1

TheparasiticwormproductES-62normalisesthegutmicrobiota/bonemarrow

axisininflammatoryarthritis

RunningTitle:ES-62normalisesintestinalbacteria

JamesDoonana,AnuradhaTarafdarb,MiguelA.Pinedab,FelicityE.Lumba,Jenny

Croweb,AneesahM.Khanb,PaulA.Hoskissona,MargaretM.Harnettb*andWilliam

Harnetta*

aStrathclydeInstituteofPharmacyandBiomedicalSciences,UniversityofStrathclyde,

Glasgow,G40RE,UK

bInstituteofInfection,ImmunityandInflammation,UniversityofGlasgow,GlasgowG12

8TA,UK

*Correspondingauthors:

WilliamHarnett,StrathclydeInstituteofPharmacyandBiomedicalSciences,161

CathedralStreet,UniversityofStrathclyde,GlasgowG40RE,UK;Phone–0044-141-

548-3715;FAX:0044-141-552-2562;e.mail:w.harnett@strath.ac.uk

MargaretHarnett,InstituteofInfection,ImmunityandInflammation,CollegeofMedical,

VeterinaryandLifeSciences,UniversityofGlasgow,GlasgowG128TA,UK;Phone–

0044-141-330-8413;e.mail–Margaret.Harnett@glasgow.ac.uk

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AbstractThehumanimmunesystemhasevolvedinthecontextofourcolonisationbybacteria,

viruses,fungiandparasitichelminths.Reflectingthis,therapideradicationof

pathogensappearstohaveresultedinreducedmicrobiomediversityandgenerationof

chronicallyactivatedimmunesystems,presagingtherecentriseofallergic,

autoimmuneandmetabolicdisorders.Certainly,gastrointestinalhelminthscanprotect

againstgutandlungmucosainflammatoryconditionsbymodulatingthemicrobiome

andsuppressingthechronicinflammationassociatedwithdysbiosis.Here,weemploy

ES-62,animmunomodulatorsecretedbytissue-dwellingAcanthocheilonemaviteaeto

showthatmodulationofthegutmicrobiomedoesnotrequireliveinfectionwith

gastrointestinal-basedhelminthsnorisprotectionrestrictedtomucosaldiseases.

Specifically,subcutaneousadministrationofthisdefinedparasiticwormproduct

affordsprotectionagainstjointdiseaseincollagen-inducedarthritis,amousemodelof

rheumatoidarthritis,whichisassociatedwithnormalisationofgutmicrobiotaand

preventionoflossofintestinalbarrierintegrity.

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1. Introduction

Parasitichelminths(worms)haveevolvedtheabilitytomodulatehostimmuneand

tissuerepairresponsesinordertopromotetheirsurvivalbylimitingtheinflammation

thatwouldotherwisedrivetheirexpulsionandcausepathology1.Therecentrapid

eradicationofhelminths(andotherinfectiouspathogens)appearstohaveresultedin

over-activatedimmunesystemsandthisprovidesarationalefortheincreasing

prevalenceofchronicallergic(e.g.asthma)andautoimmune(e.g.inflammatorybowel

disease,type-1diabetes,multiplesclerosis[MS],systemiclupuserythematosus[SLE]

andrheumatoidarthritis[RA])inflammatorydisorders,aswellascontributingtothe

riseinobesityandassociatedmetabolicsyndromeco-morbiditiesincludingtype-2

diabetesandcardiovasculardisease2-6indevelopingandurbanisedcountries.Although

geneticstudiesofpatientshaveidentifiedvariantsofgenesthatareassociatedwith

theseinflammatorydiseasesinmanycasessuchasRA,thesealonedonotappeartobe

strongriskfactors.Rather,integrationwithsignalsdrivenbyenvironmentalfactors(e.g.

smokingordiet)isrequiredtotriggerdisease.Recognitionoftheimpactof

environmentalfactorshasfocusedinterestontheroleofthemicrobiota5,7,8andhence,

onhowhelminthsmayregulatethisinhealthanddisease3,9,10.Indeed,emergingdata

suggestthatcommensalbacteriaandgastrointestinal(GI)helminthsappearto

reciprocallyregulatethecompositionofthegutmicrobiome11,12.Itappearsthatsuch

crosstalkhasevolvedtohomeostaticallymaintainimmunesystemfunctioninhealth

anddisease2asGIhelminthscaninduceregulatoryresponsestolimitinflammationand

promoteintestinalbarrierintegritywhiletheintestinalbacteriaplayanessentialrolein

trainingtheimmunesystembyimpactingonstemandprogenitorcells13,14.Consistent

withtheformer,thereisincreasingevidencefromanimalmodelsthattheprotection

affordedbyGIhelminthinfection,againstinflammatorydisordersassociatedwith

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mucosaltissueslikeasthma15andinflammatoryboweldisease16,17andcoeliac

disease18,19,involvesmodulationofthegutmicrobiota.

Nevertheless,gut,lungororaldysbiosishasalsobeenimplicatedintheaetiologyofa

widerangeofsystemicandorgan-specificautoimmunediseasesincluding

musculoskeletalpathologieslikeRAandSLE20-31.Itisnotclearwhethertheprotection

affordedbyGIhelminthsagainstthesedisorderssimilarlyinvolvesinteractionwiththe

microbiome,butinfectionwithhelminthslikeHeligmosomoidespolygyrusandTrichuris

muriscanresultinincreasesinLactobacillaceaeanddecreasesinPrevotellaspecies32-35,

commensalsthatarereportedtobedysregulatedinRApatients24,31.Inanycase,

helminth-mediatedprotectionagainstautoimmunediseaseisnotlimitedtoGI-tract

parasites,withparticularlystrikingexamplesofthisinvolvingfilarialnematodeswith

respecttoRA36andSLE37beingreportedinIndia.However,todateithasbeenunclear

whethertissue-residentorblood-borneparasiticwormscanmediatetheseeffectsvia

modulationofthehostmicrobiomeandifso,whichmechanismstheyutilisetoachieve

thisprotection.

Theabilityofhelminthstoamelioratechronicinflammatorydisordershasoftenbeen

attributedtotheircapacitytoexcreteorsecretemolecules(ES)thatexert

immunoregulationtopromoteimmune-evasionandlimithostpathology2.Amongstthe

bestcharacterisedESproductsisES-62,aphosphorylcholine(PC)-containing

glycoproteinsecretedbytherodentfilarialnematodeAcanthocheilonemaviteaethatwe

haveshowntopreventinitiationandprogressionofpathologyinmousemodelsof

certainallergic(asthma,contactdermatitis)andautoimmune(RA,SLE)inflammatory

diseases1,2,38-43.Collectively,ourstudieshaveidentifiedaunifyingmechanismofaction

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thatallowseffectiveprotectionirrespectiveoftheinflammatoryphenotype:thus,by

subvertingTLR4signallingtodownregulateaberrantMyD88-responses,ES-62actsto

homeostaticallyresettheregulatory:effectorimmunecellbalance,primarilytorestore

levelsofIL-10+regulatoryBcells(Bregs)andsuppresspathologicalIL-17-driven

inflammation1,2,38-44.InexperimentalmodelsofRAandhumandisease,perturbationof

themicrobiotahasbeenshowntodisruptthebalanceofpathogenicTh17cellsandthe

counter-regulatoryBregsandTregsthatacttohomeostaticallyresolve

inflammation20,24-26.Thus,ouraimherewastoinvestigatewhethertheactionsofES-62

reflectedanabilitytoimpactonthemicrobiome.Consistentwiththis,wenowshow

thatwhilstjointdiseaseinthecollagen-inducedarthritis(CIA)mousemodelofRAis

precededbydisturbanceofthegutmicrobiomewithaccompanyingintestinal

inflammationandlossofbarrierintegrity,ES-62actstonormalisethemicrobiomeand

maintainguthealthandfunction.Furthersupportingourhypothesisthatsuchlocalgut

pathologyandinflammationdrivesjointdisease,prophylacticdepletionofthegut

microbiotawithbroad-spectrumantibiotics(ABX)wasfoundtoreducetheconsequent

severityofarthritisinmiceundergoingCIA.SuchABX-treatmentalsoreducedthelevel

ofprotectionaffordedbyES-62,withtheseanimalsexhibitinganintermediate

phenotypeofdisease,comparabletothatofthecontrolABX-treatedmiceundergoing

CIA.Thesedatathereforeindicatethata“normalised”microbiomeisalsorequiredfor

thefullinductionoftheimmunoregulatoryactionsofES-62.

