismmightcausechangesinaltered c-fiberefferentactivity[31–33]andtheexpressionofsome proteinreceptors[34–37].Ourelectrophysiologicalassayhas shownthatSCDEscanpromotetherecoveryofnerveconduction

functionafterSCIbyinhibitinginflammation,whichmayinvolve animprovementintheactivityofC-afferent fibers.Inaddition, somereceptorsonthebladderwallordetrusorplayanimportant roleininhibitingbladderwall fibrosis.Someofthekey

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componentscontainedinSCDEsadministeredthroughthetail veinarelikelytoactonsomereceptors(suchasVEGF,FGF,and PDGF)throughsystemicblood,thuspromotingtherecoveryof bladderfunction.However,furtherexperimentsarestillneededto

Fig.6 MFG-E8wasthekeycomponentofSCDEsintheregulationofinflammatoryresponses.A Thedifferentiallyexpressedproteins betweenSCDEsandSCswereanalyzedbyproteomics,andtheup-regulatedordown-regulatedproteinsofSCDEswerescreenedand comparedwithSCs. B RepresentativewesternblotsshowingtheknockoutofMFG-E8intheSCs(shNC)andSCs(shMFG-E8-KO)group. C QuantitativeanalysisoftheMFG-E8/VINCULINratiointheSCs(shNC)andSCs(shMFG-E8-KO)group(n = 3). D Representative fluorescence imagesofSCs(green)aftertheMFG-E8genewasknockedout.Scalebar:100 μm. E Representativeimmunofluorescencestainingimagesof Iba-1(red)andMFG-E8(green).NucleiwerelabeledwithDAPI(blue)ineachgroup(n = 5).Scalebar:20 μm. F Theimageanalysisresultswere presentedastherelativemeanintensityofthe fluorescenceofMFG-E8.Datawerepresentedasmean±SEM.Resultswereanalyzedby Student ’st-testandOne-wayANOVA.Significance:**P <0.01,***P <0.001. J.Renetal.

verifytheexactmechanism.Agrowingnumberofstudieshave confirmedthatexosomeshaveapositiveeffectinreducing inflammation[38–40].However,fewstudieshavebeenfocusedon thekeyproteininSCDEsthatiscloselyrelatedtoinflammation inhibitionanditsspecificmechanismforSCIrepair.Thepresent studyhasidentifiedMFG-E8asthekeycomponentofSCDEsin improvingtheanti-inflammatoryeffectsandpromotetheM2 polarizationofmacrophage/microgliacausedbySCDEstreatment. MFG-E8isasecretedmultifunctionalglycoprotein.Itcanbe expressedinvariouscellsandplayanimportantroleintreating differentdiseases[ 41 ].Anincreasingamountofresearchhas foundthatMFG-E8playsaneuroprotectiverolebyinhibiting neuronalapoptosisandreversinganti-in fl ammatoryeffectsin variousneurodegenerativedisea sesandtraumaticbraininjury [ 42 ].MFG-E8caninhibitthereleaseofpro-in fl ammatory mediatorstoregulateimmuneresponsesandpromotethe reprogrammingofmicroglial ,andconvertthemicroglia phenotypetotheM2phenotype[ 25 – 28 ].ElodieSeguraetal. haverevealedthatmaturedendriticcellscanalsosecrete exosomescontainingMFG-E8byconductingproteomicand biochemicalanalyses[ 43 ].However,ithasnotbeenstudied thatSchwanncell-derivedexosomalMFG-E8acrosstheBBBis appliedforSCI[ 24 ].Herein,ithasbeenveri fi edthatMFG-E8is thekeycomponentofSCDEsinimprovingthein fl ammatory responsebyproteomicsequenc ing.Besides,SCDEstreatment withMFG-E8knockouthasattenuatedtheanti-in fl ammatory effectwithahighernumberofED1-positivecellsand suppressedtheM2polarizationwithalowernumberof CD206-positivecells.Similarly,Shietal.havefoundthatMFGE8couldregulatethealteration ofmicrogliaM1/M2phenotypes inAD[ 27 ].Wuetal.HavefoundthatM2phenotypemarkeris reducedandM1phenotypemarkerisincreasedafterMFG-E8is knockedoutinmicroglia[ 28 ].Neuronalapoptosisisanother importantmolecularpathologicalfeatureinSCI[ 2 ].The interactionsbetweenmicroenvironmentalin fl ammation responsesandneuronalapoptosisarecomplex[ 44 ].Many studieshaveshownthatexosomescanalsoplayarolein inhibitingapoptosis[ 45 ].OurstudyhasprovedthatSCDEscan inhibitapoptosisandtheknockoutofMFG-E8canreversethe positiveeffectinvivoandinv itro,furtherexplainingthe mechanismofSCDEsinrepairingSCIfromanotherperspective. MacrophagepolarizationinCNShasemergedduringthepast decadesandislargelygeneratedbythelocalmicroenvironmental signals[46].Itmeansthatmacrophagesareabletoswitchfrom pro-inflammatoryM1phenotypetoanti-inflammatoryM2phenotype[5, 6].M2macrophagesareconsideredtobecriticalin attenuatingtheinflammationresponseandpromotingtissue remodeling.Thetargetedinterventionshaveagreatapplication prospectforthetreatmentofSCI[9],suchasblockingthe recruitmentandproliferationofmacrophages,inhibiting theactivationpathwayofM1phenotype,andpromotingthe transformationtoM2macrophages.Apreviousstudyhasfound thatincreasedexpressionofMFG-E8candownregulatethe phosphorylationofSTAT3andupregulatetheexpressionofSOCS3, thenpromoteM2polarizationandexertananti-inflammatory effectaftersubarachnoidhemorrhage[47].Nevertheless,itisstill unclearwhetherthemechanismofMFG-E8inSCDEsmediatesthe

shiftofmacrophage/microgliatoM2afterSCI.Inourstudy,theKO ofMFG-E8inSCDEsmayinducetheactivationofSOCS3and interferewiththephosphorylationofSTAT3tostimulateM2 polarizationandreduceinﬂammation.

