Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
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:[email protected]
MargaretHarnett,InstituteofInfection,ImmunityandInflammation,CollegeofMedical,
VeterinaryandLifeSciences,UniversityofGlasgow,GlasgowG128TA,UK;Phone–
0044-141-330-8413;e.mail–[email protected]
2
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.
3
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
4
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
5
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.
6
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-
7
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.
8
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.
9
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
10
(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
11
gplotspackageinR.SupervisedheatmapsweregeneratedusingGraphPadPrism7
software.Indicatorsofsignificanceinclude*=p<0.05,**=p<0.01and***=p<0.001.
12
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.
13
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
14
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
15
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
16
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
17
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
9. References
1 Harnett,W.Secretoryproductsofhelminthparasitesasimmunomodulators.Molecularandbiochemicalparasitology195,130-136,doi:10.1016/j.molbiopara.2014.03.007(2014).
2 Harnett,M.M.&Harnett,W.CanParasiticWormsCuretheModernWorld'sIlls?Trendsinparasitology33,694-705,doi:10.1016/j.pt.2017.05.007(2017).
3 Chen,X.,Liu,S.,Tan,Q.,Shoenfeld,Y.&Zeng,Y.Microbiome,autoimmunity,allergy,andhelminthinfection:Theimportanceofthepregnancyperiod.AmJReprodImmunol,doi:10.1111/aji.12654(2017).
4 Liu,A.H.Revisitingthehygienehypothesisforallergyandasthma.TheJournalofallergyandclinicalimmunology136,860-865,doi:10.1016/j.jaci.2015.08.012(2015).
5 Bach,J.F.Thehygienehypothesisinautoimmunity:theroleofpathogensandcommensals.Naturereviews18,105-120,doi:10.1038/nri.2017.111(2018).
6 deRuiter,K.etal.Helminths,hygienehypothesisandtype2diabetes.Parasiteimmunology39,doi:10.1111/pim.12404(2017).
7 Smits,H.H.etal.Microbesandasthma:Opportunitiesforintervention.TheJournalofallergyandclinicalimmunology137,690-697,doi:10.1016/j.jaci.2016.01.004(2016).
8 Blander,J.M.,Longman,R.S.,Iliev,I.D.,Sonnenberg,G.F.&Artis,D.Regulationofinflammationbymicrobiotainteractionswiththehost.Natureimmunology18,851-860,doi:10.1038/ni.3780(2017).
9 Zaiss,M.M.&Harris,N.L.Interactionsbetweentheintestinalmicrobiomeandhelminthparasites.Parasiteimmunology38,5-11,doi:10.1111/pim.12274(2016).
10 Giacomin,P.,Agha,Z.&Loukas,A.HelminthsandIntestinalFloraTeamUptolmproveGutHealth.Trendsinparasitology32,664-666,doi:10.10164/j.pt.2016.05.006(2016).
11 Gause,W.C.&Maizels,R.M.Macrobiota-helminthsasactiveparticipantsandpartnersofthemicrobiotainhostintestinalhomeostasis.CurrOpinMicrobiol32,14-18,doi:10.1016/j.mib.2016.04.004(2016).
12 Filyk,H.A.&Osborne,L.C.TheMultibiome:TheIntestinalEcosystem'sInfluenceonImmuneHomeostasis,Health,andDisease.EBioMedicine13,46-54,doi:10.1016/j.ebiom.2016.10.007(2016).
13 Jennewein,M.F.,Abu-Raya,B.,Jiang,Y.,Alter,G.&Marchant,A.Transferofmaternalimmunityandprogrammingofthenewbornimmunesystem.SeminImmunopathol,doi:10.1007/s00281-017-0653-x(2017).
14 Gourbal,B.etal.Innateimmunememory:Anevolutionaryperspective.Immunologicalreviews283,21-40,doi:10.1111/imr.12647(2018).
15 Zaiss,M.M.etal.TheIntestinalMicrobiotaContributestotheAbilityofHelminthstoModulateAllergicInflammation.Immunity43,998-1010,doi:10.1016/j.immuni.2015.09.012(2015).
16 Ramanan,D.etal.Helminthinfectionpromotescolonizationresistanceviatype2immunity.Science(NewYork,N.Y352,608-612,doi:10.1126/science.aaf3229(2016).
