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Raiders of the CNSVainchtein, Ilia Davidovich

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Chapter6MHCIIposmicrogliainchronicEAEphagocytoseperipheral

immuneinfiltrates

IliaD.Vainchtein,SabrinaJacobs,ErikW.G.M.BoddekeandBartJ.L.Eggen

DepartmentofNeuroscience,SectionMedicalPhysiology,UMCG,Groningen,TheNetherlands

Highlights:

Microglia are most strongly activated in the spinal cord, this correlates with

immuneinfiltration.

SpinalcordmicrogliaexpressMHCIIandAXLduringEAE.

Immuneinfiltratesarephagocytosedbyspinalcordmicroglia.

Manuscriptinpreparation.

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Abstract

Microglia,theresidentimmunecellsofthecentralnervoussystem(CNS),areknown

fortheirsurveillanceofthebrainparenchyma,capacitytotuneneuronalnetworksand

tocleardebris.Here,thereactionofmicrogliatoinfiltrationofperipheralimmunecells

anddemyelinationwasstudiedinexperimentalautoimmuneencephalomyelitis(EAE),

amousemodelformultiplesclerosis.EAEisaninducedautoimmunereactioninthe

CNS against myelin oligodendrocyte glycoprotein. In EAE, peripheral immune cells

infiltrate theCNSand inducedemyelination, andalthough ithasbeen reported that

microgliaremainimmunesuppressedduringthisprocess,theirexactroleinEAEorMS

isstillunclear.Here,weinvestigatedtheputativecellclearanceactivityofmicrogliain

detail by focusing on MHCII expressing cells and we performed a transplantation

experimentwithDsRed‐expressing cells to trace the fate of immune infiltrates. Our

results indicated that during EAE, the number ofmicroglia increases and that they

becomeactivated inthespinalcord,hindbrainandforebrain.Microgliaproliferation

and activation was most prominent in the spinal cord that also contained most

infiltrated peripheral immune cells. Microglia upregulated apoptotic cell clearance

molecules;theAxlreceptorandGas6attheRNAlevel, inconjunctionwithincreased

expressionofthelipiduptakereceptorsLgals3andCd36.DuringacuteEAE,IBA1posLy‐

6CnegmicrogliawereobservedcloselysurroundingLy‐6CandIL‐1βpositivecells,while

atthechronicstage,densemicroglianodulesweredetectedwithonlyfewinfiltrates.

Retro‐orbital transplanted DsRedpos cellsmigrated to the CNS and DsRed DNAwas

detected in microglia, primarily during the acute phase. These data indicate that

microglia do become activated, expand in numbers, clear debris including myelin

fragments,andmost likelyalsophagocytoseapoptotic, infiltratedimmunecells.This

emphasizestheroleofmicrogliaasimportantcellsforCNShomeostasis,evenunder

theconditionofsevereneuro‐inflammationanddemyelination.

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Introduction

 Microgliaarecrucial tomaintainahealthyand“debris”‐freecentralnervoussystem

(CNS).Asidefromtherecentlydiscoveredfunctionsinsynapticpruningandneuronal

networkformation,theirmaintasksencompassthephagocytosisofdebrisanddead

cells(Kreutzberg,1996;Paolicellietal.,2011;Nayaketal.,2014).Thisprocesshasbeen

studiedextensivelyandindicatedthatmicrogliacanphagocytosedeadneuronsandare

involvedintheuptakeofβ‐amyloid(Sierraetal.,2010;Morschetal.,2015;Wangetal.,

2015).Microgliaareversatilecells,capableofengulfingabroadrangeofantigensand

structures encountered in their environment. The study of microglia under

demyelinatingandneurodegenerativeconditionsthatincludeextensiveinfiltrationof

peripheral immune cells remains challenging.While previous studies have revealed

that microglia remain rather immune suppressed in experimental autoimmune

encephalomyelitis (EAE), aMSmodelhallmarkedbyCNSdamageand infiltrationof

peripheralimmunecells,theirexactroleisunknown(Vainchteinetal.,2014;chapter

2). In fact,microgliaphagocytosevery littlemyelinduringEAEandarenot actively

involvedindemyelination(Yamasakietal.,2014;Vainchteinetal.,2014;chapter2).

Microglia become activated and upregulate genes involved in phagocytosis during

demyelinatingconditions, likeAxl,which ismostly linked to theuptakeofapoptotic

cells(Olahetal.,2012;chapter5).Inaddition,microglianoduleswereobservedduring

the remission phase of EAE in Biozzi ABH mice, when peripheral infiltrates had

disappeared (chapter 5). Therefore, we hypothesized that peripheral immune

infiltrates, that undergo apoptosis, might be one of the targets of microglial

phagocytosis.Many immunecells, likemacrophagesandneutrophils,areshort‐lived

andrapidlygointoapoptosis(Leyetal.,2011;RansohoffandBrown,2012).Thus,the

rapidexpansionandnoduleformationofmicrogliamightbeareactiontotheextensive

infiltrationthatoccursduringEAE(Ponomarevetal.,2005;chapter5thisthesis).

Here,wehaveusedafluorescenceactivatedcellsorting(FACS)basedapproachto

sort microglia and to determine their activation status, using MHCII expression, in

different CNS regions and correlating it to immune cell infiltration. In addition, we

performed a DsRed labeled‐cell transplantation experiment to trace CNS infiltrated

immunecellsandstudywhethertheywerephagocytosedbymicroglia.

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MethodsandMaterials

EAEinductionandscoring

Female C57BL/6 mice (C57BL/6OlaHsd, Harlan Laboratories, The Netherlands) or

B6.Cg‐Tg(CAG‐DsRed*MST)1Nagy/J(DsRed+fromin‐housebreeding)micewereused

forEAEinductions.EAEwasinducedusingapre‐madeinductionkit(EK‐2110)from

HookeLaboratories,USA. Inbrief,mice received twosubcutaneous injectionsat the

upper and lower back each ±100 µg myelin oligodendrocyte glycoprotein 35‐55

(MOG35‐55; sequenceMEVGWYRSPFSRVVHLYRNGK) emulsified in completeFreund’s

adjuvant containing 200 – 500 µg killed mycobacterium tuberculosis H37Ra.

Subsequently, intraperitoneally ±250ngpertussis toxin (PTX)dissolved inPBSwas

injectedandrepeated24hlater.Miceweredailymonitored,inthefirst7daysonlyfor

weight,andafterwardsforEAEscoreandweight.A6‐pointscoringsystemwasapplied:

0=noobviouschanges(normal),1=limptail,2=limptailandimpairedrightingreflex,

3=limptailandpartialparalysisofhindlegs,4=limptailandcompleteparalysisof

hindlegs,5=moribund,6=death.Ingeneral,5micewerehousedpercageandwhen

thefirstmousereachedscore2,extracarewasgivenbyaddingspecialbinswithwater,

soliddrinks (solidifiedwater)andsoft food(pelletpowderdissolved inwater).The

exactnumberofmiceusedisstatedbelowandfurtherdetailedinthefigurelegends.All

experiments were performed according to local and international guidelines and

approved by the Animal Welfare Committee of the University of Groningen, The

Netherlands (DEC number: 6756B/C). In addition, the experimental design and

relevantanimalswereaccordingtotheARRIVEguidelines(Kilkennyetal.,2010;Amor

etal.,2012;Bakeretal.,2012).