Thereareseverallimitationstoourstudy.Thisresearchis mainlyfocusedonthein fl ammationmodulationofSCDEsfor SCItreatmentwithoutanalyzingtheneuralproliferationand differentiationandaxonalrege neration.Inaddition,thestudy hasonlycon fi rmedthatMFG-E8caninduceM2polarization withoutexploringthemultipleactivatedM2subtypes,includingM2a,M2b,andM2c.Furtherstudiesinvolvedinknockout rats/micearerequiredtoverifytheabove fi ndings,sinceonly shRNAswereusedtoknockoutMFG-E8inourstudy.

Inconclusion,SCDEscanexertananti-inflammatoryeffectby inhibitingM1polarizationandpromotingM2polarizationof macrophage/microglia,andreduceneuronalapoptosisand improvetherecoveryofmotorfunctionafterSCI,whichmaybe ascribedtotheMFG-E8inSCDEsmediatedbyregulatingSOCS3/ STAT3signalingpathway.These findingscanenrichtheantiinflammatorymechanismofSCDEsinrepairingSCIandprovide potentialapplicationprospectsandnewinsightsfortheclinical translationofSCDEsforSCI.

MATERIALSANDMETHODS

Preparationofanimalsandexperimentalgroups

AlladultfemaleWistarrats(thebodyweightrangingfrom200to220g, n = 200)weresuppliedbyBeijingVitalRiverLaboratoryAnimal TechnologyCo.,Ltd.(Beijing,China,PermissionNumber:SCXK(Jing)2016-0011).AllfemaleWistarratswerehousedinthesameenvironments(thetemperaturerangingfrom22to24°C,thehumidityranging from60to80%).Allanimalexperimentalprotocolswereapprovedby theEthicsCommitteeoftheInstituteofRadiationMedicine,Chinese AcademyofMedicalSciences(Tianjin,China,ApprovalNumber:IRMDWLL-2021180).Ratswererandomlydividedintothefollowinggroups: Sham,SCI,SCI + SCDEs,SCI + SCDEs(shMFG-E8-KO),andSCI + SCDEs (shNC).Thetreatmentgroupswereinjectedwith250uLofSCDEs(0.1 ug/uL)transfectedwithlentivirus,andtheSCIgroupwasinjectedwith thesamevolumeofDPBS.Halfanhourafterthespinalcordinjury modelwasestablished,SCDEswereinjectedthroughthetailveinof rats.Weinjectedratsusedintheacutephase(3days)withSCDEsonce aday.Ratsinthesubacute(2weeks)andpostacutephases(4weeks) wereinjectedwithSCDEsviathetailveinthreetimesperweek.

Cellculture

BonemarrowwasextractedfromthefemurandtibiaofafemaleWistar rat.ItwasincubatedintheinductionmediumcontainingDMEM/F-12 (Gibco,#11320033),FBS(10%,Gibco,#10099141C),penicillin/streptomycin(1%,Gibco,#15070063),andM-CSF(20ng/ml,#SRP3247,SigmaAldrich)at37°Cina5%CO2 incubatorfor7days.After7daysofinduction, BMDMscouldbedeﬁnedasM0macrophagesandsetasthecontrolgroup, whichcouldbeusedforsubsequentexperiments.M0macrophageswere incubatedundertheconditionofLPS(1000ng/ml,#L3129,Sigma-Aldrich) for24handsetastheLPSgroup.M0macrophageswereincubatedunder theconditionsofLPSandSCDEs(2µg/ml)for24handsetasthe SCI + SCDEsgroup.

PrimarySCswereextractedfromthesciaticnerveofrats.Thesciatic nervewasisolatedandminced,digestedwith0.3%oftypeIIcollagenase (Solarbio,#C8150,Beijing,China)andTrypLE(Gibco,#2277057,Denmark). ThecellswereculturedinSCpuriﬁcationmedium(10%ofFBS,10 μMof

arabinosidehydrochloride(Ara-C,Solarbio,#C1768,Beijing,China),1%of penicillin/streptomycin,DMEM/F-12)at37°Cina5%CO2 incubatorfor 24htoremove ﬁbroblasts.ThenSCgrowthmediumwasusedat37°Cina 5%CO2 incubatortocontinuetheculture.

