Toll-like receptor 4, but not neutrophil extracellular Traps, Promote IFN Type I expression to enhance Th2 responses to Nippostrongylus brasiliensis

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FrontiersinImmunology|www.frontiersin.org 1 November2017|Volume8|Article1575 Editedby:

JoaoP.B.Viola,I nstitutoNacionaldeCâncer (INCA),Brazil Reviewedb y : ClaudiaIdaBrodskyn,Centr odePesquisaGonçaloMoni z-FIOCRUZ/BA,Brazil JohnP.Vasilakos, 3MCom pany,U ni t ed St at es

*Correspondence:

FrancaRonchesefronchese@

malaghan.org.nz

Specialtysection:

Thisarticlewassubmittedt oMolecularInnateImmunity, asectionofthejournal FrontiersinImmunology Received:21August2017 Accepted:02November2017 Published:16November2017 Citation:

PellefiguesC,TangS- C,SchmidtA,WhiteRF,LamiableO,C onnorLM,RuedlC,DobruckiJ,LeGros GandRoncheseF(2017)Toll- LikeReceptor4,butNotNeutrophilExt racellularTraps,PromoteIFNTypeIEx pressiontoEnhanceTh2Responsesto Nippostrongylusbrasili ensi s.

Front.Immunol.8:1575.doi:

10.3389/fimmu.2017.01575

Originalresearchpub lished:16November2017doi:10.338 9/fimmu.2017.01575

Toll-

liker e c e p t o r 4 , b u t n o t ne utrophilextracellularTraps,Pr omoteiFnTypeiexpressiontoe nhanceTh2responsestoNip postrongylusbrasiliensis

ChristophePellefigues¹,Shiau-

ChootTang¹,AlfonsoSchmidt¹,RubyF.White¹,OlivierLamiable¹,LisaM.Connor¹,Christia neRuedl²,JurekDobrucki³,GrahamLeGros¹and

FrancaRonchese¹*

1Malaghan

InstituteofMedicalResearch,Wellington,NewZealand,²School

ofBiologicalSciences,NanyangTechnologicalUn iversity,Singapore,Singapore,³Faculty

ofBiochemistry,BiophysicsandBiotechnology,DepartmentofCellBiophysics, JagiellonianUniversity,Kraków,Poland

TheinductionofTh2responsesisthoughttobemultifactorial,andemergefromspecificpath waysd i s t i n c t f romt h os e a s s o c i a t e d w i t h a n t a g o n is t ic a n t i b a c t e r ia l o r a n t i v ir a l T h 1 responses.H e re,w e s h o w t h a t t h e recognitiono f n o n -

v i a b l e N i p p o s t r o n g y l u s b r a s i l -

iensis(Nb) intheskininduc es a s trongrecruitmentofmon ocytesa nd neu trophilsandt hereleaseofneutrophilextracellulartraps(NETs).Nbalsoactivatestoll-

likereceptor4(TLR4)signalingwithexpressionofIfnbtranscriptsintheskinandthedevelopmento fanIFNtypeIsignatureonhelminthantigen-

bearingdendriticcellsindraininglymphnodes.Co-

injectiono f N b t o g e t h e r w i t h a b o u t 1 0 , 0 0 0 G r a m -

n e g a t i v e b a c t e r i a a m p l i f i e d t h i s TLR4-dependentb u t N E T- independentI F N t y p e I responsea n d e n h a n c e d t h e d e v e l -

opmentofTh2responses.Thus,alimitedactivationofantibacterialsignalingpathways isabletoboostantihelminthicresponses,suggestingaroleforbacterialsensinginth eoptimalinductionofTh2immunity.

Keywords:Nippostrongylusbrasiliensis,helminth,dendriticcells,toll-likereceptor4,iFn- i,neutrophilextracellulartraps,Th2response,skinimmunity

inTrODUcTiOn

Diverseimmuneresponseshavebeenassociatedwithdifferentclassesofpathogenorinsult,andwitht hes pecializationofC D 4^+Thelperc ellstowardt hesecretionofa c e rtainsetofe f fectorcytokines:Th1cellss ecretingInterferon-

γ(IFNγ)aregeneratedinantitumoral,antibacterial,andanti-“intracellularpathogen”responses,Th2ce llssecretinginterleukin4(IL4),IL5,and/orIL13aregeneratedinanti-venom/toxin/irritantandanti- macroparasite(includinghelminthsandticks)responses,andTh17/22cellssecretingIL17orIL22aregene

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FrontiersinImmunology|www.frontiersin.org 2 November2017|Volume8|Article1575

rateduponantifungalandantib acterialresponses(1–3).