17 Leonardi,I.etal.PreventiveTrichurissuisova(TSO)treatmentprotectsimmunocompetentrabbitsfromDSScolitisbutmaybedetrimentalunder
44
conditionsofimmunosuppression.Scientificreports7,16500,doi:10.1038/s41598-017-16287-4(2017).
18 Giacomin,P.etal.Experimentalhookworminfectionandescalatingglutenchallengesareassociatedwithincreasedmicrobialrichnessinceliacsubjects.Scientificreports5,13797,doi:10.1038/srep13797(2015).
19 Giacomin,P.etal.Changesinduodenaltissue-associatedmicrobiotafollowinghookworminfectionandconsecutiveglutenchallengesinhumanswithcoeliacdisease.Scientificreports6,36797,doi:10.1038/srep36797(2016).
20 Wu,H.J.etal.Gut-residingsegmentedfilamentousbacteriadriveautoimmunearthritisviaThelper17cells.Immunity32,815-827,doi:10.1016/j.immuni.2010.06.001(2010).
21 Chervonsky,A.V.Influenceofmicrobialenvironmentonautoimmunity.Natureimmunology11,28-35,doi:10.1038/ni.1801(2010).
22 Chervonsky,A.V.Intestinalcommensals:influenceonimmunesystemandtolerancetopathogens.Currentopinioninimmunology24,255-260,doi:10.1016/j.coi.2012.03.002(2012).
23 Brusca,S.B.,Abramson,S.B.&Scher,J.U.Microbiomeandmucosalinflammationasextra-articulartriggersforrheumatoidarthritisandautoimmunity.Currentopinioninrheumatology26,101-107,doi:10.1097/BOR.0000000000000008(2014).
24 Scher,J.U.etal.ExpansionofintestinalPrevotellacopricorrelateswithenhancedsusceptibilitytoarthritis.eLife2,e01202,doi:10.7554/eLife.01202(2013).
25 Rosser,E.C.etal.RegulatoryBcellsareinducedbygutmicrobiota-driveninterleukin-1betaandinterleukin-6production.Naturemedicine20,1334-1339,doi:10.1038/nm.3680(2014).
26 Abdollahi-Roodsaz,S.etal.StimulationofTLR2andTLR4differentiallyskewsthebalanceofTcellsinamousemodelofarthritis.TheJournalofclinicalinvestigation118,205-216(2008).
27 Manasson,J.&Scher,J.U.Spondyloarthritisandthemicrobiome:newinsightsfromanancienthypothesis.CurrRheumatolRep17,10,doi:10.1007/s11926-014-0487-7(2015).
28 Costello,M.E.etal.Intestinaldysbiosisinankylosingspondylitis.ArthritisRheumatol,doi:10.1002/art.38967(2014).
29 Rojo,D.etal.Rankingtheimpactofhumanhealthdisordersongutmetabolism:systemiclupuserythematosusandobesityasstudycases.Scientificreports5,8310,doi:10.1038/srep08310(2015).
30 Vieira,S.M.,Pagovich,O.E.&Kriegel,M.A.Diet,microbiotaandautoimmunediseases.Lupus23,518-526,doi:10.1177/0961203313501401(2014).
31 Clemente,J.C.,Manasson,J.&Scher,J.U.Theroleofthegutmicrobiomeinsystemicinflammatorydisease.BMJ(Clinicalresearched360,j5145,doi:10.1136/bmj.j5145(2018).
32 Reynolds,L.A.etal.Commensal-pathogeninteractionsintheintestinaltract:lactobacillipromoteinfectionwith,andarepromotedby,helminthparasites.Gutmicrobes5,522-532,doi:10.4161/gmic.32155(2014).
33 Walk,S.T.,Blum,A.M.,Ewing,S.A.,Weinstock,J.V.&Young,V.B.AlterationofthemurinegutmicrobiotaduringinfectionwiththeparasitichelminthHeligmosomoidespolygyrus.InflammBowelDis16,1841-1849,doi:10.1002/ibd.21299(2010).
45
34 Holm,J.B.etal.ChronicTrichurismurisInfectionDecreasesDiversityoftheIntestinalMicrobiotaandConcomitantlyIncreasestheAbundanceofLactobacilli.PLoSONE10,e0125495,doi:10.1371/journal.pone.0125495(2015).
35 Houlden,A.etal.ChronicTrichurismurisInfectioninC57BL/6MiceCausesSignificantChangesinHostMicrobiotaandMetabolome:EffectsReversedbyPathogenClearance.PLoSONE10,e0125945,doi:10.1371/journal.pone.0125945(2015).