CellDeathandDisease(2023)14:70

PC12cellswereobtainedfromShanghaiInstitutesforBiologicalScience, ChineseAcademyofSciences.Thecellsweregrowninamedium containingDMEM(Gibco,#12430054),FBS(10%),andpenicillin/ streptomycin(1%)at37°Cina5%CO2 incubatorculturedunder

Fig.7 MFG-E8knockoutsuppressedtheM2polarizationinvitroandvivo.A RepresentativeimmunofluorescencestainingimagesofiNOS (red)andF4/80(green)invitro.NucleiwerelabeledwithDAPI(blue)ineachgroup(n = 5).Scalebar:20 μm. B Representative immunofluorescencestainingimagesofCD206(red)andF4/80(green)invitro.NucleiwerelabeledwithDAPI(blue)ineachgroup(n = 5). Scalebar:20 μm. C, D TheresultsoftheiNOSandCD206relativemeanintensityofthe fluorescence(n = 5). E Representative immunofluorescencestainingimagesofiNOS(green)andED1(red)invivo.NucleiwerelabeledwithDAPI(blue)ineachgroup(n = 3).Scale bar:20 μm. F RepresentativeimmunofluorescencestainingimagesofCD206(green)andED1(red)invivo.NucleiwerelabeledwithDAPI (blue)ineachgroup(n = 3).Scalebar:20 μm. G, H QuantitativeanalysisofthepositivecellsofED1,iNOS,andCD206(n = 3). I, J Flow cytometryassaydetectedCD86+/CD11b + M1phenotype,indicatingthatMFG-E8knockoutpromotedtheM1polarization(n = 3). K, L Flow cytometryassaydetectedCD206+/CD11b + M2phenotype,showingthatMFG-E8knockoutinhibitedtheM2polarization(n = 3).Datawere presentedasmean±SEM.ResultswereanalyzedbyOne-wayANOVA.Significance:*P <0.05,**P <0.01,***P <0.001.

conditions.TostudytheeffectofBMDMspolarizationonneuronal apoptosis,weco-culturedBMDMswithPC12cellsina0.4 μm-pore Transwell® coculturesystem(Corning,USA).BMDMswereseededinthe upperchamber(1×10^5perchamber),whichwerethentreatedwithPBS, LPS,LPS + SCDEs,LPS + SCDEs(shNC),orLPS + SCDEs(shMFG-E8-KO)for 24hours.TheinsertsweregentlywashedthreetimeswithPBSandplaced abovethePC12cells(1×10^5perwell)in24-wellplates.Finally,PC12 cellswerecollectedafter48hoursforapoptosisanalysis.

Thepreparation,puriﬁcation,andidentiﬁcationofSCDEs

WhenSchwanncellswereculturedtotheP3-P5,andexosome-freeserum wasusedtopreparetheSchwanncellmedium.Duringthecellculture process,themediumwaschangedeverythreedays.Themediumwas collectedintothecentrifugetubeandcentrifugedat4°Cand300× g for 5mintoremovedeadcellsfromthemedium.Then,thesupernatantofthe mediumwasplacedat 20°Cforuse.AftertheSchwanncellculture mediumwascollected,Schwanncellexosomeswereobtainedthrough thisexperimentthroughultracentrifugation.Inthe firststep,centrifugation wasperformedat4°Cand2000× g for20mintoremovecellresiduesand organelles,andthesupernatantwascollected.Inthesecondstep, centrifugationwasperformedat4°Cand10000× g (Centrifuge5810R, Eppendorf,Germany)for60min.Subsequently,thecollectedsupernatant was filteredwitha0.22-μmcell filter,andthesupernatantwascollectedfor thethirdtime,centrifugedat130,000×gandat4°C(BeckmanOptimal100XP,BeckmanCoulter,Germany)for70min.Afterthe finalultracentrifugation,SCDEswereresuspendedinsterilewaterforthefollowing experiments.Thetotalproteinconcentrationofexosomeswasquantified byBCAassay(ThermoScientific,#23225MA,USA).Afteraseriesofsample preparationprocesses,themorphologyofexosomeswasobservedby usingahigh-resolutiontransmissionelectronmicroscope(TEM,Hitachi HT7700,Tokyo,Japan).TheSCDEssuspensionwasdiluted1000-fold,and thesampleswereexaminedbyNanoSight(Malvern,UK,NS300)at25°C. Westernblotwasperformedtoidentifyspecificexosomesurfaceprotein markers,suchasAlixCD9andCD63.

SCDEsuptakenbyBMDMs

AccordingtotheinstructionsofthereagentinstructionsofthePKH26Red FluorescentCellLinkerMiniKit(MINI26-1KT,#MKCM1863,Sigma-Aldrich), SCDEswereincubatedwithexcessdyeatroomtemperaturefor4hand thenremovedbyultracentrifugationat100,000× g for1h.AfterbeingcoculturedwithPKH26-labeledexosomesfor16h,BMDMswere ﬁxed, penetrated,blocked,andincubatedwithCD11bantibody(1:400;Abcam, #1211MA,USA)overnightat4°C,andthenincubatedwithsecondary antibody.Afterbeingwashed,theslideswereremovedfromthe24-well plateandmountedwithMountingMediumwithDAPI(Abcam,#104139, MA,USA).PKH26-labeledSCDEsbeinguptakenbyprimarybonemarrowderivedmacrophageswasobservedbytheultra-highresolutionlaser confocalmicroscope(ZEISSLSM900,Germany).

Lentivirusproductionandcelltransfection

LV-MFGE8-EGFPinhibitorvectorwasconstructedfromthelentiviralvector (GeneChem,shanghai,China).NegativecontrolwasconstructedbyLV emptylentivirus(LV-NC-EGFP).TheshorthairpinRNA(shRNA)sequences ofMFG-E8protein(shRNA-MFG-E8)andnegativecontrol(shRNA-NC)were designedandsynthesizedbyGeneChem(Shanghai,China).HitransGP InfectionEnhancementReagent(25×)wasusedtotransfectcellsatthe multiplicityofinfection(MOI)valueof50.AftertheculturedprimarySCs wereinfectedwiththeselentiviruses,theSCscellgrowthmediumwas replaced.Theculturewascontinuedfor72htoobservethetransfection efficiency.Brightfieldand fluorescenceimagesweretakenwitha

ﬂuorescencemicroscope.Theknockoutefﬁciencyofthetargetprotein wasevaluatedviawesternblot.