CellsuspensionsfromCNS,eyes,spleen,bloodandmuscles

Micewereplacedunderisofluraneanesthesiaandblood(+/‐500µl)wascollectedby

a heart puncture using a 1 ml syringe (Braun) in EDTA blood collection tubes

(MiniCollect,Greinerbio‐one).Thereafter, themicewereperfusedwith0.9%saline.

CNS(brainandspinalcord),eyes,spleenandmuscleswereplacedinisolationmedium

withphenolred(iMed+,HBSS(1x)withphenolred(Gibco);15mMHEPES1M(Lonza);

0.6%glucose45%(Sigma‐Aldrich)).Allfollowingprocedureswereperformedoniceif

notstatedotherwise.ForCNSisolations;thebrainswerekeptasawholeorsplitinto

forebrain and hind brain (cerebellum and brainstem) with a scalpel. As described

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6

before(deHaasetal.,2008;Vainchteinetal.,2014;chapter4),theCNStissueswere

mincedandmechanicallydissociatedwithatissuehomogenizerandfilteredthrougha

70µmcell strainer (BDFALCON).The suspensionwas centrifuged for 10min (300

rcf/g,acc:9,brake:9,4oC)andthesupernatantremoved.Thepelletwasresuspended

inasolutionof22%Percoll (GEHealthcare),40mMNaCland77%myelingradient

buffer(5.6mMNaH2PO4*H20,20mMNa2HPO4*2H20,140mMNaCl,5.4mMKCl,11mM

Glucose,pH7.4),layeredwith1mLofPBSandcentrifugedfor20min(950rcf/g,acc:

4,brake:0,4oC).Thesupernatant,containingthemyelinlayer,wasremovedandthe

cell pellet resuspended in isolationmediumwithout phenol red (iMed‐; HBSS (1x)

without phenol red (Gibco); 15mMHEPES1M (Lonza); 0.6%glucose 45% (Sigma‐

Aldrich); 1 mM EDTA 0.5 M pH=8.0 (Invitrogen). For spleen, eye and lymph nodeisolations; the tissues were mechanically dissociated in a tissue homogenizer with

iMed+ and filtered through a 70 µm cell strainer (BD FALCON). Afterwards, the

suspension was centrifuged for 10 min (300 rcf/g, acc: 9, brake: 9, 4 oC) and the

supernatantremoved.Spleencellsuspensionpelletswereresuspendedin1mloflysis

buffer (155 mmol/L ammonium chloride, 10 mmol/L potassium bicarbonate, 0.1

mmol/L sodium edetate), incubated for 5 min on ice, filled up with iMed+ and

centrifugedfor10min(300rcf/g,acc:9,brake:9,4oC).Thesupernatantwasdiscarded.

Forhindlegmuscleisolations;thehindlegswereplacedinaPetridish,themuscleswere

dissected from the bones with scalpels and the tendons removed. Afterwards, the

muscleswerecutintosmallpiecesandcollectedin1mlofiMed+.Theyweremovedto

a 2 mL eppendorf and after 5 min the supernatant was removed. 1.5 mL iMed+

containing1xCollagenasetypeIV(Gibco)and1xTrypsin/EDTA(MPBiomedicals)was

addedtothetubeandplacedona37oCshaker(IKSincubators)for45min.Afterwards

10%FCSwasaddedandthetubeswereplacedonice.GlassPasteurpipets(VWR)were

usedtofullydissociatethetissues.Thetubeswerecentrifugedfor10min(300rcf/g,

acc:9,brake:9,4oC)andsupernatantwasremoved.Forbloodisolations;thebloodwas

moved to a 1.5mL tube and 1 volume of iMed‐was added. Aftermixing gently the

suspension was layered on top of 3 mL of 75% Percoll solution (75% Percoll (GE

Healthcare);10%HBSS(10x)(Gibco);15%PBS(Gibco)).Aftercentrifugationfor25

min(880rcf/g,acc:4,brake:0,4oC)theinterfacelayerwascollectedwithaglasspipet

anddissolvedin10mlofiMed‐.Aftercentrifugationof10min(300rcf/g,acc:9,brake:

9,4oC)thesupernatantwasremoved.

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StudyofmicrogliafromdifferentCNSregions

13‐15weeksoldEAEC57BL/6miceatacutescore4(n=3)orchronicphase(4‐5days

afterreachingscore4,themicereachscore3.75)(n=3)and13‐15weeksoldnaïvemice

(n=3; pool from n=6)were terminated. Their spinal cord, lymph nodes and brains,

separated in fore brain and hind brain, were processed as stated before. Also, an

additional 3 mice per condition were terminated and perfused with PFA for

immunohistochemistry.Afteracquiringthecellpellets,theywereresuspendedandFc

receptorblockedwithanti‐mouseCD16/CD32(eBioscience)iniMed‐for10minonice.

Subsequently,thecellsuspensionswereincubatedwithCD11bPE(eBioscience),CD45

FITC(ebioscience),Ly‐6CAPC(Biolegend)andLy‐6GPE/Cy7(Biolegend)for30min.

Thesampleswerecentrifugedfor3min(300rcf/g,4oC,acc:9,brake:9)andcollected

inroundbottomtubes(with35μmnylonmesh(BDBiosciences)).FACSwasperformed

onaMoFlo®Astrios™(BeckmanCoulter)andgatingforviablecellswasdonebasedon

4',6‐diamidino‐2‐phenylindole (DAPI; 0.5 µM; Sigma‐Aldrich) wheremicroglia were

definedasCD11bpos CD45intLy‐6Cneg andmyeloid infiltratesasCD11bpos CD45posLy‐

6Cpos.ThecellswerecollectediniMed‐andafterwardsspundownfor10min(500rcf,

4oC,tabletopcentrifuge).ThesupernatantwasremovedandcellswerelysedinRLT+

buffer(Qiagen).

StudyofMHCIIposmicrogliafromspinalcords

13‐15weeksoldEAEC57BL/6miceatacutescore4(n=4)orchronicphase(4‐5days

afterreachingscore4,themicereachscore3.75)(n=7)and13‐15weeksoldnaïvemice

(n=3;poolfromn=6)wereterminated.Thespinalcordswereremovedandprocessed

asstatedbefore.Afteracquiringthecellpellets,theywereresuspendedandFcreceptor

blockedwith anti‐mouse CD16/CD32 (eBioscience) in iMed‐ for 10min on ice. Cell

suspensionswereincubatedwithCD11bBV421(Biolegend),CD45FITC(ebioscience),

Ly‐6CAPC(Biolegend),Ly‐6GAPC/Cy7(Biolegend)andMHCIIPE/Cy7(Biolegend)for

30min.Thesampleswerecentrifugedfor3min(300rcf/g,4oC,acc:9,brake:9)and

collectedinroundbottomtubes(with35μmnylonmesh(BDBiosciences)).FACSwas

performedonaMoFlo®Astrios™(BeckmanCoulter)andgating forviablecellswas

done based on Propidium Iodide (PI; 0.5 µM; Sigma‐Aldrich)wheremicrogliawere

definedasCD11bpos CD45intLy‐6CnegMHCIIpos orCD11bpos CD45intLy‐6CnegMHCIIneg.