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2. MaterialsandMethods

2.1. CollageninducedArthritis(CIA)

MaleDBA/1micewerepurchasedat6-8weeksofage(Envigo;Bicester,UK)andthen

housedandmaintainedintheCentralResearchFacilityoftheUniversityofGlasgow.All

experimentswereapprovedby,andconductedinaccordancewith,theAnimalWelfare

andEthicalReviewBoardoftheUniversityofGlasgow,UKHomeOfficeRegulationsand

LicensesPPLP8C60C865,PILI518666F7,PIL1675F0C46andPILICEBDB864.

CIAwasinducedusingbovineCollagenTypeII(CII-100μg)emulsifiedwithcomplete

Freud’sadjuvant(MDBiosciences)injectedintradermallyonday0.Micewere

challengedwith200μgCIIinPBSintraperitoneallyonday21.Animalsweretreated

withPBSorpurifiedendotoxin-freeES-62(2µg/injection)subcutaneouslyondays-2,0

and21andjointinflammationanddamage(articularscore)determinedasdescribed

previously39,45,46.Gripstrengthwasrecordedaspermanufacturer’sinstruction(Ugo

basile®,Italy)usingaGrip-StrengthMeter,whichmeasuredthegripstrength(peak

forceandtimeresistance)oftheforelimbsofthemice.Theanimalswereplacedovera

baseplateandgrippedaT-shapedgraspingbarwhichwasconnectedtothepeak

amplifierthatautomaticallydetectstheanimal’sresponse.Threemeasurementswere

takenandtheaveragegripstrengthwascalculated.Inordertoinvestigatetheimpactof

gutmicrobiomeperturbationoninitiationandprogressionofinflammatoryarthritis,

animalsweregivendrinkingwatercontaining[ornot]acocktailofantibiotics(500

mg/LVancomycin,1g/LNeomycinand1g/LMetronizadole)toeliminateGram-positive,

Gramnegativeandanaerobicmicroorganisms257dayspriortotheinductionofCIA

andthereaftercontinuouslythroughouttheexperiment.Bloodwassampledusing

endotoxin-freeneedlesandsyringesandtheresultingserumisolatedandstoredat-

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20oCinendotoxin-freeEppendorftubes.Paws,ileumandcolontissuewerefixedin4%

paraformaldehyde;ileumandcolonfaecalcontentswerecollectedinsterileRNAlater

(Sigma)andstoredat-80oC.

2.2. FlowCytometry

Spleenandbonemarrow(BM)cellsweresuspendedinFACSbuffer(2.5%BSA;0.5mM

EDTA,inPBS)followingredbloodcell-lysis(eBioscience).BMcellswerelabelledwitha

cocktailofPE-labelledantibodiesspecificforCD3,B220andTer119toexcludeanalysis

oflymphocytesanderythroidcellpopulationsusingadumpchannel,andmonocytes

wereidentifiedbylabellingwithantibodiesagainstCD11b(FITC),Ly6C(PerCPCy5.5)

andLy6G(APC)43.Lymphocytesfromthespleenwerelabelledwithantibodiesspecific

forCD19(AF700)andIL-10(PEorAPC).Allantibodieswerepurchasedfrom

BioLegend,UK.Fixableviabilitystain(APC-ef780;ThermoFisherScientific)wasusedto

selectforlivecellsandforanalysisofIL-10+regulatoryBcells(Bregs),lymphocytes

werestimulatedwithPMA,ionomycin,BrefeldinAandLPSasdescribedpreviously41,44.

DatawereacquiredusingaFACSCantoorBDLSRIIflowcytometerandpopulations

weregatedasdescribedpreviouslyusingisotypeandfluorescenceminusone(FMO)

controlsusingFlowJo,LLCanalysissoftware(TreeStar/BD)41,44,43.

2.3. Histology

Ileum,colonandjoint(paw)tissuesfromindividualmiceineachtreatmentgroupwere

fixedin4%paraformaldehydefor24hoursbeforeguttissueswereembeddedinOCT

andpawjointsweredecalcifiedandsubsequentlyparaffinembedded.Paraffinsections

(6µm)andOCTcryosections(9-10µm)werepreparedandstandardH&Ehistological

stainingwasperformedonalltissuesforidentificationofmorphologicalchanges39,43,46.

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Ileum(villithickness)andcolon(numberoflesions)pathologywasquantitatedby

ImageJanalysis.Jointpathologywasscoredaccordingtothegradingsystemof0forno

inflammation,1formildinflammation,pannusformationandbonedamage,uptoa

scoreof4representingahighlevelofinflammation,pannusinfiltrationandboneand

cartilagedestruction,aspreviouslydescribed47.

2.4. Osteoclastdifferentiation

OCsweredifferentiatedfromBMobtainedfromthehindlimbsofexperimentalanimals

aspreviouslydescribed43.Briefly,followingremovalofadherentcells,BMcellswere

culturedinαMEMmediumsupplementedwith30ng/mlM-CSFand50ng/mlRANKL

(Peprotech,London,UK)andthenassessedforOCdifferentiationbyTRAPstaining

(LeukocyteAcidPhosphataseKit,Sigma-Aldrich,UK)onday5.Imageswereobtained

usinganEVOSFLAutoCellImagingSystem.TRAP+cellswith≥3nucleiwere

enumeratedandImageJsoftwarewasusedtocalculatetheaveragesizeof

multinucleatedOCsperfieldofview(FoV)43.

2.5. SerumcytokineandantibodyELISAs

Interleukin-6(IL-6)andIL-10expressionwasmeasuredbyELISAaccordingtothe

manufacturer’sinstructions(BDBiosciences,Oxford,UK).Fordeterminationofcollagen

typeII(CII)-specificIgG1andIgG2aantibodiesinserum45,highbinding96wellELISA

plateswerecoatedwithCII(5µg/ml)overnightat4oCbeforewashingandblocking

withBSA/PBS.Serumwasdiluted1:100andthenseriallydilutedthree-folduntil

1:218700andincubatedwithHRP-conjugatedgoatanti-mouseIgG1orIgG2a

(1:10,000)in10%FBS/PBSpriortodevelopingwithTMBand2Msulphuricacidand

readatanopticaldensityof450nm.

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2.6. qRT-PCR

BMcells(106)werelysedinRLTLysisBuffer,whilesplenictissuewaslysedinTrizol

reagent(Sigma),priortomRNAextractionusingRNeasyPlusMinikit(Qiagen,

Germany)orphenol-chloroformextraction,respectively,accordingtothe

manufacturer’sinstructions.TheHighCapacitycDNAReverseTranscriptasekit

(AppliedBiosystems,LifeTechnology,UK)wasusedtogeneratecDNAforusewith

StepOnePlus™real-timePCRsystem(AppliedBiosystems,UK)andKiCqStart®qPCR

ReadyMix(Sigma-Aldrich).Pre-designedKiCqStart™Primers(Sigma-Aldrich)were

purchasedtoevaluateRANK(tnfrsf11a;forward–GAAATAAGGAGTCCTCAGGG,reverse

-GAAATAAGGAGTCCTCAGGG),OPG(tnfrsf11b;forward–

GAAGATCATCCAAGACATTGAC,reverse-TCCTCCATAAACTGAGTAGC),MyD88(myd88;

forward–GAAGATCATCCAAGACATTGAC,reverse-TCCTCCATAAACTGAGTAGC)andβ-

actin(actb;forward-GATGTATGAAGGCTTTGGTC,reverse-

TGTGCACTTTTATTGGTCTC).Datawerenormalisedtothereferencegeneβ-actinto

obtaintheΔCTvaluesthatwereusedtocalculatethefold-changefromtheΔΔCT

followingnormalisationtobiologicalcontrolgroup.