Contusionmodelofspinalcordinjuryinrats

Themodi fi edAllenmethodwasusedtoconstructthecontusionmodel ofSCIinrats,andstabilityanduniformityofthemodelwerewell maintained[48].Afterbeingweighed,theratswereanesthetizedwith 5%ofiso fl urane(RWDlifescience,Shenzhen,China).AdorsallaminectomywasperformedontheT10vertebralbodytoexposethespinalcord. Theimpactbarwasplacedonthespinalcord,anda10gnode(the diameterof2.5mm)wasfreelydroppedfromaheightof2.5cmto createcontusioninjurywithNYUImpactorModelIII(W.M.KeckCenter forCollaborativeNeuroscience,Rutgers,theStateUniversityofNew Jersey,UnitedStates).Afterhemostasis,themuscleandskinatthe incisionsiteissutured.SuccessfulSClexhibitsthefollowingcharacteristicsintherats:formationoftailswayre fl ex,spinalcordischemia,leg swings,andparalysis [49].Ifratsdidnotshowtheabovesymptomsaftera singleblowandthemotorfunctionoftheirhindlimbremainednormal ontheseconddayaftersurgery,theywereconsideredunsuccessfulin theconstructionofaspinalcordinjurymodelandwouldbeexcluded fromthescopeofthisstudy.Within3daysafterSCI,cefuroximesodium wasusedtopreventwoundinfection.Within7daysafterSCI,the bladderwassqueezedthrougharti fi cialassistancetovoid3timesaday untilthere fl exivecontrolofbladderfunctionrecovered.

HematoxylinandEosin(H&E)staining

Aftertheparafﬁnsectionswereprepared,theywereheatedinaconstant temperatureovenat60°Cfor4h.Then,thesectionswereplacedinxylene IandIIfor10minandplacedinalcoholsolutionswithdifferent concentrations,including100%,95%,90%,80%,and70%,for5mineach time,andrinsedindistilledwaterfor1min.Thesectionswerestainedwith thehematoxylin(Solarbio,#G1120,Beijing,China)stainingsolutionfor 2min,differentiatedwiththedifferentiationsolution(1%ofhydrochloric acid),washedindistilledwateragainfor2min,andthendyedwith0.5%of theeosinstainingsolution(Solarbio,#G1120,Beijing,China)for1min. Finally,inordertocompletethedehydration,thetissuesectionswere successivelyplacedin70%,80%,90%,and100%gradientethanolsolution. TheslideswereplacedinxyleneIandIIfor5minandweresealedwith neutralresin(Solarbio,#G8590,Beijing,China).Sampleswereobserved andphotographedonafullyautomatedtissueinsitumulti-label landscapequantiﬁcationanalyzer(VectraPolaris,PerkinElmer).

Behavioralevaluation

TheBBBfunctionalscorewasevaluatedtoassesstherecoveryofmotor function.Thescorewasevaluatedatthesametimebeforethesurgery,on thedayaftersurgery,onday3,onday7,andonthesamedayeveryweek. Thetestwasperformedbytwotrainedresearchers(blindedtotreatment) toobserveandassessthemotorfunctionoftherats.TheCatwalk-assisted gaitanalysis(NoldusInformationTechnologyB.V,Netherlands)was performedtoevaluatethegaitdynamics.Onthe28thdayafterSCDEs treatment,theCatWalksystemwasusedtotesttheratsineachgroup.The gaitparameterswereautomaticallycalculatedbytheanalysissoftware (CatWalkXT10.6).Thisexperimentmainlyevaluatedtheeffectofthe followinggaitparametersonrelatedbehavioralchangesafterSCI, includingregularityindex,printposition,andstancesofthehindlimb. Anelectrophysiologicaldevice(YRKJ-G2008;ZhuhaiYiruikejiCo,Ltd, Guangdong,China)wasusedtoanalyzemotorevokedpotential(MEP)in ratsfourweeksafterSCItoevaluatetherecoveryofnerveconduction function.ThelatencyandamplitudeofMEPwererecordedandanalyzedin theexperiment.

Fig.8 Improvedinflammatorymicroenvironmentinhibitedneuronalapoptosis.A Schematicrepresentationofco-cultureofPC12cellsand BMDMs. B, E RepresentativewesternblotsofCleavedCaspase-3andBcl-2inPC12cells.QuantitativeanalysisoftheCleavedCaspase-3/ GAPDHratioandBcl-2/GAPDHratioinPC12cells(n = 3). C, F FlowcytometryassaydetectedapoptosisofPC12cellsineachgroup,showing thatSCDEsinhibitedLPS-inducedapoptosisandthattheknockoutofMFG-E8reversedthepositiveeffect(n = 3). D, G TheTUNELstainingof PC12cellsshowedthatSCDEsreducedTUNEL + cellswhileMFG-E8knockoutincreasedTUNEL + cells(n = 3).Scalebar:50 μm.Datawere presentedasmean±SEM.ResultswereanalyzedbyOne-wayANOVA.Significance:ns-notsignificant,*P <0.05,**P <0.01,***P <0.001.