ThecellswerecollectediniMed‐andafterwardsspundownfor10min(500rcf,4oC,

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6

tabletopcentrifuge).ThesupernatantwasremovedandcellswerelysedinRLT+buffer

(Qiagen).

ProofofprincipleexperimentforDsRedposcells

Splenocyteswere isolated (as stated below) from one 14weeks old B6.Cg‐Tg(CAG‐

DsRed*MST)1Nagy/Jmouse,andtransplantedto12weeksoldC57BL/6mice(n=4)by

retro‐orbital injections,each2.75millionsplenocytes in250µl.Group1ofacceptor

C57BL/6mice(n=2)receivedanLPS(Lipopolysacharide)injectedof0.25mg/kgin20

µl inoneof thehind legs (uppermuscle;Gracilis)30minsprior to transplantation,

whereasgroup2(n=2)underwentthesameprocedure24houraftertransplantation.

Group1was terminated24hoursafter transplantationandgroup2;48hoursafter

transplantation. After termination, the CNS (brain + spinal cord), blood, spleen and

muscleswerecollectedandprocessedasstatedbefore.Afteracquiringthecellpellets,

they were resuspended and Fc receptor blocked with anti‐mouse CD16/CD32

(eBioscience) in iMed‐ for 10 min on ice. Subsequently, the cell suspensions were

incubated with CD11b BV421 (Biolegend), CD45 FITC (ebioscience), Ly‐6C APC

(Biolegend)andLy‐6GPE/Cy7(Biolegend)for30min.Thesampleswerecentrifuged

for3min(300rcf/g,4oC,acc:9,brake:9)andcollectedinroundbottomtubes(with35

μmnylonmesh(BDBiosciences)).FlowcytometrywasperformedonaBDLSRIIFACS

analyzerandDAPI(Sigma‐Aldrich)wasusedtogateonviablecells.

DsRedposcellstransplantationsinEAEmice

EAEwasinducedin14weekoldB6.Cg‐Tg(CAG‐DsRed*MST)1Nagy/Jmice(n=4)and

14weeksoldC57BL/6mice(n=14).After10days(nodiseasesymptoms),theB6.Cg‐

Tg(CAG‐DsRed*MST)1Nagy/J mice were terminated and their spleens collected.

Splenocyteswereisolatedasstatedbelowand±10millionweretransplantedbyretro‐

orbital injection (250µl) in each acceptorC57BL/6mousewhereEAEwas induced

(n=14) and acceptor naïve 14weeks old C57BL/6mice (n=6). Acceptormicewere

terminated:1dayaftertransplantation;2xEAEC57BL/6miceand2xnaïveC57BL/6

mice,4daysaftertransplantation;7xEAEC57BL/6miceand2xnaïveC57BL/6mice,7

days after transplantation; 5x EAE C57BL/6 mice and 2x naïve C57BL/6 mice

irrespectiveofEAEscore.Aftertermination,theCNS,bloodandspleenwerecollected

and processed as stated before. From each brain, half (sagittal) was fixed in 4%

paraformaldehyde(PFA).Afteracquiringthecellpellets,theywereresuspendedand

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Fcreceptorblockedwithanti‐mouseCD16/CD32(eBioscience)iniMed‐for10minon

ice.Subsequently,thecellsuspensionswereincubatedwithCD11bBV421(Biolegend),

CD45FITC(ebioscience),Ly‐6CAPC(Biolegend),andLy‐6GAPC/Cy7(Biolegend)for

30min.Thesampleswerecentrifugedfor3min(300rcf/g,4oC,acc:9,brake:9)and

collectedinroundbottomtubes(with35μmnylonmesh(BDBiosciences)).FACSwas

performedonaMoFlo®Astrios™(BeckmanCoulter)andgating forviablecellswas

donebasedonPI(Sigma‐Aldrich)wheremicrogliaweredefinedasCD11bposCD45intLy‐

6Cneg DsRedneg, infiltrated macrophages as CD11bpos CD45pos Ly‐6Chigh Ly‐6Gneg and

neutrophilsasCD11bposCD45posLy‐6CintLy‐6Gpos.ThecellswerecollectediniMed‐and

afterwardsspundownfor10min(500rcf,4oC,tabletopcentrifuge).Thesupernatant

wasremovedandcellswerelysedinRLT+buffer(Qiagen).Ofday4terminatedmice,

1/15 of the cell suspension (n=3) was set aside and incubatedwith CD11b BV421

(Biolegend),CD45FITC(ebioscience),Ly‐6CAPC(Biolegend),fixatedwith1%PFAfor

10min,andpermeabilizedwith0.1%saponininPBS.Thesampleswereincubatedwith

Ly‐6CPE/Cy7(Biolegend)with0.1%saponin/PBSfor20min,washed,centrifugedfor

3min(300rcf/g,4oC,acc:9,brake:9)andmeasuredonaBDLSRIIFACSanalyzer.

Quantitativereal‐timePCR(qPCR)

TheRNAwasextractedwiththeRNeasyPlusMicrokit(Qiagen)orAllPrepDNA/RNA

Micro kit (Qiagen) when both DNA and RNA were needed according to the

manufacturer’sprotocol.cDNAwasmadebyreverse transcriptionwithamixtureof

random hexamers, dNTPS, M‐MLV buffer, RibolockTMRNase Inhibitor and

RevertAidTMM‐MuLVReverseTranscriptase(Fermentas).qPCRwasperformedin384

well plates (Applied Biosystems) with iQTM SYBR Green Supermix (Bio‐Rad) on an

ABI7900HTmachine (AppliedBiosystems).All theprimers (supplementary table1)

weredesignedwithNCBIPrimer‐BlastandorderedfromBiolegio(TheNetherlands).

Data were quantified using the 2‐ΔΔCt method where Hmbs (hydroxymethylbilane

synthase)wasusedasahousekeepinggene(Livaketal.,2001).

Immunohistochemistry

Micewereperfusedwithsalinefollowedby4%PFA,thespinalcolumnwasremoved

and post fixed overnight with the same solution. Afterwards, the spinal cord was

dissectedfromthecolumnandplacedin1%PFAovernight.Tissueswerewashedwith

PBSandtransferredtoa20%sucrosesolution.After1day, theywereembedded in

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TissueTekoptimalcuttingtemperature(OCT;Sakura)and50µmsagittalsectionswere

cut with a cryostat (CM3050S, Leica Biosystems). Immunohistochemistry was

performedonfree‐floatingsections.Inshort,sectionswerewashedandblockedfor1h

with 10% normal donkey serum (NDS) in PBS with 0.1% TritonX‐100 (PBST).