2.7. Metagenomics

GenomicDNAfromtheileumandcolonfaecalmatterwaspurifiedusingQIAampDNA

StoolMiniKit(Qiagen,UK)andstoredat-20oC.FormetagenomicanalysisusingtheIon

TorrentPGM™platform,samplesfromthreeindividualmicepergroupwerepooledand

between10and100ngofthepooledDNAwasfragmented(NEBFastDNA

Fragmentation&LibraryPrepSetforIonTorrent,NEBInc,UK)andbarcoded

(IonXpressBarcodeAdaptersKit,ThermoFisherScientific,UK).Barcodedlibrarieswere

quantifiedusingQubitFluorometer(ThermoFisherScientific,UK)andbioanalyser

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(HighSensitivityDNAanalysisKit,Agilent,UK).Uptothreebarcodedlibrarieswere

combinedperIon316™ChipKitv2followinglibrarypreparationusingtheIonPGM™

Hi-Q™ViewOT2andIonPGM™Hi-Q™ViewSequencingKits(ThermoFisherScientific,

UK).DatawereextractedasFASTQfilesandanalysedusingMG-RASTtogenerate

taxonomicdatafromsequencingreads48.Thenumberofreadsperphylum,class,order,

family,generaorspeciesofinterestwereexpressedasacompositionofallbacteria

presenttonormaliseforvariationbetweensequencingruns.Sequencingrunscanbe

accessedusingMG-RASTIDs;mgm4777616.3,4777615.3,4777614.3,4777613.3,

4777481.3,4777480.3,4777479.3,4777478.3,4767994.3,4767993.3,4767992.3,

4767991.3,4767990.3,4767989.3,4767988.3,4767987.3,4767986.3,4738191.3,

4738190.3,4738025.3,4737887.3,4737053.3and4737052.3.qPCRwasusedto

validatechangesinbacterialpopulationsusingprimersspecificforBacteriodetes

(forward-GTTTAATTCGATGATACGCGAG,reverse-TTAAGCCGACACCTCACGG)

Firmicutes(forward-GGAGCATGTGGTTTAATTCGAAGCA,reverse-

AGCTGACGACAACCATGCAC)andButyrivibrio(forward–GCGAAGAAGTATTTCGGTAT,

reverse-CCAACACCTAGTATTCATC)andwerenormalisedtothetotallevelsofbacteria

usingpan-bacterialprimers(forward-CGGTGAATACGTTCCCGG,reverse-

TACGGCTACCTTGTTACGACTT).

2.8. Statistics

AlldatawereanalysedusingGraphPadPrism6softwareusingOneorTwo-WayANOVA

withFishersLSDpost-testsforparametricdataorKruskal-WallistestandDunn’spost-

testfornon-parametricdata.UnsupervisedhierarchicalclusteringwithEuclidean

distancewasperformedonthecolonsamplesusingtheheatmap.2functionofthe

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gplotspackageinR.SupervisedheatmapsweregeneratedusingGraphPadPrism7

software.Indicatorsofsignificanceinclude*=p<0.05,**=p<0.01and***=p<0.001.

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3. Results

3.1ES-62protectionagainstCIAisassociatedwithnormalisationofthegut

microbiome

Asreportedpreviously,ES-62amelioratesCIAintermsofarticularscore(Fig.1a)and

reflectingthisprotectionagainstjointmorbidity,wenowshowthatitpreventsthe

resultinglowgripstrengthfoundinCIA-mice(Fig.1b).Indeed,treatmentwithES-62

maintainsgripstrengthinCIA-miceatasimilarleveltothatfoundinhealthy,Naive(not

subjectedtoCIA)DBA/1mice(Fig.1b).Commensalbacteriahaveincreasinglybeen

proposedtoplayaroleinRApathogenesis(reviewedin49-51)andthedeclineingrip

strengthduringageinghasbeenassociatedwithchangesinthegutmicrobiome52.

Interestingly,inadditiontobeinganindicatoroffrailty,gripstrengthhasbeenshown

tobeapredictorofawiderangeofadversehealthoutcomes53,e.g.,cardiovascular

disease,whichRApatientsareatincreasedriskofdeveloping54andthatareimpacted

bythemicrobiome49,55.Thus,toaddresswhethertheprotectionaffordedbyES-62is

associatedmodulationofthegutmicrobiota,ametagenomicapproachwasusedto

profilethebacterialpopulationspresentintheintestinesofCIA-mice,exposedornotto

ES-62.InitiationofRA(andCIA)pathogenesisisassociatedwithdisruptionofthe

balanceofeffector:regulatoryimmunesystemcellsandsowecharacterisedthe

bacterialchangespertainingduringestablishedarthritisintheileumandcolon,

intestinalsiteswherethemicrobiomeandthemetabolicmicroenvironmentplaykey

rolesinshapingTh17(ileum-associated)andregulatory(colon-associated)immune

responses25,56,57.

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Anoverviewofthemicrobiotaoftheileumandcolonatthephylumlevelshows

substantialchangesbetweenhealthyNaiveDBA/1miceandthosewithestablished

arthriticdisease(PBS;Fig.1c).FirmicutesandBacteriodetesarethepredominantphyla

inalltreatmentgroupsbutwithrespecttoCIA-mice,whereastheyexhibitoutgrowths

ofFirmicutesandProteobacteriaintheileum,bycontrasttheyexhibitdecreasedlevels

ofFirmicuteswithacompensatoryoutgrowthofBacteriodetesinthecolon(Fig.1c).ES-

62essentiallyhelpstomaintainthehealthydiversityofthemicrobiotaobservedin

Naivemice,whichwasreducedintheCIA-mice(Fig.1c).Deeperanalysisillustratesthe

differentialdiversitysignaturesexhibitedbyhealthyandarthriticmice,aswellasthe

impactofES-62treatment(Fig.1d):drillingdownonthemodulationoftheGram-ve

Bacteroidetesphylumrevealssomekeydifferentialsignaturesthroughouteachofthe

predominantBacteroides,PorphyromonasandPrevotellagenerabetweenthecolon

contentsofNaiveandCIAmice,andidentifyingthosenormalisedbyexposuretoES-62

(Fig.1e).Strikingly,atthespecieslevel,B.vulgatuswasfoundtobesignificantly

increasedinthecolonofCIArelativetoNaiveorES-62-CIAmice(Fig.1e).Bycontrast

abundanceofmembersoftheRikenellaceaefamilyandspecifically,Alistipesputredinis,

werereducedinthecolonofCIAmicewhencomparedtoNaiveandES-62-treatedCIA

mice(Fig.1e).Similarly,inspiteofthefactthatdifferentialprofilesamongstthe

treatmentgroupswereobservedthroughoutmajorgenera(e.g.Bacillus,Staphyloccus,

Streptococcus,EnterococcusandClostridium)ofGram+veFirmicutes,themostdramatic

changesobservedwithestablishedCIAoccurredwithintheClostridialesorder.In

particular,decreasesintheClostridiaceae(Fig.1f)andtheLachnospiraceae(Fig.1f)

familieswerenotedwithES-62promotingmaintenanceoftheRuminococcus,

Faecalibacterium,BlautiandErysipelotrichaceaegeneraandbutyrate-producingDorea

andRoseburiaspecies,thelatterofwhichhavebeenimplicatedinguthealthand

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inflammationhomeostasis58.IntermsoftheProteobacteria,CIAwasassociatedwith

outgrowthofmembersoftheHelicobacter,specificallyHelicobacterhepaticus

(Epsilonproteobacteria)andEscherichia,particularlyEscherichiacoli

(Gammaproteobacteria)genera(Fig.1g)andagainthiswasnormalisedbyES-62.

PerturbationofthemicrobiomewasalsoobservedintheileumofCIAmiceandagain,

exposuretoES-62actedtonormalisethistowardsthehealthycommunity(Fig.1c).In

termsofprotectivesignatures,despitetherelativepaucityofbacteriaintheileum

relativetothecolon,ES-62clearlypromotedgrowthoftheClostridiales,again

particularlyClostridaceaeandLachnospiraceaespeciesbutinthiscasegenerally

promotingtheiroutgrowthbeyondthelevelsfoundinhealthymice(Fig.1h).This

presumablyreflectsthatwhilsttreatmentofnaive,healthyDBA/1micewithES-62for

thedurationoftheCIAmodelhadlittleeffectonthecolonmicrobiota,itpromoted

expansionofClostridialesspeciesintheileum(Fig.S1aandb),perhapsexplainingthe

increaseinspeciesdiversityobservedintheileum(Fig.S1c)butnotthecolon(Fig.S1d)

ofCIA-micetreatedwithES-62.Additionally,reflectingtheabilityofCIAtoperturb,and

ES-62tonormalise,thegutbacteria,functionalmetagenomicanalysisalsoshowedthat

ES-62generallyactedtonormalisethemetaboliccapacityofthecolonicmicrobiome

(Fig.1i).