36 Panda,A.K.,Ravindran,B.&Das,B.K.Rheumatoidarthritispatientsarefreeoffilarialinfectioninanareawherefilariasisisendemic:commentonthearticlebyPinedaetal.Arthritisandrheumatism65,1402-1403,doi:10.1002/art.37883(2013).
37 Panda,A.K.&Das,B.K.Absenceoffilarialinfectioninpatientsofsystemiclupuserythematosus(SLE)infilarialendemicarea:apossibleprotectiverole.Lupus23,1553-1554,doi:10.1177/0961203314546019(2014).
38 Melendez,A.J.etal.InhibitionofFcepsilonRI-mediatedmastcellresponsesbyES-62,aproductofparasiticfilarialnematodes.Naturemedicine13,1375-1381(2007).
39 Pineda,M.A.etal.TheparasitichelminthproductES-62suppressespathogenesisincollagen-inducedarthritisbytargetingtheinterleukin-17-producingcellularnetworkatmultiplesites.Arthritisandrheumatism64,3168-3178,doi:10.1002/art.34581(2012).
40 Rzepecka,J.etal.Thehelminthproduct,ES-62,protectsagainstairwayinflammationbyresettingtheThcellphenotype.Internationaljournalforparasitology43,211-223,doi:10.1016/j.ijpara.2012.12.001(2013).
41 Rodgers,D.T.etal.TheParasiticWormProductES-62TargetsMyeloidDifferentiationFactor88-DependentEffectorMechanismstoSuppressAntinuclearAntibodyProductionandProteinuriainMRL/lprMice.ArthritisRheumatol67,1023-1035,doi:10.1002/art.39004(2015).
42 Coltherd,J.C.etal.Theparasiticworm-derivedimmunomodulator,ES-62anditsdrug-likesmallmoleculeanaloguesexhibittherapeuticpotentialinamodelofchronicasthma.Scientificreports6,19224,doi:10.1038/srep19224(2016).
43 Doonan,J.etal.ProtectionagainstarthritisbytheparasiticwormprojectES-62,anditsdrug-likesmallmoleculeanalogues,isassociatedwithinhibitionofosteoclastogenesisFrontiersinimmunology9,1016,doi:10.3389/fimmu.2018.011016(2018).
44 Rodgers,D.T.etal.Protectionagainstcollagen-inducedarthritisinmiceaffordedbytheparasiticwormproduct,ES-62,isassociatedwithrestorationofthelevelsofinterleukin-10-producingBcellsandreducedplasmacellinfiltrationofthejoints.Immunology141,457-466,doi:10.1111/imm.12208(2014).
45 McInnes,I.B.etal.Anoveltherapeuticapproachtargetingarticularinflammationusingthefilarialnematode-derivedphosphorylcholine-containingglycoproteinES-62.JImmunol171,2127-2133(2003).
46 Pineda,M.A.,Rodgers,D.T.,Al-Riyami,L.,Harnett,W.&Harnett,M.M.ES-62protectsagainstcollagen-inducedarthritisbyresettinginterleukin-22towardresolutionofinflammationinthejoints.ArthritisRheumatol66,1492-1503,doi:10.1002/art.38392(2014).
47 Rzepecka,J.etal.ProphylacticandtherapeutictreatmentwithasyntheticanalogueofaparasiticwormproductpreventsexperimentalarthritisandinhibitsIL-1βproductionviaNRF2-mediatedcounter-regulationofthe
46
inflammasome.Journalofautoimmunity60,59-73,doi:10.1016/j.jaut.2015.04.005(2015).
48 Meyer,F.etal.ThemetagenomicsRASTserver-apublicresourcefortheautomaticphylogeneticandfunctionalanalysisofmetagenomes.BMCBioinformatics9,386,doi:10.1186/1471-2105-9-386(2008).
49 Abdollahi-Roodsaz,S.,Abramson,S.B.&Scher,J.U.Themetabolicroleofthegutmicrobiotainhealthandrheumaticdisease:mechanismsandinterventions.NatRevRheumatol12,446-455,doi:10.1038/nrrheum.2016.68(2016).
50 Chen,B.,Sun,L.&Zhang,X.Integrationofmicrobiomeandepigenometodecipherthepathogenesisofautoimmunediseases.Journalofautoimmunity83,31-42,doi:10.1016/j.jaut.2017.03.009(2017).