Subsequently,thesectionswereincubatedwithrabbitanti‐IBA1(1:1000,WAKO)and

ratanti‐Ly‐6C(1:200,ADbSerotec)orgoatanti‐IL‐1β(1:100,R&DSystems)inPBST

with 1% NDS overnight. The next day, after washing, they were incubated with

secondaryantibodiesdonkeyantirabbit488(MolecularProbes)anddonkeyantirat

Cy3 (JIR Laboratories) or donkey anti goat Cy3 (JIR Laboratories) (all 1:400). After

extensivewashingsectionswereincubatedwithHoechst(1:1000)inPBSfor5minand

mountedwithMowiol.ImagesweretakenwithaLeicaTCSSP8confocalmicroscope

(LeicaMicrosystems).

Graphsandstatisticalanalysis

BargraphsweremadeinPrism5(Graphpad)andorganizedinAdobeIllustratorCC

(Adobe).FACSplotanalysis andcalculationswereperformedwithTreeStarFlowJo

softwarev10.StatisticalanalysiswasconductedwithSPSS22(IBM)usingtheKruskal‐

Wallis test to identify differences between groupswith a deeper group comparison

usingtheMann‐WhitneyUtest.Differenceswereclassifiedassignificantwhenthep‐

valuewasbelow0.05.

Results

Microgliaactivationwasmostpronouncedinthespinalcordwhereinfiltrationof

peripheralimmunecellswasmostsevere

Todeterminethecharacteristicsofmicrogliaactivation,C57BL/6micewereusedthat

displayanEAEprogressionthatincludesanacutephaseofscore4and4to5dayslater

becomeschronicatanaverageEAEscoreof3.75(fig.1A).Whentheacutephasewas

compared with the chronic phase, a striking reduction in the number of infiltrates

duringthechronicphasewasobserved.EAE‐relatedinfiltrationwasmostlydescribed

inthespinalcord(chapter2and5thisthesis).Inthisstudy,infiltrationwasobserved

inallmeasuredregions;forebrain,hindbrainandspinalcord(fig.1B,D;suppl.fig.1A).

Infiltrationismostextensiveinthespinalcord,andtoalesserextentintheforebrain.

MicroglianumbersincreasedduringEAE,mainlyinspinalcordandhindbrain(fig.1D,

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suppl.fig.1A),butupregulationofCD11bandCD45occurredalsointheforebrain(fig.

1C; hindbrain not shown). This indicates that microglial activation is a CNS‐wide

phenomenon,andmostprominentinthespinalcord.

In order to studymicroglia at the RNA level, we sorted these cells as CD11bhigh

CD45intLy‐6CnegbyFACS(fig.1B).Microgliadisplayedamorerobustupregulationof

genes in thespinalcord than in the forebrain (fig.2). Interestingly, theoverallgene

expressionpatternsinthehindbrainandspinalcordwerequitesimilar(Cox2andCd36

Figure1.DuringEAE,microgliaactivationwasmostprominent in the spinal cord correlatingwith

infiltrationofimmuneinfiltrates.A)EAEprogressioncurve,approximately16daysafterEAEinductionmost

micereachacutescore4(acutephase)and4to5dayslaterbecomechronic(n=48atday12,n=13atday21;

EAEmicefrommultiplechaptersarecombinedinthisgraph).B)ExamplesofFACSplotsfromforebrainand

spinalcordattheacutephaseforCD11bvsCD45,insetsarefromcontrolandchronicphase.TheseFACSplots

indicatethatinfiltrationofimmunecellsoccursnotonlyinthespinalcord,butalsointheforebrainduringEAE.

Insetsofmicrogliagatedisplaythesortingstrategy,wheremicrogliaweresortedasCD11bhighCD45intLy‐6Cneg.

Oneexampleisshownoutof3micepercondition.C)Quantificationsofthemeanfluorescenceforthesurface

expressionofCD11bandCD45onmicrogliainforebrainandspinalcord.MicrogliaupregulateCD11bandCD45

inbothregionsduringEAE(n=3percondition).Allvaluesarenormalizedtothecontrolconditionofeachregion,

setto1.D)Thenumberofsortedmicroglia(CD11bhighCD45intLy‐6Cneg)andmyeloidinfiltrates(CD11bposCD45pos

Ly‐6Cpos)cellsfromforebrainandspinalcordbyFACS.Microglianumberssignificantlyincreaseinthespinal

cord,wheremostinfiltrationofmyeloidcellsoccurs(n=3percondition).Co=controls,Ac=acutephase,Ch=

chronicphase.*/#:p≤0.05.*representsasignificantdifferencewiththecontrols,#adifferencewithEAEacute

phase.ErrorbarsareSEM.

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areshownasexample;suppl.fig.1B).Thisindicatesthat,althoughmicroglialactivation

inthespinalcordismorepronouncedthaninotherregions,thereisagradedlevelof

activation from spinal cord to forebrain, probably correlating with the amount of

peripheral immunecell infiltration.MicrogliaupregulatedMHCII (H2Aa),anddown‐

regulated scavenger receptor Fcrls a gene shown to be downregulated in activated

microglia(fig.2,suppl.fig.1B)(Butovskyetal.,2014).ExpressionofCox2,ageneoften

linkedtopro‐andanti‐inflammatoryactivity,wasupregulatedduringEAEinmicroglia,

andmostprominentattheacutephase,indicatingthatmicrogliaareactivatedduring

EAE.

Figure2.UpregulationofMHCII,apoptotic cell clearanceandmyelinuptake receptors inmicroglia,

mostlyinthespinalcord.Microgliaupregulatedactivation‐relatedgeneslikeMHCII(H2Aa)andCox2,butalso

myelin/lipiduptakereceptorsLgals3andCd36andreceptorsandligandsinvolvedinapoptoticcellclearance

(AxlandGas6)weremostprominentinthespinalcord(n=3).Thespinalcordmicrogliafromcontrolmiceare

setto1.Co=controls,Ac=acutephase,Ch=chronicphase.*/#:p≤0.05.*representsasignificantdifference

withthecontrols,#adifferencewithEAEacutephase.ErrorbarsareSEM.

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Interestingly,AxlandGas6arebothupregulatedinmicroglia.AXLisinvolvedinthe

uptakeofapoptoticcells,andGas6isaligandforAXL(fig.2).Otherapoptoticcelluptake

receptors,liketheTAMreceptorsMertkandTyro3,andTrem2weredownregulatedor

unaffected (suppl. fig. 1B). Myelin/lipid uptake receptors Lgasl3 and Cd36 were

upregulatedinspinalcordmicroglia,althoughotherlipiduptakereceptorslikeSirpa

andLrp1weredownregulated(fig.2,suppl.fig.1B).Cd80wasdownregulatedandIl1b

mildlyupregulated,aswasreportedbefore(chapter2and5).Concluding,asubsetof

genesincludingAxL,Cd36andH2Aa,involvedinuptakeandpresentationofantigens,

areincreasedinexpressioninmicrogliaduringEAE.