CellDeathandDisease(2023)14:70

Fig.9 M2polarizationinducedbyMFG-E8inSCDEswasupregulatedviaSOCS3/STAT3signalingpathway.A Representativewesternblots showediNOSwasreduced,andCD206wasincreased,whileMFG-E8knockoutreversedtheeffectsofSCDEsinvitro. B Quantitativeanalysisof theiNOS/GAPDHandCD206/GAPDHratio(n = 3). C RepresentativewesternblotsshowingthereductionofiNOSandtheincreaseofCD206, whileMFG-E8knockoutreversedtheeffectsofSCDEsinvivo.RepresentativewesternblotsshowedthatSOCS3/STAT3signalingwasactivated viaMFG-E8inSCDEs,whileMFG-E8knockoutreducedtheSOCS3andincreasedtheSTAT3invivo. D QuantitativeanalysisoftheiNOS/GAPDH andCD206/GAPDHratioinvivo(n = 3). E QuantitativeanalysisoftheMFG-E8/GAPDH,SOCS3/GAPDH,andSTAT3/GAPDHratioandCD206/ GAPDHratioinvivo(n = 3).Datawerepresentedasmean±SEM.ResultswereanalyzedbyOne-wayANOVA.Signiﬁcance:ns-notsigniﬁcant, *P <0.05,**P <0.01,***P <0.001.

Westernblot

TheproteinconcentrationwasmeasuredbyusingtheBCAassay (ThermoScienti ﬁ c,#23225,MA,USA).Targetproteinswereseparatedby

12%SDS-PAGEandthentransferredtoaPVDFmembrane.Afterbeing blockedwith5%nonfatmilkinTBST,thePVDFmembranewas incubatedwiththeprimaryantibodyovernightandthenincubated

Fig.10 ThemechanismofSCDEscontainingMFG-E8modifymacrophage/microglialpolarizationthroughSOCS3/STAT3pathwayafter SCI. SpinalcordinjuryinducedactivationofSTAT3phosphorylationinmacrophages/microglia,ledtoM1polarizationandexacerbated inflammatorydamageatthesiteofspinalcordinjury.AftertreatmentwithSCDEs,theywerephagocytosedbymacrophages/microgliaatthe injurysiteandbegantoreleaseMFG-E8.ThisincreasedtheproteinlevelsofSOCS3,whichinhibitedthephosphorylationofSTAT3topromote theconversionofthemacrophage/microgliaphenotypetoM2foralleviatinginflammatorydamage.Moreover,theimprovedinflammatory microenvironmentattheSCIsitereducedneuronalapoptosisandpromotednerveregeneration.

withthesecondaryantibody.Proteinbandswerevisualizedbyusing ECLreagents(ThermoScienti ﬁ c,#35050,MA,USA)andexposedby usingtheChemiDocXRSSystem(BioRad,USA).Quantitativeanalysis wasperformedwithImageJsoftware.

Theantibodieswerelistedasfollows:rabbitanti-CD9(1:1000;Cell SignalingTechnology,#98327,USA),rabbitanti-Alix(1:1000,CellSignaling Technology,#92880,USA),rabbitanti-CD63(1:200;SantaCruz,#sc-5275, USA),rabbitantiCleavedCaspase-3(1:1000;CellSignalingTechnology, #9661,USA),mouseanti-Bcl-2(1:1000;Abcam,#196495,USA),rabbitantiiNOS(1:1000;Abcam,#178945,MA,USA),rabbitanti-mannosereceptor (1:1000;Abcam,#300621,MA,USA),mouseanti-MFG-E8(1:500,SantaCruz, #sc-271574,USA),rabbitanti-SOCS3(1:1000;Abcam,#280884,MA,USA), rabbitanti-p-STAT3(1:1000;CellSignalingTechnology,#9145,USA),rabbit anti-STAT3(1:1000;CellSignalingTechnology,#12640,USA),rabbitantiglyceraldehyde3-phosphatedehydrogenase(GAPDH)(1:1000;Abcam, #181602,MA,USA),mouseanti-vinculin(1:10000,Proteintech,#66305-1Ig),anti-rabbitIgG,HRP-linkedAntibody(1:1000;CellSignalingTechnology,#7074,USA),anti-mouseIgG,HRP-linkedAntibody(1:1000;Cell SignalingTechnology,#7076,USA).

Immunoﬂuorescencestaining

Concerningimmunoﬂuorescencestainingofcells:cellswere ﬁxed, penetrated,andblocked.Thentheywereincubatedwithprimaryantibody at4°Covernightandwithsecondaryantibody.PC12cellswereincubated withTUNELreactingmixturefromtheTUNELapoptosisdetectionkit (Roche,Germany)for1h.Finally,thecellswereobservedwiththeultrahighresolutionlaserconfocalmicroscope(ZEISSLSM900,Germany).

Concerningimmunoﬂuorescencestainingofspinalcordtissue,thespinal cordsegmentcontainingtheinjurysitewastakenand ﬁxedwith4% paraformaldehydeovernight.Thespinalcordtissuesweredehydratedin 30%sucrosesolutionfor3days,embeddedwithOCT,andcutinto10-µm thickslices.Thefrozenspinalcordsectionswerepermeatedandblocked. Thetissuesectionswereincubatedwithprimaryantibodiesandsecondary

antibodies.Finally,thetissuesectionswereobservedwiththeultra-high resolutionlaserconfocalmicroscope(ZEISSLSM900,Germany).