51 Steves,C.J.,Bird,S.,Williams,F.M.&Spector,T.D.TheMicrobiomeandMusculoskeletalConditionsofAging:AReviewofEvidenceforImpactandPotentialTherapeutics.Journalofboneandmineralresearch:theofficialjournaloftheAmericanSocietyforBoneandMineralResearch31,261-269,doi:10.1002/jbmr.2765(2016).
52 Buigues,C.etal.EffectofaPrebioticFormulationonFrailtySyndrome:ARandomized,Double-BlindClinicalTrial.Internationaljournalofmolecularsciences17,doi:10.3390/ijms17060932(2016).
53 Celis-Morales,C.A.etal.Associationsofgripstrengthwithcardiovascular,respiratory,andcanceroutcomesandallcausemortality:prospectivecohortstudyofhalfamillionUKBiobankparticipants.BMJ(Clinicalresearched361,k1651,doi:10.1136/bmj.k1651(2018).
54 Charles-Schoeman,C.Cardiovasculardiseaseandrheumatoidarthritis:anupdate.CurrRheumatolRep14,455-462,doi:10.1007/s11926-012-0271-5(2012).
55 Brown,J.M.&Hazen,S.L.Thegutmicrobialendocrineorgan:bacteriallyderivedsignalsdrivingcardiometabolicdiseases.AnnuRevMed66,343-359,doi:10.1146/annurev-med-060513-093205(2015).
56 Grigg,J.B.&Sonnenberg,G.F.Host-MicrobiotaInteractionsShapeLocalandSystemicInflammatoryDiseases.JImmunol198,564-571,doi:10.4049/jimmunol.1601621(2017).
57 Zeng,H.&Chi,H.MetaboliccontrolofregulatoryTcelldevelopmentandfunction.Trendsinimmunology36,3-12,doi:10.1016/j.it.2014.08.003(2015).
58 Tamanai-Shacoori,Z.etal.Roseburiaspp.:amarkerofhealth?FutureMicrobiol12,157-170,doi:10.2217/fmb-2016-0130(2017).
59 Rosser,E.C.&Mauri,C.Aclinicalupdateonthesignificanceofthegutmicrobiotainsystemicautoimmunity.Journalofautoimmunity74,85-93,doi:10.1016/j.jaut.2016.06.009(2016).
60 Rogier,R.etal.AberrantintestinalmicrobiotaduetoIL-1receptorantagonistdeficiencypromotesIL-17-andTLR4-dependentarthritis.Microbiome5,63,doi:10.1186/s40168-017-0278-2(2017).
61 Rogier,R.etal.Alterationoftheintestinalmicrobiomecharacterizespreclinicalinflammatoryarthritisinmiceanditsmodulationattenuatesestablishedarthritis.Scientificreports7,15613,doi:10.1038/s41598-017-15802-x(2017).
62 Harnett,M.M.etal.ThephosphorycholinemoietyofthefilarialnematodeimmunomodulatorES-62isresponsibleforitsanti-inflammatoryactioninarthritis.AnnRheumDis67,518-523,doi:10.1136/ard.2007.073502(2008).
47
63 Maseda,D.,Bonami,R.H.&Crofford,L.J.RegulationofBlymphocytesandplasmacellsbyinnateimmunemechanismsandstromalcellsinrheumatoidarthritis.Expertreviewofclinicalimmunology10,747-762,doi:10.1586/1744666X.2014.907744(2014).
64 Craft,J.E.FollicularhelperTcellsinimmunityandsystemicautoimmunity.NatRevRheumatol8,337-347,doi:10.1038/nrrheum.2012.58(2012).
65 Lucas,S.etal.Short-chainfattyacidsregulatesystemicbonemassandprotectfrompathologicalboneloss.Naturecommunications9,55,doi:10.1038/s41467-017-02490-4(2018).
66 Sjogren,K.etal.Thegutmicrobiotaregulatesbonemassinmice.Journalofboneandmineralresearch:theofficialjournaloftheAmericanSocietyforBoneandMineralResearch27,1357-1367,doi:10.1002/jbmr.1588(2012).
67 Luo,Y.etal.MicrobiotafromObeseMiceRegulateHematopoieticStemCellDifferentiationbyAlteringtheBoneNiche.CellMetab22,886-894,doi:10.1016/j.cmet.2015.08.020(2015).