MicrogliaexpressedMHCIIduringEAE

AsMHCIIRNAlevelswerehighlyupregulatedduringEAE,MHCIIproteinexpressionat

thecellmembranewasdeterminednext.Microgliadisplayedanincreasedexpression

ofMHCII(fig.3A),controlmicrogliawereMHCIInegandupregulatedMHCIIduringEAE,

reachingamaximumatthechronicphase.ToanalyzeMHCIInegandMHCIIposmicroglia

inmoredetail,MHCIIposandMHCIInegcellswereFACSsortedattheacuteandchronic

phase(fig.3A;rightFACSplot).AttheRNAlevelthemaindifferencebetweenMHCIIpos

andMHCIInegmicrogliawasinMHCII,asseenwithH2Aa,otherquantifiedgeneslike

Cd80,Fcrls,Il1b,AxlandIl10showedmarginaldifferences,althoughMHCIIposmicroglia

haveaslightlyhigherexpressionofLgals3.Summarizing,anincreaseinRNAexpression

ofthegenesshowninfigure2wasobservedinallmicroglia,andthedifferencebetween

MHCIIposandMHCIInegmicrogliaismainlyintheirMHCIIexpressionlevels.

MicrogliawerelocalizedincloseproximitytoLy‐6CposandIL‐1βposcells

Genesinvolvedinphagocytosisandantigenpresentationwereupregulatedinmicroglia

during EAE. To determine their target cells, immunohistochemistry was performed

withantibodiesdirectedagainstIBA1,Ly‐6CandIL‐1β.Ibaposmicrogliaarelocalizedin

closeproximitytoLy‐6CposandIL‐1βposimmuneinfiltrates(fig.4).Attheacutephase,

IBA1posmicrogliasurroundedandseemtoconnectwithtoLy‐6Cposinfiltrates(fig.4A,

B). The samewas observed for IL‐1βpos cells (fig. 4C).While this phenomenonwas

observedattheacutephase,atthechronicphasefewinfiltratescouldbeidentified,but

IBA1pos nodules appeared (fig. 4D). This indicates that microglia are most likely

engulfingtheseinfiltrates,basedonimmunohistochemistryandRNAdata.

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TransplantedDsRedpossplenocytesmigratedintotheCNSduringEAE

Tostudywhethermicrogliacanphagocytoseinfiltrates,DsRed+splenocytesfromEAE

DsRedposmiceweretransplantedinEAEC57BL/6mice.First,DsRed+splenocyteswere

retro‐orbitally injectedintoC57BL/6acceptormicethatreceivedanLPSinjectionin

thelefthindleglowermuscle(Gracilis)30minpriortotransplantation(group1)or

receivedanLPSinjection1dayaftertransplantation(group2)(suppl.fig.3).Group1

was terminated 1 day after transplantation and the second group 2 days after

transplantation.DsRedposcellswereidentifiedbyflowcytometryintheblood,lymph

nodes,spleenandlefthindlegmuscles,but,asexpected,notintheCNS.Around0.40%

Figure3.ThemajorityofspinalcordmicrogliawereMHCIIpositiveduringEAE.A)Left;representative

pictureoftheMHCIIsurfaceexpressionofspinalcordmicroglia.Middle;meanMHCIIfluorescenceintensityper

conditionofspinalcordmicroglia.Right;FACSstrategyforsortingofMHCIIposandMHCIInegmicroglia.Controls

aren=3,acuten=4and chronicn=7.B)TheMHCIIpos spinal cordmicrogliadiffermainly inMHCII (H2Aa)

expressionandnotinothergenesasshownhere(Cd80,FcrlsandLgals3)fromMHCIInegmicrogliaduringeach

phase.Allvaluesarenormalizedtothecontrolmicrogliaexpression levels,setto1.Co=controls,Ac=acute

phase,Ch=chronicphase.*/#:p≤0.05.*representsasignificantdifferencewiththecontrols,#adifference

withMHCIInegmicrogliafromthesameEAEphase.ErrorbarsareSEM.

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of the cells in the muscle samples expressed DsRed, while the highest percentage

DsRedposcellswasobservedinthespleen(suppl.fig.3).Theseexperimentsindicated

thattransplantedDsRedpossplenocyteswereviable,homedtolymphoidorgans,andto

asiteofLPS‐inducedinflammation.

Figure 4. At the acute phase, spinal cordmicrogliawere proximal to Ly‐6Cpos infiltrates and IL‐1β

positivecellsandformnodulesatthechronicstage.A),B)andC)Nearsmallinfiltrateclusters,IBA1posare

detected that have physical connections with Ly‐6C or IL‐1β expressing cells,most likely infiltrates (best

examplesinwhitecircles).D)Atthechronicphase,whereminimalinfiltrationispresentasseenbytheLy‐6C

staining,microglia formnodules.Representativepictureswere chosen from3x50µm slicespermouseper

condition,atotalof3micepercondition.Scalebaris20µm.

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Next, DsRedpos splenocyteswere transplanted 10 days after EAE induction from

DsReddonorstoEAEornaïveC57BL/6recipientmiceandthemicewereterminated

after1,4and7days(fig.5A).DsRedposcellsweredetectedinthespinalcordsandbrains

of acceptor EAE mice while the CNS of naïve mice that received EAE DsRedpos

splenocytesdidnotcontainDsRedposcells(fig.5B;suppl.fig.4A).DsRedposcellswere

presentbothinEAEandnaïvemiceinthespleen,lymphnodesandblood,butnotatthe

injection site behind the eye (fig. 5B; suppl. fig. 4A). In the spinal cord, the highest

percentageofDsRedposcellswasobservedafter4daysanddeclinedat7days(fig.5C).

Almost all DsRedpos cellswere CD11b and CD45positive in the spinal cord and the

myeloid infiltrates consisted mostly out of Ly‐6Gpos and Ly‐6Cpos cells; the largest

DsRed+ fraction was Ly‐6Cneg cells, mostly CD45high and CD11blow indicating

lymphocytes(fig.5D)(chapter2and5).TheseDsRed+cellswerealsodetectedinbrain

sections. Sections from the cerebellum of acceptor EAEmice sacrificed 4 days after

transplantationhadDsRedposcellslocatedwithinthecharacteristicinfiltrateclustersin

thewhitematterareas(fig.6A).Although,thenumberofinfiltrateswasloweratthe

chronicphase,inthelymphnodestheirnumbersincreasedincomparisontotheacute

phase(fig.6B).ThisindicatesthatalthoughsomeDsRedposcellsmightremainorgointo

apoptosisintheCNS,aninfluxintothelymphnodesfromtheCNSislikely.