3.2.Perturbationofthegutmicrobiomewithbroad-spectrumantibioticsboth

amelioratesCIAandimpactsontheprotectionaffordedbyES-62

ToaddresswhethertheperturbationofthemicrobiomeobservedinCIAplaysarolein

theinitiationandprogressionofinflammatoryarthritis,weinvestigatedtheeffectof

continuousexposureofmicetoacocktailofbroad-spectrumantibiotics(ABX;500

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mg/LVancomycin,1g/LNeomycinand1g/LMetronizadole)administeredintheir

drinkingwaterfromoneweekpriortoinitiationofCIA.SuchABXtreatmenthadno

obviouseffectontheoverallhealthoftheanimalsas,afteracharacteristicinitialdip,

therewasnosignificantdifferenceinbodyweightamongsttheCIAgroupsatcull(Fig.

S2a).Nevertheless,thisregimenessentiallyeliminatedthebacterialmicrobiotaofall

animalsirrespectiveoftreatmentgroup(Fig.S2b),whilstmetagenomicanalysis

showedtheresidualgutcommunitytobealmostentirelycomprisedofproteobacteria

(Fig.S2candS2d).Aspredictedfrompreviousstudiesinavarietyofinflammatory

arthritismodels25,59-61,ABXtreatmentreducedtheincidence(PBS,65.2%;PBS-ABX,

36.3%,asmeasuredbyarticularscore≥1)andseverityofjointpathologyinCIA-mice,

bothintermsofarticularscore(Fig.2a)andhistopathology(Fig.2bandc).Inaddition,

theprotectionaffordedbyES-62wasreducedinABX-treatedanimalssuchthatthe

articularscoreofsuchmicewasnotsignificantlydifferentfromPBS-CIAmicereceiving

ABXornot(Fig2a).Thus,prophylacticadministrationofABXresultedinan

intermediatephenotypeofCIAirrespectiveofwhetherthemiceweretreatedwithPBS

orES-62.

ES-62-mediatedprotectionagainstCIAisassociatedwithrestorationofthehomeostatic

balanceofregulatory:effectorBcellresponsesviadown-regulationofaberrantMyD88

signalling2,44.Typically,ES-62actstoreducepathogenicanti-CIIIgG2abutnotIgG1

antibodyproduction45,62.ReflectingtheABX-drivenameliorationofCIApathology,it

wasnotedthatanti-CIIIg2alevelsinPBS-ABXmicearenotsignificantlydifferentfrom

thelowerlevelsoftheseantibodiespertaininginES-62-CIAmicewhilstthoseinES-62-

ABXmicearenolongersignificantlyreducedrelativetothoseinPBS-CIAmice(Fig2d).

Nodifferencesweredetectedamongstanyofthegroupintermsofanti-CIIIgG1

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antibodies(Fig.2e).ConsistentwiththeeffectsofABXoneffectorBcellresponses,

analysisofsplenicIL-10+CD19+“regulatory”Bcellsshowedthatboththedecreasein

thesecellsoccurringduringCIA(PBS-CIA)andalsothemaintenanceofhealthylevelsin

ES-62-CIAmice2,44werelostinABX-treatedanimals,withthelevelsinES-62-ABXmice

mirroringthosereducedlevelsfoundinPBS-CIAanimals(Fig.2f).Moreover,whilstES-

62actstoincreaseserumIL-10levelsinCIAmice,thisregulatorycytokineisfoundat

similarlylowlevelsinPBS-CIA,PBS-ABXandES-62-ABXmice(Fig.2g).Atthesametime,

ES-62-mediatedsuppressionofserumlevelsofIL-6,acytokinethatpromotesBcell

(auto)immunity63,64,islostfollowingABXtreatmentsuchthatES-62-ABXmicedisplay

thehighlevelsofthiscytokinefoundinPBS-CIAmice(Fig.2h).

Inadditiontomaintainingimmunesystemhomeostaticregulationandpromoting

resolutionofinflammationthatbecomesdysregulatedinCIAmice,ES-62alsoactsto

suppressthefunctionalmaturationofosteoclasts(OC)43thatdirectlycauseerosivejoint

damage.Interestingly,changesintheintestinalmicrobiomehavebeenshowntoimpact

onbonemass65-67andwethereforeinvestigatedwhethertheintermediatephenotypeof

jointpathologyoccurringinABX-treatedCIAmicereflectedmodulationof

osteoclastogenesisresultingfromperturbationofthegutmicrobiota.Asshown

previously,exposuretoES-62invivohadnoeffectperseonthenumbersofOC

differentiatedfrombonemarrow(BM)progenitorsexvivo(Fig.3a)butrather,it

blockedtheirfusiontolarge,activemultinucleatedcells(Fig.3a-c)thatresorbbone43.

ConsistentwithitsameliorationofCIApathology,ABXadministrationresultedina

decreaseinlargemultinucleatedOCandacorrespondingincreaseinthetotalnumbers

ofOCsdetectedinBMfromCIAmice(Fig.3a-c).ES-62rewiresosteoclastogenesisby

modulatingtheRANK/OPGboneremodellingaxis43andthisisevidencedagainhereby

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itsabilitytosignificantlydecreaseexpressionofRANKandincrease(albeitnot

significantly)expressionofthedecoyreceptor,OPGinBMrelativetothatseeninBM

fromPBS-CIAmice(Fig.3dande).ThisaxisisindeedtargetedbyABXtreatment,with

theelevatedRANKexpressionobservedinPBS-CIABMbeinglostfollowingABX

treatmentsuchthatthelevelsinBMfrombothPBS-ABXandES-62-ABXmicewerenot

beingsignificantlydifferenttothosedetectedinES-62-CIABM.Inaddition,OPG

expressionwasessentiallythesameinPBS-andES-62-ABXmiceandsimilartothatin

PBS-CIABM(Fig.3dande):thesechangeswouldresultinsimilarlevelsofRANKL-

drivenosteoclastogenesisconsistentwiththeintermediateCIAphenotypeobservedin

PBS-andES-62-ABXanimals.SupportingtheseABX-changesinosteoclastogenesisand

consequently,bonedamageinCIA,PBS-ABXandES-62-ABXanimalsexhibitsimilargrip

strengths(70.8±5.4%and58.8±6.0%,respectively)thatareintermediatetothose

displayedbyPBS-(42.3±6.2%)andES-62-CIA(90.1±9.6%,relativetonaivemice)(Fig.

1).Ourdataarereminiscentofrecentstudiesshowingthatcommensalbacteriadonot

impactondifferentiationofmonocytestotheosteoclastlineageperse,butrather

enhancefunctionalmaturationofOCsasevidencedbytheirgreatersize(x2.5fold)and

boneresorptioncapabilities.Moreover,thisappearstobeachievedbycommensal

bacteriamodulatingtheRANKL:OPGratiotoresultinincreasedandsustainedRANK

signalling68.

3.3.ES-62protectsagainstgutpathologyinCIA

Intestinaldysbiosishasbeenassociatedwithlossofgutintegrityandchronic

inflammationinconditionssuchasobesitythatareknowntopromoteautoimmune

disorderslikeRAandcardiovascularcomorbidities69,70.Ourmetagenomicanalysis

showedthatestablishedCIAwasassociatedwithchangestothegutmicrobiome,

18

particularlywithrespecttoareductioninbutyrate-producingspecies(Fig.1g,i,jand

S1)thathavebeenimplicatedinthemaintenanceofepithelialintegrityandgut

health50,58.Strikingly,wehavefoundthatmicewithCIAdisplayseveregutpathology

andinflammation,thelevelsofwhichdirectlycorrelatewithseverityofCIA(Fig.4a).