68 Novince,C.M.etal.CommensalGutMicrobiotaImmunomodulatoryActionsinBoneMarrowandLiverhaveCatabolicEffectsonSkeletalHomeostasisinHealth.Scientificreports7,5747,doi:10.1038/s41598-017-06126-x(2017).
69 Jin,C.,Henao-Mejia,J.&Flavell,R.A.Innateimmunereceptors:keyregulatorsofmetabolicdiseaseprogression.CellMetab17,873-882,doi:10.1016/j.cmet.2013.05.011(2013).
70 Versini,M.,Jeandel,P.Y.,Rosenthal,E.&Shoenfeld,Y.Obesityinautoimmunediseases:notapassivebystander.Autoimmunityreviews13,981-1000,doi:10.1016/j.autrev.2014.07.001(2014).
71 Kim,J.J.&Khan,W.I.Gobletcellsandmucins:roleininnatedefenseinentericinfections.Pathogens2,55-70,doi:10.3390/pathogens2010055(2013).
72 Birchenough,G.M.,Johansson,M.E.,Gustafsson,J.K.,Bergstrom,J.H.&Hansson,G.C.Newdevelopmentsingobletcellmucussecretionandfunction.MucosalImmunol8,712-719,doi:10.1038/mi.2015.32(2015).
73 Golan,L.,Gonen,E.,Yagel,S.,Rosenshine,I.&Shpigel,N.Y.EnterohemorrhagicEscherichiacoliinduceattachingandeffacinglesionsandhemorrhagiccolitisinhumanandbovineintestinalxenograftmodels.DisModelMech4,86-94,doi:10.1242/dmm.005777(2011).
74 Charles,J.F.,Ermann,J.&Aliprantis,A.O.Theintestinalmicrobiomeandskeletalfitness:Connectingbugsandbones.Clinicalimmunology(Orlando,Fla159,163-169,doi:10.1016/j.clim.2015.03.019(2015).
75 Christ,A.etal.WesternDietTriggersNLRP3-DependentInnateImmuneReprogramming.Cell172,162-175e114,doi:10.1016/j.cell.2017.12.013(2018).
76 Pineda,M.A.,Eason,R.J.,Harnett,M.M.&Harnett,W.Fromthewormtothepill,theparasiticwormproductES-62raisesnewhorizonsinthetreatmentofrheumatoidarthritis.Lupus24,400-411,doi:10.1177/0961203314560004(2015).
77 Harnett,M.M.etal.FromChristiandeDuvetoYoshinoriOhsumi:Moretoautophagythanjustdiningathome.BiomedJ40,9-22,doi:10.1016/j.bj.2016.12.004(2017).
78 Abdollahi-Roodsaz,S.etal.Destructiveroleofmyeloiddifferentiationfactor88andprotectiveroleofTRIFininterleukin-17-dependentarthritisinmice.Arthritisandrheumatism64,1838-1847,doi:10.1002/art.34328(2012).
48
79 Abdollahi-Roodsaz,S.,vandeLoo,F.A.&vandenBerg,W.B.Trappedinaviciousloop:Toll-likereceptorssustainthespontaneouscytokineproductionbyrheumatoidsynovium.Arthritisresearch&therapy13,105,doi:ar3287[pii]10.1186/ar3287(2011).
80 Zhang,X.etal.Theoralandgutmicrobiomesareperturbedinrheumatoidarthritisandpartlynormalizedaftertreatment.Naturemedicine21,895-905,doi:10.1038/nm.3914(2015).
81 Rogier,R.,Koenders,M.I.&Abdollahi-Roodsaz,S.Toll-LikeReceptorMediatedModulationofTCellResponsebyCommensalIntestinalMicrobiotaasaTriggerforAutoimmuneArthritis.Journalofimmunologyresearch2015,527696,doi:10.1155/2015/527696(2015).
82 Chen,J.etal.Anexpansionofrarelineageintestinalmicrobescharacterizesrheumatoidarthritis.GenomeMed8,43,doi:10.1186/s13073-016-0299-7(2016).
83 Thomas,V.,Clark,J.&Dore,J.Fecalmicrobiotaanalysis:anoverviewofsamplecollectionmethodsandsequencingstrategies.FutureMicrobiol10,1485-1504,doi:10.2217/fmb.15.87(2015).
84 Donaldson,G.P.,Lee,S.M.&Mazmanian,S.K.Gutbiogeographyofthebacterialmicrobiota.NatRevMicrobiol14,20-32,doi:10.1038/nrmicro3552(2016).