MicrogliaphagocytosedperipheralimmuneinfiltratesduringEAE

Todeterminewhethermicrogliaareabletophagocytoseinfiltratedimmunecells,EAE

andnaïveC57BL/6micewereretro‐orbitallytransplantedwithDsRedpossplenocytes

from DsRed EAEmice. Afterwards, microglia (CD11bhigh CD45int Ly‐6Cneg DsRedneg),

macrophages(CD11bposCD45posLy‐6ChighLy‐6Gneg)andneutrophils(CD11bposCD45pos

Ly‐6CintLy‐6Gpos)weresortedfromEAEandnaïveacceptormice.GenomicDNAwas

isolatedandaqPCRwasruntodetermineifthesecellscontainedDsRedDNA.Asthe

FACSplotsdepicted(fig.5D),themacrophageandneutrophilsamplescontainedDsRed

DNAbasedoncorrectdissociationcurves(fig.6C,D,E).Ascontrols,samplescontaining

onlywaterorCNSDNAfromnon‐transplantedmicegavenoorincorrectdissociation

curves(fig.6C).SomeofthemicrogliasamplesalsocontainedDsRedDNA,butnotall

PCRreactionsfromthesamesamplewerepositive(fig.6D,E).Thissuggeststhatthe

amountofDsRedDNAinmicrogliawasverylowandsometimesbelowthedetection

limit.Microglia fromtransplantednaïvemice rarelydisplayeda correctdissociation

curve(fig.6D).Whilesortedmacrophagesandneutrophilshadthehighestnumberof

correctdissociationcurves(70‐80%),55.1%ofmicrogliafromEAEmicewerepositive

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compared to 6.9% of control microglia (fig. 6E). This indicates that during EAE,

microgliatakeupDsRedDNAprobablybyphagocytosinginfiltratingDsRedposimmune

cells.Tofurtherconfirmthisobservation,cellsuspensionsfromthespinalcordofthe

acceptormiceatday4wereincubatedwiththestandardsetofantibodies(CD11b‐

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Figure5.TransplantedDsRedposcellsinfiltratedthespinalcordduringEAE.A)Schematicdepictingthe

transplantation scheme,EAEmice (n=14) ornaïve C57BL/6 (n=6)micewere transplantedwith10million

DsRedposEAEsplenocytes10daysafterEAEinductionandterminated1,4or7daysaftertransplantation.B)

FACSplotswithSSCvsDsReddepictingspinalcord,lymphnodes,spleenandbloodfromEAEorcontrolsmice4

daysaftertransplantation.EAEmicehaveDsRedposinfiltratespresentinthespinalcord,buttheyareabsentin

controlmice.RepresentativeFACSplotsareshownfor1xEAE(totalofn=7)and1xcontrolmouse(totalofn=2)

after4days.C)CalculationofpercentageDsRedposcellsinthespinalcordforCo=controls(totalof6mice),EAE

D1=EAEmiceterminatedafter1day(n=2),EAED4=EAEmiceterminatedafter4days(n=7)andEAED7=

EAEmiceterminatedafter7days(n=5).*representsasignificantdifferencewiththecontrols.Errorbarsare

SEM.D)FACSplotsforCD11bvsCD45fromspinalcordatday4aftertransplantation.DsRednegandDsRedpos

cellsareshown(left)andDsRednegandDsRedposcellsseparatedinLy‐6Gpos(Ly‐6Cint),Ly‐6ChighandLy‐6Cneg.In

addition,the3groups(n=7)ofDsRedposcellsareshownaspercentageinthebargraph.Thelargestgroupisthe

Ly‐6CnegthatareCD45highandCD11bint.*representsasignificantdifferencewiththeLy‐6Cneg.

BV421, CD45 FITC and Ly‐6C APC). After fixing the samples with 1% PFA and

permeabilizationwith0.1%saponin, thesampleswere incubatedwithLy‐6C,witha

PE/Cy7 label. 20% of the CD11bhigh CD45int Ly‐6C APCneg microglia contained

intracellularLy‐6C(PE/Cy7pos;suppl.fig.4B).Thesedatafurthersupportthenotion

thatmicrogliatakeupLy‐6Cposcells,mostlikelymacrophagesorneutrophils.

Discussion

Microglial activation is present in many neurodegenerative disorders and is

characterizedbyupregulationofCD11bandIBA1andaswitchfromaramifiedtoan

amoeboid‐likemorphology(Kettenmannetal.,2011;Meliefetal.,2012).Inaddition,

microglia upregulate MHCII expression in the cuprizone mouse model (Olah et al.,

2012).We aimed to studymicroglial activation in the context of demyelination and

inflammationandusedtheEAEmousemodel.Thismodelinvolvesdemyelinationand

infiltrationofimmunecells,amodelinducedandstartedintheperiphery,thatresults

inmicroglialactivation(chapter2and5).OurresultsindicatethatduringEAEmicroglia

becomeactivatedCNS‐wide,astheyupregulateCD11bandCD45inthespinalcord,but

alsoinlessaffectedregionsliketheforebrain.Also,microglianumbersincreaseinthe

spinal cordandhindbrain, regions thatdisplay thehighest infiltrationofperipheral

immunecells.Substantialinfiltrationisalsopresentintheforebrain,butlesssothanin

otherregions.

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Previouslyitwasshownthatmicrogliaareactivated,butnotpro‐inflammatoryin

EAE(Yamasakietal.,2014;Vainchteinetal.,2014). Instead, theycleardebriswhile

macrophagesarethemaindemyelinatingandneurodegenerativecells.Whilemyelin

debrisuptakemightbeoneoftheireffectorfunctions,weaimedtodeterminewhat

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Figure6.MicrogliacontainedDsRedDNA,indicatingthattheyhavephagocytosedDsRedposinfiltrates

duringEAE.A)ImmunohistochemistryofcerebellumfromEAEmice4daysaftertransplantation.The50µm

thick sliceswereonly incubated forHoechst (blue).Hoechstdense clusters, indicating infiltrates, sometimes

containDsRed+cells.B)DuringEAE,attheacuteandchronicphasemoreCD11bposCD45posLy‐6Cposcellsappear

inthelymphnodes.IndicatingapossibleinfluxfrominfiltratesoutoftheCNSasaftertheacutephasethenumber

increasedatthechronicphase(n=3foreachcondition).*representsasignificantdifferencewiththecontrols.

C) After FACS,macrophage samples display the correct dissociation curve,whilewater orDsRed negative

samples(brainfromC57BL/6mousewithouttransplantation)havenoorincorrectcurveswhenmeasuredwith

qPCR.D)Examplesshownofdifferentmicrogliasampleswheremultiplewells(left),all(middle),ornone(right)

ofthewellsdisplaytherightdissociationcurve.E)SummaryofalltheqPCRreactions.Rowsindicatesamples

from different termination days; 1, 4 or 7 days after transplantation. Columns indicate the cell type; EAE

microglia, control microglia (microglia from control mice), EAE macrophages and EAE neutrophils

(macrophagesandneutrophilsfromEAEmice).Eachcolumnhasleft;thetotalPCRreactions,middle;number

ofcorrectDsRedposdissociationcurves,right;percentageofcorrectdissociationcurves.Atthebottomthetotal

numberandpercentageofcorrectDsReddissociationcurvesisdisplayed.Totalsamplesused:11xEAEmicroglia,

6xcontrolmicrogliaand3xEAEmacrophagesandneutrophils.

otherfunctionsmightberelatedtothisactivatedstate.MicrogliaupregulatedMHCII

(H2Aa)andCox2,anddownregulatedFcrls.BothMHCIIandCOX‐2havebeen linked

withactivatedimmunecells,whileFcrlsdownregulationoccursspecificallyinactivated

microglia(Butovskyetal.,2014).Inaddition,myelin/lipiduptakereceptorsCd36and

Lgals3were elevated inmicroglia. Scavenger receptor CD36 and Lgals3 (Galectin‐3,

Mac‐2)areimportantfortheuptakeoflipidsandmyelinbutalsocrucialformicroglial

effectorfunctionsandphenotyperegulations(Etoetal.,2003;Hoyosetal.,2014;Liet

al.,2015).Thisupregulation ismostlypresent in thespinalcordandminimal in the

forebrain, indicating that lipid uptake mostly occurs in the spinal cord. Regional

differencesformicrogliahavebeenshownbeforeinthebrain,cerebellarmicrogliaare

moreimmuneactivethanmicrogliafromotherregions(Grabertetal.,2016).Lrp1and

Sirpa were both downregulated in microglia, although previous studies have

highlightedtheir involvement inmyelinuptake(Gaultieretal.,2009).Possibly their

down‐regulationisduetonegativefeedbackaftermyelinuptake,modulatedbySirp‐α

(Gitiketal.,2011).