Theusedprimary/secondantibodiesarelistedasfollows:rabbitantiiNOS(1:200;Abcam,#178945MA,USA),rabbitanti-mannosereceptor (1:100;Abcam,#64693,MA,USA),ratanti-F4/80(1:400;Abcam,#300421, MA,USA),mouseanti-GFAP(1:200;CellSignalingTechnology,3670,USA), rabbitanti-CD68(1:200;Abcam,#125212,MA,USA),mouseanti-CD68/ED1 (1:200;Abcam,#31630,MA,USA),rabbitanti-Iba-1(1:200;Abcam,#178846, MA,USA),mouseanti-MFG-E8(1:100,SantaCruz,#sc-271574,USA),rabbit anti-neuroﬁlamentheavypolypeptide(1:200;Abcam,#8135MA,USA), rabbitanti-cholineacetyltransferase(1:200;Abcam,#181023,MA,USA), goatanti-rabbitIgGH&LAlexaFluor488(1:400,Abcam,#150077,MA, USA),goatanti-rabbitIgGH&LCy3(1:400,Abcam,#6939,MA,USA),goat anti-mouseIgGH&LAlexaFluor488(1:400;Abcam,#150113,MA,USA), andgoatanti-mouseIgGH&LCy3(1:400;Abcam,#97025,MA,USA).

Flowcytometry

ThetreatedBMDMscellswere ﬁlteredthrougha70-μ mcellstrainerto obtainasinglecellsuspension,thenwereincubatedwithFITCanti-rat CD11bantibody(Biolegend,#201805,USA)andPEanti-ratCD86 antibody(Biolegend,#200308,USA)for30min.Cellswerepermeabilized byCyto-Fast™ Fix/PermBufferSet(Biolenged,#426803,USA)for15min. Subsequently,cellswereincubatedwithCD206(MannoseReceptor) antibody(SantaCruz,#sc-58986,USA)for30minatroomtemperaturein thedarkandwereincubatedwithgoatanti-mouseIgGH&L(AlexaFluor® 647/APC)(Abcam,#150115,MA,USA).BDLSRFortessa ﬂ owcytometer(BD Biosciences,US)wasusedforsamplecollection,andFlowJowasusedfor dataanalysis.

ThetreatedPC12cellswereoperatedaccordingtothestaining instructionsofFITCAnnexinVapoptosisdetectionkitI(BDBiosciences, #556547,USA),andthecellapoptosisratewasdetectedimmediatelybya BDFACSVerseanalytical ﬂowcytometer(BDBiosciences,US)afterstaining, anddataanalysiswasperformedbyusingFlowJo.

Statisticalanalysis

GraphPadPrism9.2.0software(GraphPadSoftware,SanDiego,CA,USA)was usedforstatisticalanalysis.Dataanalysisbetweendifferentgroupswascarried outviaStudent’st-testandone-wayanalysisofvariance(ANOVA),whichwere followedbyTukeymultiplecomparisonposthoctest.Thelevelofsigniﬁcant differencebetweengroupswasdeﬁnedas:*p <0.05,**p <0.01,***p <0.001, ****p <0.0001.Eachexperimentwasrepeatedatleastthreetimes,andthe resultswereshownasthestandarderror±mean(SEM).

DATAAVAILABILITY

Theauthorsconﬁrmthatthedatasupportingtheconclusionsinthepaperare presentedinthearticleanditsSupplementaryMaterial.Additionaldatarelatedto thispaperareavailablefromthecorrespondingauthor.

REFERENCES

1.GBD2016TraumaticBrainInjuryandSpinalCordInjuryCollaborators.Global, regional,andnationalburdenoftraumaticbraininjuryandspinalcordinjury, 1990-2016:asystematicanalysisfortheGlobalBurdenofDiseaseStudy2016. LancetNeurol.2019;18:56 –87.

2.HuangH,ChenL,MovigliaG,SharmaA,AlZoubiZM,HeXJ,etal.Advancesand prospectsofcelltherapyforspinalcordinjurypatients.JNeurorestoratology. 2022;10:13 –30.

3.ParkJ,DeckerJT,MargulDJ,SmithDR,CummingsBJ,AndersonAJ,etal.Local immunomodulationwithanti-inﬂammatorycytokine-encodinglentivirus enhancesfunctionalrecoveryafterspinalcordinjury.MolTher.2018;26:1756–70.

4.KobashiS,TerashimaT,KatagiM,NakaeY,OkanoJ,SuzukiY,etal.TransplantationofM2-deviatedmicrogliapromotesrecoveryofmotorfunctionafterspinal cordinjuryinmice.MolTher.2020;28:254–65.

5.MaSF,ChenYJ,ZhangJX,ShenL,WangR,ZhouJS,etal.AdoptivetransferofM2 macrophagespromoteslocomotorrecoveryinadultratsafterspinalcordinjury. BrainBehavImmun.2015;45:157–70.

6.KigerlKA,GenselJC,AnkenyDP,AlexanderJK,DonnellyDJ,PopovichPG. Identiﬁcationoftwodistinctmacrophagesubsetswithdivergenteffectscausing eitherneurotoxicityorregenerationintheinjuredmousespinalcord.JNeurosci. 2009;29:13435–44.

7.MosserDM,EdwardsJP.Exploringthefullspectrumofmacrophageactivation [publishedcorrectionappearsinNatRevImmunol.2010;10(6):460].NatRev Immunol.2008;8(12):958–69.

8.DavidS,KronerA.Repertoireofmicroglialandmacrophageresponsesafter spinalcordinjury.NatRevNeurosci.2011;12:388–99.

9.GenselJC,DonnellyDJ,PopovichPG.Spinalcordinjurytherapiesinhumans:an overviewofcurrentclinicaltrialsandtheirpotentialeffectsonintrinsicCNS macrophages.ExpertOpinTherTargets.2011;15:505–18.