Moreover,treatmentwithES-62protectsagainstsuchgutdamage,specificallythe

thickeningandgenerationofstubbyvilliintheileum(Fig.4bandc)andhole-like

“lesions”appearinginthecolon(Fig.4bandd).Thephysiologicalrelevanceofthese

colon“lesions”isunclearbutmayreflectthewidely-establishedsuppressionofmucus

productionbygobletcellsinresponsetobacteria71,72oralternatively,theymaybe

attachment/effacementlesionsinducedbypathogenicbacteriaincludingE.coli73.The

gutpathology(boththeileumvillithicknessandcolonlesions)observedinPBS-CIA

micewasabrogatedinsuchanimalsadministeredABX(Fig.4candd)suggestingthat

theassociatedreductioninchronicgut(andconsequentlysystemic)inflammation

contributestotheameliorationofCIAinPBS-ABXmice.Ofnote,ES-62-ABXmice

displayedincreasedpathologyandinflammatorycellinfiltrationoftheguttissue

comparedtoES-62-CIAmice,butagainthiswasalsoconsistentwithchronicgut

inflammationpromotingdiseaseseverity.

TofurtheraddresstheroleofgutpathologyinCIA,weanalysedileumandcolontissue

fromnaivemiceandthoseundergoingCIA(treatedwithPBSorES-62)atkeypoints

duringinitiationandprogressionofdisease:(i)Naivemice,(ii)thebreakingof

toleranceandinitiationofdisease,followingimmunisationatday0withCII/CFA(≤day

14),(iii)preclinical(day21,priortoboosterimmunisationwithCII)and(iv)

establisheddisease(≥day28;articularscore:PBS–5.2±0.8;ES-62-0.9±1.24)phases.

ThisanalysisrevealedthepresenceofgutpathologyinPBS-CIAmiceduringthe

19

initiationphasewithanincreaseinileumvillithicknessandasignificantincreaseinthe

numberofcoloniclesionsfollowingimmunisation(Fig.5a-c).Interestingly,theileum

pathologyoccurringduringtheinitiationstageinPBS-CIAmiceappearedtoresolvein

theseanimalsbytheendofthepre-clinicalphase(day21),althoughthickeningand

shorteningofthevilliwasagaininducedbytheboosterCIIimmunisation.Thispattern

wasnotthecaseforthecolonlesions,whichpeakedatday21andweremaintained

throughoutactivediseaseinPBS-CIAmice.AsopposedtoCIA-mice,ileumintegritywas

maintainedthroughoutallphasesofdiseaseinES-62-CIA-micewhilstthehighlevelsof

colonlesionsgeneratedbyday21ofthepre-clinicalphasewerereducedinES-62-

treatedanimals(Fig.5bandc).

Validatingourmetagenomicanalysis,qPCRanalysisshowedthatES-62actstoprevent

theenrichmentofBacteriodetesandmaintainlevelsofFirmicutesinthecolonofmice

duringestablishedCIA(Fig.6aandb).However,analysisatthevariouspre-clinical

phasesofthediseaseshowedamoredynamicsituation:forexample,followingthe

primaryimmunisationwithCII/CFA,thereisasignificantdecreaseinthelevelsofboth

BacteroidetesandFirmicutes,withtheriseinBacteriodetesevidentinestablished

arthritisonlyoccurringfollowingtheboosterimmunisation(Fig.6aandb).Incontrast,

ES-62actstomaintain“healthy”levelsofFirmicutesinCIA-micethroughoutbutmost

stronglyduringtheinitiationandpreclinicalphasewhereitalsoactedtomaintainthe

lowestlevelsofBacteroidetes(Fig.6aandb).Moreover,althoughES-62treatment

promotedenrichmentofbutyrate-producingbacteria(Butyrivibrio)atallstagesof

disease,thiswasmostevidentintheinitiationphaseofdisease,andpresumablythis

promotesandmaintainsgutintegrity(Fig.6c).

20

Commensalbacteriaplaykeyrolesineducatingtheimmunesystemandbonemarrow

progenitorsandhence,lossofdiversityinthemicrobiomecanimpactonboth

inflammationandbonehomeostasis68,74.Such“training”involvesinteractionsofPattern

RecognitionReceptors(PRR;e.g.TLRsandNODs)withthegutmicrobiome2,5,13,75.

Consistentwiththis,ES-62canrewireBMprogenitorsandstromalcellsfromCIAmice

toananti-inflammatory,regulatoryortissuerepairphenotype2,39,43,46,76,77by

subvertingTLR4signallingtopreventtheup-regulationofthekeyTLRsignal

transducer,MyD88observedduringchronicinflammation78,79.Interestingly,therefore,

giventheincreasedincidenceandseverityofCIAinES-62-ABXmice,weshowthatES-

62dampeningofaberrantMyD88expressionisabolishedbyABXtreatmentandindeed,

thatMyD88isexpressedatequivalentlevelsinBMfromPBS-CIA,PBS-ABXandES-62-

ABXmice(Fig.6d).

CommensalbacteriacanalsodynamicallyeducateOCprogenitor(OCP)maturation68,74:

asOCsnormallyactinconcertwithosteoblasts(OBs)tohomeostaticallymaintain

healthybone,theenhancedfunctionalcapacityofOCselicitedbycommensalbacteria

mayactuallyrenderhostsmoresusceptibletobonedamageduringchronic

inflammation,aconditionthatpromotesboneremodelling,particularlyascommensal

bacteriaalsoappeartoacttosuppressOBfunction68,74.Reflectingthedynamicand

differentialchangesincolonicmicrobiota,gutinflammationandpathology,thereisa

strongincreaseinthemonocytepopulationscontainingOCPsduringtheearly

inflammatoryinitiationphaseofCIAthathasresolvedbytheendofthepreclinical

phaseonlytoincreaseagainintheestablishedphaseofarthritisfollowingthebooster

CIIimmunisation.ConsistentwithourdatathatES-62doesnotfundamentallysuppress

OCdifferentiation,therearenodifferencesinthepercentageofmonocytesbetweenthe

21

PBS-CIAandES-62-CIAgroups(Fig.6e).However,BMfromCIAmicefollowingboth

primaryandboosterimmunisations,showsenhancedcapacityforfunctionalOC

maturationthatissuppressedbyinvivoexposuretoES-62(Fig.6fandg).

22

4. Discussion

Thisstudyisthefirsttodemonstratethatadefined,systemically-actingparasiticworm-

derivedproductcanimpacton,andharness,themicrobiometoexertitstherapeutic

effectsagainstchronicinflammationintargetorgansdistaltothegutsuchasthejoints.

Collectively,ourdatasuggestthatES-62-mediatedprotectionagainstinflammatory

jointdiseaseisassociatedwithnormalisationofthegutdysbiosisobservedinarthritic

micetowardsthe“healthy”microbiomeobservedinnaivemice.This“normalisation”is

highlyreminiscentofthatseenfollowingtreatmentofRApatientswithDMARDs80and

lendsfurtherweighttoincreasingevidencethatchangesinmicrobiomestatusmay

contributetopathogenesisinthismultifactorialdisease31,49,74,81,82.Indeed,perturbation

ofthemicrobiotahasbeenshowntoinfluenceboththegenerationofpathogenicTh17

cellsandalsothehomeostaticinductionofinflammation-resolvingBregsandTregsin

experimentalmodelsofRAandhumandisease20,24-26.Moreover,andconsistentwiththe

ideathatmicrobiomestatuscanpromotediseaseinsusceptibleindividuals,

developmentofarthritisinanumberofanimalmodelsislostunder“Germ-Free”(GF)

conditionsorfollowingdepletionofthemicrobiotabytreatmentwithbroad-spectrum

ABX25,49,59,61.