Apart from myelin, other phagocytic targets of microglia are apoptotic cells,

important for the resolution of inflammation and remyelination (Grigoriadis et al.,

2015). TAM receptors, consisting of Tyro3, AXL and MERTK are receptors that

recognize Gas6 and Protein S on apoptotic cells (Rothlin et al, 2015). Axl was

upregulatedinmicrogliaduringEAEalthoughMertkandTyro3weredownregulatedor

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unaffected. Other studies have shown thatMertk is normally upregulated in brain

inflammation (Grabert et al., 2016).Recently, itwas shown thatMertkandAXLare

importantregulatorsofmicroglialphysiology(Fourgeaudetal.,2016).AXLknockout

micehavemoresevereEAE,presumablyduetolackofdebrisclearance(Weingeretal.,

2011).Microgliaareprobablynecessaryfordebrisclearance,asthisisthefirststartto

functionalrepairoftheCNS(Neumannetal.,2009).Strikingly,theligandforAXL,Gas6

wasalsoupregulatedinspinalcordmicroglia.GeneticdeletionofGas6inmiceledto

moreactivatedmicroglia,moresevereEAEandmacrophageinfiltration,indicatingthat

Gas6likelyhasaprotectiveeffectduringEAE(Binderetal.,2008;Gruberetal.,2014).

Another receptor for apoptotic cell uptake,Trem2, was initially downregulated and

subsequentlyupregulatedatthechronicphase.Trem2isimportantforphagocytosisof

apoptoticcells,mostlyneurons,bymicrogliaandinEAEoverexpressionofTREM2by

microglialeadstomorephagocytosisandlessinflammation(NeumannandTakahashi,

2007).TREM2knockoutmicedevelopmoresevereEAEandtissuedamage(Piccioet

al.,2007;Kawaborietal.,2015).Interestingly,phagocytosisbyTREM2occurswithout

inflammation (Takahashi et al., 2005). Although it should be mentioned that some

TREM2consequencesattributedtomicroglia,mightbemorerelevantformacrophages

(Jayetal.,2015).

As MHCII is strongly upregulated in microglia, we aimed to study whether

subgroupsofmicrogliaexistthatdifferinMHCIIexpressionlevels.Flowcytometryfor

MHCIIwasperformedandMHCIIposandMHCIInegmicrogliaweresorted.Spinalcord

microgliafromnaïvemicewereMHCIIneg,andduringEAEthemajorityofthepopulation

becameMHCIIpos.WhileMHCIIpossortedmicrogliahadasignificantlyhigherexpression

ofMHCII(H2Aa),nootherexpressiondifferencescouldbefound(Cd80,Fcrls,Lgals3,

Il1b,Axl and Il10). This indicated thatmicroglia become activated and change gene

expressionasawholepopulation.Apparently,thereisagradationinMHCIIexpression,

indicatingthatsomemicrogliamoreactivelypresentantigens.

Whenexaminingmicrogliaandinfiltrateswithimmunohistochemistry,itappeared

thatmicrogliainthevicinityofinfiltrateclustersareincontactwithLy‐6Cposcells.This

suggeststhatmicrogliamightbephagocytosingthesecells.Atthechronicphase,when

very few infiltratescanbedetected, IBA1posmicroglianodulesappear.Todetermine

whether microglia indeed phagocytose immune infiltrates, a transplantation

experiment to track infiltrates entering the CNS was performed. First, a proof of

principalexperimentinnaïvemicewasperformed.TransplantedDsRedpossplenocytes

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to“non‐labeled”micesurvived,migratedandinfiltratedhindlegmusclewhereLPSwas

injected, indicatingtheyretainedanintacthomingcapacity.Weperformedthesame

experimentbytransplantingEAEDsRedpossplenocytestopre‐clinicalEAEC57BL/6or

naïveC57BL/6miceandthenterminatedthemafter1,4or7dayswhenEAEsymptoms

arepresent. Interestingly,asmall fractionof theDsRedpossplenocytes infiltratedthe

CNSofEAEmiceandreachedamaximumlevelafter4days.Asexpected, incontrol

mice,noDsRedinfiltratesweredetectedintheCNSconfirminganintactBBBinnaïve

C57BL6 mice. Most of these DsRedpos infiltrates were CD45high Ly‐6Cneg cells,

lymphocytes,whiletherestwereLy‐6GposneutrophilsorLy‐6Chighmacrophages.When

examiningthesortedmicrogliafromtheEAEmice,DsRedDNAwasdetectedinsomeof

the PCR samples. In addition, intracellular Ly‐6C staining was observed in these

microglia, suggesting that microglia phagocytosed Ly‐6Cpos and DsRedpos infiltrates

during EAE. While it cannot be excluded that a minor fraction of transplanted

splenocytes have silenced their DsRed expression and contaminated the microglia

population while sorting, the finding of intracellular Ly‐6C in microglia strongly

suggestsphagocytosisofinfiltratesbymicroglia.Macrophagesarehighlyregulatedby

lipiduptakeandoverallhaveashortlifespanafterphagocytosis.Wheninflammation

resolves,neutrophilsgointoapoptosiswhilemacrophagesfollowthesamepatternor

draintothelymphnodes(Bellinganetal.,1996;Kolaczkowskaetal.,2010).Inaddition,

apoptotic macrophages are often phagocytosed by other macrophages (Ley et al.,

2011). Therefore, microglia might be phagocytosing apoptotic neutrophils and

macrophages.

Inconclusion,weshowthatmicrogliabecomeactivatedduringEAE,mostlyinthe

spinalcord,upregulateMHCII, lipiduptakereceptorsLgals3andCd36andapoptotic

celluptakereceptorAxl.DuringtheacuteEAEphase,manymicrogliaarelocalizedin

close proximity to infiltrates and phagocytose infiltrates. At the chronic phase,

microglia nodules are observed in the spinal cord and very few infiltrates remain.

Therefore,theextensivedebrisclearanceactivitiyofmicrogliaisprobablyimportant

for resolving inflammation during acute EAE and are interesting targets for MS

medication.