10.GenselJC,ZhangB.Macrophageactivationanditsroleinrepairandpathology afterspinalcordinjury.BrainRes.2015;1619:1–11.

11.KalluriR,LeBleuVS.Thebiology,function,andbiomedicalapplicationsofexosomes.Science2020;367:eaau6977.

12.TkachM,ThéryC.Communicationbyextracellularvesicles:whereweareand whereweneedtogo.Cell2016;164:1226–32.

13.ELAndaloussiS,MägerI,Breake ﬁ eldXO,WoodMJ.Extracellularvesicles: biologyandemergingtherapeutico pportunities.NatRevDrugDiscov. 2013;12:347 – 57.

14.TerstappenGC,MeyerAH,BellRD,ZhangW.Strategiesfordeliveringtherapeuticsacrosstheblood-brainbarrier.NatRevDrugDisco.2021;20:362–83.

15.SunGD,LiGQ,LiD,HuangWJ,ZhangRW,ZhangH,etal.hucMSCderived exosomespromotefunctionalrecoveryinspinalcordinjurymiceviaattenuating inﬂammation.MaterSciEngCMaterBiolAppl.2018;89:194–204.

16.JessenKR,MirskyR,LloydAC.Schwanncells:developmentandroleinnerve repair.ColdSpringHarbPerspectBiol.2015;7:a020487.

17.WoodhooA,SahniV,GilsonJ,FranklinRJM,BlakemoreWF,MirskyR,etal. Schwanncellprecursors:afavourablecellformyelinrepairintheCentralNervousSystem.Brain2007;130:2175–85.

18.ChingRC,WibergM,KinghamPJ.Schwanncell-likedifferentiatedadiposestem cellspromoteneuriteoutgrowthviasecretedexosomesandRNAtransfer.Stem CellResTher.2018;9:266.

19.WongFC,YeL,DemirIE,KahlertC.Schwanncell-derivedexosomes:Janus-faced mediatorsofregenerationanddisease.Glia2022;70:20 –34.

20.PanDY,ZhuSB,ZhangWX,WeiZJ,YangFH,GuoZL,etal.Autophagyinducedby Schwanncell-derivedexosomespromotesrecoveryafterspinalcordinjuryin rats.BiotechnolLett.2022;44:129–42.

21.PanDY,LiYJ,YangFH,LvZH,ZhuSB,ShaoYX,etal.Increasingtoll-likereceptor2 onastrocytesinducedbySchwanncell-derivedexosomespromotesrecoveryby

inhibitingCSPGsdepositionafterspinalcordinjury.JNeuroinﬂammation. 2021;18:172.

22.RenYF,LiuWM,ZhangL,ZhangJ,BiJB,WangT,etal.MilkfatglobuleEGFfactor 8restoresmitochondrialfunctionviaintegrin-medicatedactivationoftheFAKSTAT3signalingpathwayinacutepancreatitis.ClinTranslMed.2021;11:e295.

23.LiED,NodaM,DoiY,ParajuliB,KawanokuchiJ,SonobeY,etal.Theneuroprotectiveeffectsofmilkfatglobule-EGFfactor8againstoligomericamyloid β toxicity.JNeuroinﬂammation.2012;9:148.

24.CheyuoC,AzizM,WangP.Neurogenesisinneurodegenerativediseases:roleof MFG-E8.FrontNeurosci.2019;13:569.

25.DeroideN,LiX,LerouetD,VréEV,BakerL,HarrisonJ,etal.MFGE8inhibits inﬂammasome-inducedIL-1β productionandlimitspostischemiccerebralinjury. JClinInvest.2013;123:1176–81.

26.LiuF,ChenYJ,HuQ,LiB,TangJJ,HeY,etal.MFGE8/Integrin β3pathway alleviatesapoptosisandinﬂammationinearlybraininjuryaftersubarachnoid hemorrhageinrats.ExpNeurol.2015;272:120–7.

27.ShiXL,CaiXY,DiW,LiJ,XuXT,ZhangAW,etal.MFG-E8selectivelyinhibited Aβ-inducedmicroglialM1polarizationviaNF-κBandPI3K-Aktpathways.Mol Neurobiol.2017;54:7777–88.

28.WuJ,YangHC,ChengJJ,ZhangL,KeYL,ZhuY,etal.Knockdownofmilk-fat globuleEGFfactor-8suppressesgliomaprogressioninGL261gliomacellsby repressingmicroglialM2polarization.JCellPhysiol.2020;235:8679–90.

29.BahramSanganiN,GomesAR,CurfsLMG,ReutelingspergerCP.Theroleof extracellularvesiclesduringCNSdevelopment.ProgNeurobiol.2021;205:102124.

30.NagatomiJ,DeMiguelF,TorimotoK,ChancellorMB,GetzenbergRH,SacksMS. Earlymolecular-levelchangesinratbladderwalltissuefollowingspinalcord injury.BiochemBiophysResCommun.2005;334:1159–64.

31.OzsoyO,OzsoyU,SteinG,SkourasE,SchempfG,WellmannK,etal.Functional deﬁcitsandmorphologicalchangesintheneurogenicbladdermatchtheseverity ofspinalcordcompression.RestorNeurolNeurosci.2012;30:363–81.

32.KarnupSV,deGroatWC.PropriospinalneuronsofL3-L4segmentsinvolvedin controloftheratexternalUrethralSphincter.Neuroscience2020;425:12–28.