Certainly,potentiallypathogenicchangesinthegutmicrobiomehavebeenreported

followinganalysisofstoolfaecesfrompatientswithnew-onsetdiseaseaswellasthose

withestablishedarthritis.However,differential,andevencontradictory,associations

havebeenreportedthatperhapsreflectthedifficultiesinstandardisingstoolsamples

ortheirfailuretofullyrecapitulatethecontentsofthegutmicrobiotaandthespecific

communitiesshapedbytheintestinalmicroenvironment31,83-85.Nevertheless,with

respecttotheBacteroidetes,whoselevelswefoundtobegenerallyelevatedduring

23

establishedCIA,Prevotellacoprihasbeenreportedtobeenrichedinstoolsamplesof

new-onsetpatients24,86.Moreover,transferofsuchfaecalmattercontainingPrevotella

copriintoGFarthritis-proneSKGmiceleadstoenhancedlevelsofzymosan-stimulated

intestinalTh17cellsandmoreseverearthritis86.However,SKGmiceareincreasingly

perceivedtobeamodelofSpondyloarthritisratherthanRA31andperhapsreflecting

this,althoughwefoundPrevotellaceaespeciestobeupregulatedinthecolonofmice

withestablishedarthritis,thiswasneitherrestrictedtoP.coprinorwasitreversedby

treatmentwithES-62.Moreover,P.histocolahasbeenreportedtobeprotectiveagainst

CIAinmicetransgenicfortheRA-riskgeneHLA-DQ887,whilstatthegenuslevel,

Prevotellaspecieswerefoundtobeunder-representedintheIL-1rn-/-modelof

autoimmunearthritis60.IntermsofthepathogenicpotentialofBacteroidetesinCIA,we

foundB.vulgatustobesignificantlyenrichedandA.putredinis(Rikenellaceaefamily)to

bedepletedinthecolonofCIArelativetoNaiveorES-62-CIAmice.Interestingly,a

similarinversepatternofB.vulgatusandRikenellaceaespecieswasobservedinrats

transgenicforHLA-B27,ahumanisedrodentmodelforrheumatologicalconditionssuch

asAnkylosingSpondylitis,ReactiveArthritisandPsoriaticArthritis88.

Colonisationwithsegmentedfilamentousbacteria(SFB)fromtheFirmicutesphylum

haslikewisepreviouslybeenshowntopromote(IL-22-dependent89)Th17responses

andarthritisinGFK/BxNmice20.However,asalsoreportedfortheIL1rn-/-model60,we

wereunabletodetectanyOTUsthatcouldbeidentifiedasSFB(e.g.Candidatus

arthromitus)inourmetagenomicanalysis:moreover,wedetectednosignificant

differencesbyqPCRinthelevelsofSFBfoundineitherthecolonandileumbetween

PBS-andES-62-treatedCIAmiceatanyphaseofdisease(datanotshown).Ourfindings

thereforeresonatewiththereportthatdespitebeingpresentinallcages,SFBcould

24

onlybedetectedin2/10miceundergoingCIApriortotheboosterCIIimmunisation61

andofrelevancetoRA,doesnotappeartobepresentinthegutmetagenomicanalysis

ofthelargenumbersofadulthumanfaecalsamplessequencedfortheHuman

MicrobiomeProject85.Indeed,andfurtherquestioningtheroleforSFBintheK/BxN

model,arecentstudyreportsthatIL-17actuallyappearsdispensableforarthritisin

K/BxNmiceandproposesthatthegutmicrobiotaregulatesjointdiseaseviaimpacting

onTfollicularhelper(Tfh)ratherthanTh17cells90.

FurtherfaecalprofilingofFirmicuteshaslinkedchangesintheClostridiaceae,

CoriobacteriaceaeandLachnospiraceaefamiliestopatientswithestablisheddisease80,82.

Ourmetagenomicanalysisofluminalcolonandileumfaecalmattershowsthe

protectiveactionsofES-62tobemoststronglyassociatedwithmaintenanceofthe

Clostridiaceae,LachnospiraceaeandRuminococcaceaefamilies,particularlythose

associatedwithbutyrateproduction(Blautia,Roseburia,DoreaandButyrvibrioand

Ruminococcus).Interestingly,Ruminococcusspecieswerefoundtobedepletedinthe

IL1rn-/-modelofinflammatoryarthritissupportingthesuggestionthatmaintenance

and/orenrichmentofbutyrate-producingbacteriaareprotective91.Reflectingthis,

administrationofbutyratewasfoundtoameliorateseverityofCIA,notablyintermsof

reducedinflammatorycellinfiltrationofthejoint,pannusformationandcartilageand

bonedestruction91.Incontrast,administrationofbutyrateexacerbatedantibody-

inducedarthritis,amodelwhereintheadministrationofserumfromK/BxNmice

(containinganti-glucose-6-phosphateisomeraseautoantibodies)toC57BL/6mice

bypassestheinitiationandadaptiveimmunityphasesofdisease.Theseapparently

conflictingdatacanbereconciledbytheproposalthatbutyrateneedstoactduringthe

preclinicalphasesofdiseasetoexhibititsprotectiveactions91.Perhapsconsistentwith

25

this,whilstES-62protectsagainstthedepletionofbutyrate-producingspeciesobserved

inmicewithestablisheddisease,wealsofindButyrivibriotobemostenrichedbyES-62

intheinitiationphaseofCIA.Collectively,thesedatasuggestthatdepletionofbutyrate-

producingbacteriaassociatedwiththeonsetofCIAmaycontributetothegutpathology

promotingandperpetuatingtheinflammationthatdrivesthebreakdownofimmune

toleranceandconsequentautoimmunejointdisease.

Certainly,wefindgutpathologytoprecedeonsetofjointdisease,beingdetectable

within6daysoftheprimaryCIIimmunisationandthelossofcolonbarrierintegrity

peakingbytheendofthepreclinicalphase(d21)ofCIA.Moreover,suchgutpathology

isaccompaniedbydynamicchangesinthemicrobiomeofCIA-miceasevidencedbythe

decreaseinthecolonabundanceofBacteroidetesandFirmicutesduringtheearly

initiationphaseofdiseaseandtheenrichmentoftheformerinmiceduringestablished

arthritis.AsimilardepletionofBacteriodetesduringthepreclinicalphase(d21)ofCIA

hasbeenindependentlyreportedbutinthiscase,wasaccompaniedbyacorresponding

enrichmentofFirmicutes61.OurfailuretoobservesuchanenrichmentofFirmicutesat

thistimepointmaysimplyreflectouranalysisofcoloncontentsratherthanfaecal

matter.Rather,wefoundES-62maintainedandenhancedthecolonlevelsofFirmicutes

throughoutallphasesofCIA,likelybypromotingenrichmentofButyrate-producing

Lachnospiraceaespeciesasseeninourmetagenomicanalysisofbothileumandcolon

faecalmatterinmicewithestablishedarthritis.Inanycase,thedynamicandintestinal-

siteselectivenatureofthedysbiosisobservedinCIA,andtheprotectionagainstit

affordedbyES-62,underlinestheneedtoidentifyandvalidatepreciseprotective

microbialsignaturesandthecontextoftheircomplexbiogeographic

microenvironment84,foreffectivetherapeuticinterventionatallstagesofhumanRA.

26

Butyrateisknowntoregulategutbarrierintegrity92,93andgobletcellproductionof

MUC294andhencedepletionofbutyrate-producingspeciesduringCIAlikelypromotes

andperpetuatesdysbiosisandlossofgutbarrierintegrity.Onepotentialconsequence

ofthisgutpathologyistheaberrantsystemiccolonisationofpathogenicbacteriaas

illustratedhereinCIAbytheoutgrowthofProteobacteria[particularlyE.coliandH.

hepaticus,thelatterofwhichhasalsobeenreportedtobeenrichedintheIL1rn-/-

model60]andaccompanyinggutpathologyreminiscentofthesuppressionofgobletcell

mucusproductionandattachment/effacementlesionsresultingfrompathological

entericinfections,includingE.coli71-73.Perhapsreflectingthelossofdiversityinthe

microbiotaandthebreakdownofintestinalhomeostasisinRA,inadditiontotheir

increasedfaecalabundanceofP.copri,earlyonsetRApatientsalsoexhibitP.copriin

theirsynovialfluid24,95.Moreover,E.coliandotherinfectiousmicroorganismshave

beenreportedtocommonlycoloniseRApatientsandmayexacerbatedisease96-98.

ThenormalisationofthegutmicrobiotabyES-62mayactuallyresultinadualpronged

mechanismbywhichbutyrate,inadditiontoitslocalgut-protectingactions,couldalso

impactsystemicallytoprotectmoredirectlyagainstjointpathologyinCIA.Consistent

withthisidea,butyratehasbeenreportedtosuppressosteoclastogenesis99andby

protectingagainstpathologicalboneloss,toregulatebonemass65.Intriguingly,wealso

findspikesoffunctionalmaturationofOCsintheinitiationandestablishedphasesof

CIAthatarepreventedbyES-62andareassociatedwithitsenrichmentofbutyrate-

producingspeciesinCIA-mice.ThemechanismsbywhichES-62orchestratessuch

maintenanceofthecomplexhomeostasisofthegut-bonemarrowaxisarenotclear.