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Acknowledgements

TheauthorswanttothankNieskeBrouwer,SusanneKooistra,MichaelHuang,Martha

Ritsema and Martine Stevelink for their help during the experiments and Geert

Mesander,HenkMoes,RoelofJanvanderLeifortheirtechnicalassistancewithFACS

sorting.IDVwassupportedbyaDutchMSResearchGrant(10‐723MS).

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Supplementarymaterial

TableS1.qPCRprimerinformationGenename Accessionnumber

Forwardprimer5’‐3’ Reverseprimer5’‐3’

Hmbs NM_013551.2

NM_001110251.1

CCGAGCCAAGCACCAGGATA CTCCTTCCAGGTGCCTCAGA

H2Aa NM_010378.2 CTGTCTTATCTCACCTTCATCC GGAATCTCAGGTTCCCAGTG

Cd80 NM_009855.2 GTCCATCAAAGCTGACTTCTC GGAAGCAAAGCAGGTAATCC

Il1b NM_008361.3 GGCAGGCAGTATCACTCATT AAGGTGCTCATGTCCTCAT

Mertk NM_008587.1 CTCTGGAGTGGAGGCACTG CCCTGGTAAAGGCCCTGAAA

Tyro3 NM_019392.2

NM_001290800.1

ACTGTCGAAGGTGTGCCATT AAGACAGCTGAAAAGGGGCA

Axl NM_009465.4

NM_001190974.1

NM_001190975.1

TGAAGCCACCTTGAACAGTC GCCAAATTCTCCTTCTCCCA

Gas6 NM_019521.2 CGAGTCTTCTCACACTGTGCT CCTCGAAGACTTGGTAGGCG

Trem2 NM_031254.3

NM_001272078.1

CTGGAACCGTCACCATCACTC CGAAACTCGATGACTCCTCGG

Sirpa NM_007547.4

NM_001177647.2

NM_001291019.1

NM_001291020.1

NM_001291021.1

NM_001291022.1

AACCCAGATCCAGGACACAAA GGGCTTCTTCTCTTTGGGCA

Lgals3 NM_001145953.1

NM_010705.3

CAGGATTGTTCTAGATTTCAGGAG TGTTGTTCTCATTGAAGCGG

CD36 NM_001159558.1

NM_007643.4

NM_001159555.1

NM_001159557.1

NM_001159556.1

GATGTGGAACCCATAACTGGA AGGTACAATGTAAGGTCTCTTC

AG

Lrp1 NM_008512.2 CGTGCGAGCGGACATCCCTG GGGCGCTTCACACCTGGACA

Fcrls NM_030707.3 CTCAATGTCACAGAGCCCCC TGTTCAACCTCTACGCGTCC

Cox2 NM_011198.4 CTCCCTGAAGCCGTACACAT CCCCAAAGATAGCATCTGGA

Il10 NM_010548 AAGGGTTACTTGGGTTGCCA TTTCTGGGCCATGCTTCTCTG

DsRed transgene GTGAACTTCCCCTCCGACG TCCAGCTTGGAGTCCACGTA

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Supplementary figure 1.RNA expression inmicroglia in forebrain,hindbrainand spinal cord.A)A

representativeexampleofaCD11bvsCD45FACSplotfromcellsisolatedfromthehindbrainattheacutephase

ofEAE,insetsarefromcontrolandchronicstagehindbrain.Thenumberofsortedmicroglia(CD11bhighCD45int

Ly‐6Cneg)andmyeloidinfiltrates(CD11bposCD45posLy‐6Cpos)fromhindbrainarevisualizedinbargraphs(n=3

mice).B)RNAexpressionlevelsinhindbrainmicrogliaofCox2andCd36.Allvaluesarenormalizedtocontrol

spinalcordmicroglia,assetto1.C)RNAexpressionlevelsinforebrainandspinalcordmicroglia.Apoptoticcell

clearancereceptors(Mertk,Tyro3,Trem2)andmyelin/lipiduptakereceptors(SirpaandLrp1)didnotchange

orweredecreasedduringEAE.Scavenger receptorFcrlswasdown‐regulatedduringEAE, the same for co‐

stimulatorymoleculeCd80(spinalcord).Il1bwasmodestlyupregulated.Allvaluesarenormalizedtothecontrol

spinalcordmicroglia,assetto1.Co=controls,Ac=acutephase,Ch=chronicphase.*/#:p≤0.05.*represents

asignificantdifferencewiththecontrols,#adifferencewithEAEacutephase.ErrorbarsareSEM.

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Supplementaryfigure2.RNAexpressionlevelsofIl1b,AxlandIl10inMHCIIposandMHCIInegmicroglia.

RelativeRNAexpressionforIl1b,AxlandIl10areshown.Controlsaren=3,acuten=4andchronicn=7.Allvalues

arenormalizedtothecontrolmicrogliaexpressionlevels,setto1.Co=controls,Ac=acutephase,Ch=chronic

phase.*/#:p≤0.05.*representsasignificantdifferencewiththecontrols.ErrorbarsareSEM.

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Supplementary figure 3. Proof of principal experiment forDsRedpos transplantations. C57BL/6mice

receivedaretro‐orbitalinjectionwithsplenocytes,onegroup(leftside;n=2)receivedaLPSinjectionintheleft

hindleguppermuscle30minpriortotransplantationandwasterminated1dayposttransplantation.Another

group(rightside;n=2)receivedtheLPSinjection1dayaftertransplantationandthemicewereterminated2

daysaftertransplantation.FACSplotsareshownfor1mouseoutofeachgroup,depictingtheSSCvsDsRedfor

blood,lymphnodes,spleen,CNSandlefthindlegmuscle.DsRedposcellsarepresentinthemuscles,blood,lymph

nodesandspleen,butnotintheCNS.

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Supplementaryfigure4.FACSplotsoftransplantedEAEDsRedpossplenocytesandintracellularmicroglia

Ly‐6Cstaining.A)RepresentativeFACSplotsdepictingbrain(n=2)andeye(n=2)samplesof7mice4daysafter

transplantation,illustratingthatinfiltrationofDsRedposcellsoccurredinthebrain,buttheyarenotpresentin

theeyeorretro‐orbitalsinus.B)Fourdaysaftertransplantation,cellsuspensionsofEAEspinalcord(n=3)were

incubatedwithCD11bBV421,CD45FITCandLy‐6CAPCantibodies,subsequently1%PFAfixed,permeabilized

with0.1%saponinandincubatedwithLy‐6CPE/Cy7.TheleftFACSplotshowsthefluorescenceforSSCvsLy‐6C

PE/Cy7 formicroglia(CD11bhighCD45int)thatweregatedasLy‐6CAPCnegative,Ly‐6CAPCpositivemyeloid

infiltrates(CD11bposCD45pos)andasamplethatwasfixed,permeabilizedbutnotincubatedwithantibodies.The

rightFACSplotshowsmicrogliathatareLy‐6CnegfortheAPCchannelandplottedforLy‐6CPE/Cy7.Apopulation

isidentifiedasLy‐6CposforPE/Cy7andthepercentageisplottedinthebargraph.