33.WadaN,KarnupS,KadekawaK,ShimizuN,KwonJ,ShimizuT,etal.Current knowledgeandnovelfrontiersinlowerurinarytractdysfunctionafterspinalcord injury:basicresearchperspectives.UrolSci.2022;33:101–13.

34.KwonJ,LeeEJ,ChoHJ,JangJA,HanMS,KwakE,etal.Antiﬁbrosistreatmentby inhibitionofVEGF,FGF,andPDGFreceptorsimprovesbladderwallremodeling anddetrusoroveractivityinassociationwithmodulationofC-ﬁberafferent activityinmicewithspinalcordinjury.NeurourolUrodyn.2021;40:1460–9.

35.DeFinisJH,WeinbergerJ,HouS.Deliveryofthe5-HT2Areceptoragonist,DOI, enhancesactivityoftheSphinctermuscleduringtheMicturitionreﬂexinrats afterspinalcordinjury.Biology.2021;10:68.Published2021Jan19.Koozekanani SH,ViseWM,HashemiRM,McGheeRB.Possiblemechanismsforobserved pathophysiologicalvariabilityinexperimentalspinalcordinjurybythemethodof Allen.JNeurosurg.1976;44:429–434.

36.ShangZ,JiaC,YanH,CuiB,WuJ,WQ,etal.InjectingRNAinterferencelentivirusestargetingthemuscarinic3receptorgeneintothebladderwallinhibits neurogenicdetrusoroveractivityinratswithspinalcordinjury.NeurourolUrodyn.2019;38:615–24.

37.MatsumotoY,MiyazatoM,YokoyamaH ,KitaM,HiraoY,ChancellorMB,etal. RoleofM2andM3muscarinicacetylcholinereceptorsubtypesinactivation ofbladderafferentpathwaysinspinalcordinjuredrats.Urology 2012;79:1184.e15 – 1184.e1.184E20.

38.GuoYX,JiX,LiuJ,FanDD,ZhouQB,ChenC,etal.Effectsofexosomesonpremetastaticnicheformationintumors.MolCancer.2019;18:39.

39.ChenXY,LiuBL,LiXZ,AnTT,ZhouY,LiG,etal.Identi ﬁ cationofantiin ﬂ ammatoryvesicle-likenanoparticlesinhoney.JExtracellVesicles. 2021;10:e12069.

40.KimH,WangSY,KwakG,YangY,KwonIC,KimSH.Exosome-guidedphenotypic switchofM1toM2macrophagesforcutaneouswoundhealing.AdvSci. 2019;6:1900513.

41.NiYQ,ZhanJK,LiuYS.RolesandmechanismsofMFG-E8invascularagingrelateddiseases.AgeingResRev.2020;64:101176.

42.CheyuoC,JacobA,WuR,ZhouM,QiL,DongWF,etal.Recombinanthuman MFG-E8attenuatescerebralischemicinjury:itsroleinanti-inﬂammationandantiapoptosis.Neuropharmacology2012;62:890–900.

43.SeguraE,NiccoC,LombardB,VéronP,RaposoG,BatteuxF,etal.ICAM-1on exosomesfrommaturedendriticcellsiscriticalforefﬁcientnaiveT-cellpriming. Blood2005;106:216–23.

44.RongY,JiC,WangZ,GeX,WangJ,YeW,etal.SmallextracellularvesiclesencapsulatingCCL2fromactivatedastrocytesinducemicroglialactivationandneuronal apoptosisaftertraumaticspinalcordinjury.JNeuroinﬂammation.2021;18:196.

45.FanH,ZhangK,ShanL,KuangF,ChenK,ZhuK,etal.Reactiveastrocytes undergoM1microglia/macrohpages-inducednecroptosisinspinalcordinjury. MolNeurodegener.2016;11:14.

CellDeathandDisease(2023)14:70

46.MilichLM,RyanCB,LeeJK.Correctionto:Theorigin,fate,andcontributionof macrophagestospinalcordinjurypathology.ActaNeuropathol.2019;137:799–800.

47.GaoYY,TaoT,WuD,WuD,ZhuangZ,LuY,etal.MFG-E8attenuatesinﬂammationinsubarachnoidhemorrhagebydrivingmicroglialM2polarization.Exp Neurol.2021;336:113532.

48.YaoX,ZhangY,HaoJ,DuanHQ,ZhaoCX,SunC,etal.Deferoxaminepromotes recoveryoftraumaticspinalcordinjur ybyinhibitingferroptosis[published correctionappearsinNeuralRegenRes.2019;14:532 –41.NeuralRegen Res.2019;14:1068.

ACKNOWLEDGEMENTS

ThisresearchissupportedbytheNationalKeyResearchandDevelopmentProjectof StemCellandTransformationResearch(2019YFA0112100),theNationalNatural ScienceFoundationofChina(81930070)andtheTianjinKeyMedicalDiscipline (Specialty)ConstructProject.

AUTHORCONTRIBUTIONS

JR,XMS,ZJW,andSQFconceivedanddesignedthestudy.JRandZJWanalyzedand interpretedthedata.XMS,ZJW,MW,andHDWprovidedmaterialandtechnical support.JR,BZ,YHQ,GJG,JJL,andZWPwereresponsibleforthelaboratoryresearch. JR,ZJW,BZ,GJG,andWCZwrotethemanuscript.Allauthorsapprovedthe manuscript.

COMPETINGINTERESTS

Theauthorsdeclarenocompetinginterests.Partofthe ﬁgureswerecreatedwith biorender.com.

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