However,itisintriguinggiventhatTLR4/MyD88signallingistheprimarytargetofES-

27

62inpromotingBregsandsuppressingTh17-mediatedinflammation2,39,41,44,thatthe

systemicTh17differentiationandconsequentautoimmunearthritisoccurringinthe

IL1rn-/-modelisdependentonTLR4100.Moreover,theaccompanyingdysbiosis,thaton

faecaltransfercanconferarthritis-predisposingTh17inflammationinwildtypemice,is

alsoregulatedbyTLR460.Furthermore,interestingly,11/44taxadisruptedinIL1rn-/-

micewerenormalisedinIL1rn-/-TLR4-/-animalsandtheseincludedRuminococcus

species,whicharealsopromotedbyES-6260.Inmechanisticterms,theresettingof

BregslevelsbyES-62inCIAhasalsobeenreportedtoberegulatedbythemicrobiome

inothermodelsofautoimmunearthritis25.Indeed,consistentwiththeproposalthat

Bregsarehomeostaticallyinducedtoresolvedysbiosis-inducedinflammationin

autoimmunearthritis,asindicatedbydisruptionoftheprocessbyABXtreatment25,we

havesimilarlyfoundtheES-62-mediatedrestorationofIL-10+BcellsinCIAtobe

compromisedbysuchperturbationofthemicrobiome.Collectively,thesefindings

suggestthatES-62mayachievebothitsimmunoregulatoryandmicrobiome

normalisationeffectsbytargetingMyD88.

TLR4/MyD88signallinginRAhadpreviouslybeenattributedsolelytorecognitionof

DAMPsinthejoint78,79andthuscollectively,thesefindingsshednewlightonits

pathogenicrolesininitiationandprogressionofdiseaseaswellasemphasiseits

potentialasatherapeutictargetinRA.Inparticular,theyunderscorethecomplexand

centralroleofTLR4/MyD88signallinginregulatingthegut-bonemarrowaxisin

musculoskeletalhomeostasisandit’sdysregulationresultinginsystemicinflammation,

breakingoftolerance,aberrantosteoclastogenesisandconsequentlyjointdestructionin

arthritis.Moreover,theysuggestthatES-62mayachieveitsprotectiveeffectsinCIAby

directlytargetingthiskeyregulatorynodeinordertorebalancethegut-bonemarrow

28

axisandlimitaberrantinflammationandjointdamage,byhomoestaticallyrestoring

levelsofBregsandresettingosteoclastogenesis.Thus,byexploitingES-62asaunique

tooltodissectpathogenicandprotectivemicrobialsignaturesinCIAwecould

potentiallyunderstandhowtoelicithomeostaticregulationofgutandresolve

inflammationinautoimmuneinflammatoryarthritis.

29

5. Acknowledgements

TheworkwasfundedbyanawardtoMMH,WHandPAHfromArthritisResearchUK

(21133).

6. Authorcontributions

JD,AT,MAP,FL,JCandAMKperformedtheexperimentsforthestudydesignedbyMMH,

WHandPAH.JDandFLmanufacturedES-62.MMH,WHandJDwrotethepaperandall

authorswereinvolvedinreviewingandrevisingthemanuscriptandhaveapprovedthe

finalversion.

7. ConflictsofInterest

Theauthorshavenoconflictsofinterest.

30

8. FigureLegends

Fig.1.ES-62“normalises”themicrobiomeduringCIAtoanaïvephenotype

(a)Articularscoresexpressedasmeans±SEMofPBS(n=10)orES-62(n=7)-treated

CIAanimals,withdatapooledfrom3experiments.(b)Gripstrengthexpressedasan

averageofthreemeasurementspermousemeasuredpriortocullasanindicatorof

forelimbstrengthandjointmorbidity(Naïve;n=4,PBS;n=8andES-62;n=6).(c)The

compositionofbacterialphylapresentintheileumandcolonofNaïve,PBSandES-62-

treatedCIAanimalspresentingproportionvaluesaspiechartsfromasingle

representativeexperimentusingpooledsamplesfrom3miceineachcondition.(d)

HeatmapanalysisofallbacteriapresentinthecolonofNaïve,PBSandES-62-treated

CIAanimals(n=3/group)fromarepresentativemodelisshown.(e-i)Statistically

significantchangesbetweenthePBS-CIAandES-62-CIAgroupsinBacteriodetes(e;

Bacteriodaceae;PBSversusNaïveorES-62,p<0.05),Firmicutes(f;Clostridiales;PBS

versusNaïve,p<0.01andPBSversusES-62,p<0.05)andProteobacteria(g;

Epsilonbacteria;PBSversusNaïveorES-62,p<0.05)inthecolonandFirmicutesinthe

ileum(h;Clostridiaceae;PBSversusES-62,p<0.05)aswellasfunctionalmetagenomics

ofthecolon(i;Phages;NaiveversusPBS,p<0.01,Proteinmetabolism;PBSversus

NaïveorES-62,p<0.01)arepresentedasheatmapswithchangesinbacterial

populationsinPBS-andES-62-treatedCIAanimalsnormalisedtoNaïvecontrols.Three

micepergroupwerepooledformetagenomicanalysisandthemeandatafrom3

independentexperimentsarepresented.Statisticalsignificancewasdeterminedusing

Two-way(a,e-i)andone-wayANOVA(b)withLSDFishersmultiplecomparisonsand

forlevelsofsignificancewhereindicatedbyasterisks,*=p<0.05,**=p<0.01and***

=p<0.001.

31

32

Fig.2.AntibiotictreatmentofPBS-andES-62-CIAanimalsresultsinan

intermediatediseasephenotype

(a)OneweekpriortoCIAinitiation,ABXwasprovidedinthedrinkingwaterofDBA/1

miceandmaintainedthroughoutthecourseoftheexperimenttodepletebacteria.Data

arepresentedasarticularscores(mean±SEM)andhavebeenpooledfromthree

independentexperiments(PBS;n=23,ES-62;n=19,PBS-ABX;n=22andES-62-ABX;

n=22).(b)H&Estainingofhindpawsfromrepresentativemicefromeachtreatment

groupareshownontheimagesandscalebarsrepresent200µm.(c)Blindscoringof

thejointpathologyexhibitedinexperimentalreplicatesofjointsections(Clinical

articularscoreoftheindividualpawswere,PBS≥3(n=9)ES-62=0(n=7),PBS-ABX=

0(n=3)andES-62-ABX≥3(n=3)).(dande)LevelsofcollagenII(CII)-specificIgG2a

(d;PBS;n=18,ES-62;n=13,PBS-ABX;n=16andES-62-ABX;n=14)andIgG1(e;PBS;

n=15,ES-62;n=10,PBS-ABX;n=16andES-62-ABX;n=14)antibodiesinserumwere

determinedbyELISA.(f)SplenicBregulatorycells(CD19+IL-10+cells)were

determinedbyflowcytometry(Naïve;n=13,PBS;n=21,ES-62;n=14,Naïve-ABX;n=

8PBS-ABX;n=14andES-62-ABX;n=15).(gandh)IL-10(g-PBS;n=6,ES-62;n=5,

PBS-ABX;n=6andES-62-ABX;n=6)andIL-6(h-PBS;n=24,ES-62;n=10,PBS-ABX;

n=12andES-62-ABX;n=13)concentrationsinserumweredeterminedbyELISA.

Statistics:allresultsarepresentedasmean±SEMandeachsymbolrepresentsan

individualmouse;dataarefromone(g)orpooledfromtwoorthreeindependent

experiments.Statisticalsignificancewasdeterminedusingtwo-wayANOVA(a),one-

wayANOVA(c-h)withLSDFishersmultiplecomparisonsandsignificanceindicatedby

asterisks,*=p<0.05and**=p<0.01.