85 Sczesnak,A.etal.Thegenomeofth17cell-inducingsegmentedfilamentousbacteriarevealsextensiveauxotrophyandadaptationstotheintestinalenvironment.CellHostMicrobe10,260-272,doi:10.1016/j.chom.2011.08.005(2011).
86 Maeda,Y.etal.DysbiosisContributestoArthritisDevelopmentviaActivationofAutoreactiveTCellsintheIntestine.ArthritisRheumatol68,2646-2661,doi:10.1002/art.39783(2016).
87 Marietta,E.V.etal.SuppressionofInflammatoryArthritisbyHumanGut-DerivedPrevotellahisticolainHumanizedMice.ArthritisRheumatol68,2878-2888,doi:10.1002/art.39785(2016).
88 Lin,P.etal.HLA-B27andhumanbeta2-microglobulinaffectthegutmicrobiotaoftransgenicrats.PLoSONE9,e105684,doi:10.1371/journal.pone.0105684(2014).
89 Sano,T.etal.AnIL-23R/IL-22CircuitRegulatesEpithelialSerumAmyloidAtoPromoteLocalEffectorTh17Responses.Cell163,381-393,doi:10.1016/j.cell.2015.08.061(2015).
90 Block,K.E.,Zheng,Z.,Dent,A.L.,Kee,B.L.&Huang,H.GutMicrobiotaRegulatesK/BxNAutoimmuneArthritisthroughFollicularHelperTbutNotTh17Cells.JImmunol196,1550-1557,doi:10.4049/jimmunol.1501904(2016).
91 Mizuno,M.,Noto,D.,Kaga,N.,Chiba,A.&Miyake,S.Thedualroleofshortfattyacidchainsinthepathogenesisofautoimmunediseasemodels.PLoSONE12,e0173032,doi:10.1371/journal.pone.0173032(2017).
92 Hamer,H.M.etal.Reviewarticle:theroleofbutyrateoncolonicfunction.Alimentarypharmacology&therapeutics27,104-119,doi:10.1111/j.1365-2036.2007.03562.x(2008).
93 Mathewson,N.D.etal.Gutmicrobiome-derivedmetabolitesmodulateintestinalepithelialcelldamageandmitigategraft-versus-hostdisease.Natureimmunology17,505-513,doi:10.1038/ni.3400(2016).
49
94 Burger-vanPaassen,N.etal.TheregulationofintestinalmucinMUC2expressionbyshort-chainfattyacids:implicationsforepithelialprotection.TheBiochemicaljournal420,211-219,doi:10.1042/BJ20082222(2009).
95 Pianta,A.etal.EvidenceoftheImmuneRelevanceofPrevotellacopri,aGutMicrobe,inPatientsWithRheumatoidArthritis.ArthritisRheumatol69,964-975,doi:10.1002/art.40003(2017).
96 Newkirk,M.M.,Zbar,A.,Baron,M.&Manges,A.R.DistinctbacterialcolonizationpatternsofEscherichiacolisubtypesassociatewithrheumatoidfactorstatusinearlyinflammatoryarthritis.Rheumatology(Oxford)49,1311-1316,doi:10.1093/rheumatology/keq088(2010).
97 Li,S.,Yu,Y.,Yue,Y.,Zhang,Z.&Su,K.MicrobialInfectionandRheumatoidArthritis.JClinCellImmunol4,doi:10.4172/2155-9899.1000174(2013).
98 Pretorius,E.,Akeredolu,O.O.,Soma,P.&Kell,D.B.Majorinvolvementofbacterialcomponentsinrheumatoidarthritisanditsaccompanyingoxidativestress,systemicinflammationandhypercoagulability.ExpBiolMed(Maywood)242,355-373,doi:10.1177/1535370216681549(2017).
99 Rahman,M.M.etal.Twohistonedeacetylaseinhibitors,trichostatinAandsodiumbutyrate,suppressdifferentiationintoosteoclastsbutnotintomacrophages.Blood101,3451-3459,doi:10.1182/blood-2002-08-2622(2003).
100 Abdollahi-Roodsaz,S.etal.Shiftfromtoll-likereceptor2(TLR-2)towardTLR-4dependencyintheerosivestageofchronicstreptococcalcellwallarthritiscoincidentwithTLR-4-mediatedinterleukin-17production.Arthritisandrheumatism58,3753-3764,doi:10.1002/art.24127(2008).