33

34

Fig.3.ES-62requiresthegutmicrobiometoprotecttheboneremodellingaxis

(a,bandc)Osteoclastsweredifferentiatedfrombonemarrowobtainedatculland

culturedfor5daysbeforedifferentiation(a)andsize(b)ofosteoclastswasmeasured

usingImageJanalysissoftwareanddatawasnormalisedasapercentageofNaïve

controlswithrepresentativeimages(c)provided(Naïve;n=11,PBS;n=11,ES-62;n=

6,Naïve+ABX;n=2,PBS+ABX;n=3,ES-62+ABX;n=3).Wholebonemarrowwasused

toquantifyRANK(d;PBS;n=20,ES-62;n=13,PBS+ABX;n=15,ES-62+ABX;n=15)

andOPG(e;PBS;n=17,ES-62;n=11,PBS+ABX;n=16,ES-62+ABX;n=14)mRNA

levelsusingqRT-PCRandfoldchangewascalculatedfollowingnormalisationtoNaïve

controls.Statistics:alldataarepresentedasmean±SEM.Inaandb,eachsymbol

representsexperimentalreplicatesandindandeeachsymbolrepresentsindividual

miceanddataarepooledfromthreeindependentexperiments.Statisticalsignificance

wasdeterminedusingone-wayANOVA(aandb)withLSDFishersmultiple

comparisonsorKruskal-WalliswithDunn’smultiplecomparisonstests(dande)and

significanceindicatedbyasterisks,*=p<0.05,**=p<0.01and***=p<0.001.

35

Fig.4.CIAisaccompaniedbymicrobiome-dependentgutpathology

(a)RepresentativeH&Eimages(scalebarrepresenting50µm)ofileumsectionsof

naiveandPBS-CIAmicewithdifferentialdiseaseseverityatcull(mousearticularscore

asindicated).(b)RepresentativeH&Eimagesofileum(toprow),colon(middlerow)

andPAS-stainedcolon(bottomrow)sectionsdisplayingscalebar(200µm)and

articularscoresofthemiceareshownontheimages.(c)Quantitativeanalysisofileum

villithicknesswheresymbolsrepresentmeanvaluesofreplicatesectionsasmeasured

usingImageJanalysissoftware(n=3/groupwith3–5replicates/animalanddataare

representativeoftwoindependentexperiments).(d)Lesionswereenumeratedper

colonsectionofindividualmiceusingImageJimagingsoftware(Naïve;n=6,PBS;n=8,

ES-62;n=7,Naive+ABX;n=6,PBS+ABX;n=7,ES-62+ABX;n=7).Statistics:dataare

pooledfromtwoindependentexperiments.Statisticalsignificancewasdetermined

usingone-wayANOVAwithLSDFishersmultiplecomparisonsandindicatedby

asterisks,*=p<0.05,**=p<0.01and***=p<0.001.

36

37

Fig.5.ES-62protectsagainstthegutpathologyoccurringpriortoonsetof

arthritis

(a)RepresentativeH&Eimagesofileumandcolonpathologyofmiceculledduringthe

followingphasesofCIA:Naïve,initiation(post-immunisation≤d14),preclinical(d21

priortochallenge)anddisease(establishedarthritisd≥28,articularscorePBS–

5.2±0.8;ES-62–0.9±1.24).Scalebarsare200µm.(bandc)Changesinthevillus

thickness(b;Initiation;PBS-n=5,ES-62-n=6,Pre-Clinical;n=3,Disease;PBS-n=4,

ES-62-n=5mice)andnumberofcolonlesions(c;Initiation;n=6,Pre-Clinical;n=3,

Disease;PBS-n=4,ES-62-n=5mice)werequantifiedusingImageJanalysissoftware

ateachphaseoftheexperimentanddatawerenormalisedtovaluesofnaïvemicewith

representativeimagesdisplayed.Statistics:dataarepresentedasmean±SEMof

individualmicefromoneexperiment.Statisticalsignificancewasdeterminedusingtwo-

wayANOVAstocomparePBSandES-62treatmentateachtimepoint(b-c)and

significanceisdenotedas*=p<0.05and***=p<0.001.

38

Fig.6.ES-62modulatesthegut-bonemarrowaxisduringtheearlyphasesofCIA

(a,bandc)ChangesinBacteriodetes(a),Firmicutes(b)andButyrivibrio(c)

populationsinthecolonfaecalmatterofNaïve,PBS-orES-62-treatedanimalswere

measuredateachstageofdiseasebyqPCRanddatawerenormalisedtototalbacterial

contentandpresentedas%changecomparedtoNaïvemice.(d)Wholebonemarrow

wasusedtoquantifyMyD88mRNAlevelsusingqRT-PCRandnormalisedtoNaïve

controls.(e)Theproportionofmonocytes(CD3+B220-Ter119-Ly6G-Ly6C+)inbone

marrowwasmeasuredbyflowcytometryandnormalisedtothoseinNaivecontrolmice.

(fandg)Osteoclastsweredifferentiatedfrombonemarrowobtainedatculland

culturedfor5daysandsizeofosteoclasts(f)wasmeasuredusingImageJanalysis

softwareandnormalisedtothosefromnaivecontrolswithrepresentativeimagesfor

eachdiseasestageinbothtreatmentgroupsdisplayed(g).Statistics:dataarepresented

asmean±SEMvaluesfromindividualanimals(a,bandc;Initiation;n=6,Pre-Clinical;

n=3,Disease;n=10.d;Naïve;n=4,PBS;n=8,ES-62;n=6,Naive+ABX;n=4,

PBS+ABX;n=8,ES-62+ABX;n=8.e;Initiation;n=6,Pre-Clinical;n=3,Disease;n=4)

ormean±SDfromexperimentalreplicates(f;Initiation;n=18,Pre-Clinical;n=9,

Disease;PBS–n=12andES-62–n=15).Statisticalsignificancewasdeterminedusing

two-(a,b,c,eandf)orone-way(d)ANOVAwithLSDFishersmultiplecomparisonsand

indicatedbyasterisks,*=p<0.05,**=p<0.01and***=p<0.001.

39

40

SupplementaryFig.1.TreatmentofnaïvemicewithES-62increasesbutyrate-

producingbacteriaintheileum,butnotthecolonofanimals

(aandb)Ileum(a)andcolon(b)fecalmatterfromNaïveorES-62-treatedNaïve

animalswasanalysedforchangesinbacterialpopulationsinasingleexperimentusing

samplespooledfromthreeanimalspergroupanddisplayedasHeatmaps,wherethe

ES-62-treatedsamplesarenormalisedtotheNaïvecontrols.(candd)Thenumberof

speciesdetectedduringmetagenomicanalysisoftheileum(c)andcolon(d)ofNaive,

PBS-CIAandES-62-CIAmice.Statistics:dataarepresentedasmean±SEMwherefor

metagenomicanalysis,threemicepergroupwerepooledfromthreeindependent

experimentsandnormalisedtoNaïvecontrols(candd).

41

SupplementaryFig.2.ABXtreatmenteliminatescommensalbacteriaand

enrichesresidualproteobacteriaspecies

(a)BodyweightsofindividualanimalsoverthetimecourseofCIAweremeasuredfrom

day-7(priortoABXadministration)andpresentedasmean%weight±SEM(Naïve;n

=8,PBS;n=16,ES-62;n=13,Naive+ABX;n=8,PBS+ABX;n=16,ES-62+ABX;n=15).

(b)DNAwasisolatedfromthecoloncontentsofindividualanimalsandthelevelsof16S

bacterialDNAdetectedbyqPCRwasmeasuredandnormalisedtothelevelsoftotal

DNAextracted.Symbolsrepresentvaluesfromindividualmicefromasingle

experiment.(c)RepresentativerarefactionplotsfromtheMG-RASTmetagenomic

analysisshowingthelevelsofbacterialdiversityofcolonfaecalmatterineach

treatmentgroupfromoneofthethreeexperiments.(d)Compositionofbacterialphyla

presentinPBSorPBS-ABXanimals(metagenomicdataobtainedbypoolingsamples

from3mice/groupfromasingleexperiment)representedasa%ofallbacteria

detected.Statistics:dataarepresentedasmeanormean±SEM(a,bandd).Statistical

significancewasdeterminedusingone-wayANOVAwithDunn’smultiplecomparisons

comparingNaïve,PBSorES-62-treatedgroupstotheirrespectiveABX-treatedcontrol

andindicatedbyasterisks**=p<0.01.

42

43

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