Tissueresidentdendriticcel ls(DCs)arecriticalfortheprimi ngofantigenspecificTcellresp onses.Theyareabletoshapethe polarizationofadaptiveimmu nitytowardaTh1,Th2,orTh17r esponsebyinterpretingsignals fromtheirenvironment(1).Wh ilethesensingofviralandbacte rialnucleicacidsbyintracellul arPatternRecognitionRecept ors,andofbacteriallipopolysa ccharides(LPS)byToll- likereceptor4(TLR4)havebe enshowntoinducethedevelop mentofstrongTh1responses,

(3)

TLR4ControlsIFN-I- DependentTh2Responses Pellefiguesetal.

aconsensussig nalrequiredtoinducethedevelopmentofTh2 imm uneresponseshasnotbeenidentified.Severaltypesofsig-

nalscanparticipateininducingaTh2responseincluding,butnotlimit edto,tissuederivedcytokinesandalarminssuchasTSLP,IL25,andI L33,aninnate“thirdparty”sourceofIL4,theactiv-

ityofspecificproteasesorphospholipases,somemacroparasiteglyc ansorglycolipidmotifs,andthedetectionofextremelylowamountso fLPS(1,3–

7).Understandingthesignalsthatgovernthed evelopmentofT h 2 re sponsesisofuttermosti mportanceconsideringthathelminthspoten tiallyaffectaround25%oftheworldpopulation(8,9)whileallergicd iseasescanaffectmorethan30%ofthepopulation(10),mainlyinare aswherehelmin-thiasesarenotendemic.

WeunexpectedlyidentifiedthatthedevelopmentofTh2respons estot heprototypicalhelminthNippostrongylusbrasil-

iensis(Nb)ispartlydependentonIFNtypeI(IFN-

I)signaling(11).IthasalsobeenshownveryrecentlythatIFN- IisimportantfortheinitiationofTh2responsestoSchistosomamans oniandHousedustmite(HDM)byDCs(12).Indeed,2daysafterthei njectionofnon-viableAF488-

labeledNbintotheeardermis,AF488^+DCsinfiltratingthedraining lymphnodes(dLNs)showedastrongtranscriptomicIFN-

Isignature.TheseAF488^+DCsalsoshowedaphenotypicIFN- IsignatureasdemonstratedbytheirexpressionofseveralIFN-I- dependentcellsurfacemark-

ersi ncludingbonemarrow( B M) stromalantigen2( B ST2,orCD3 17).NeutralizingIFN-

IsignalingwithαIFNAR1antibodieswasabletodiminishtheexpansi onofIL4-secretingCD4^+TcellsinresponsetoNb(11).AsIFN- Isignalingisgenerallyassociatedwiththedevelopmentofantiviral,a ntibacterial,orautoimmuneresponses(13–

15),andwithDCactivationandmaturation(16–

21),wesoughttoidentifybywhichmechanismsNbcouldinduceanI FN-Iresponse.

ThereleaseofendogenousoxidizedDNAduringcelldeathorneu trophile x t racellulart raps( N ETs)secretionisstronglyimmunog enicandi nterferogenicin variouspathophysiologicalcontexts(22 –

25).AsneutrophilsareimportantfortheimmuneresponsetoNb(26 –

28),weinvestigatedwhetherNbinjectioninducedthesecretionofN ETsintheskin.Tothisend,weusedaverycontrolledanddefinedsyste mutilizingnon-

viableL3Nblarvaeinjectedintotheeardermis(29).Thisenabledusto moni-

torthedevelopmentoftheimmuneresponseinlocaltissueandauricu lardLNsintheabsenceofpotentialinterferingfactorssuchaslocaltiss uedamageorinfection-relatedsystemiceffects.

Weshowthattheinjectionofnon-

viableL3Nblarvaeintothee ardermisinducesrecruitmentofneutrop hilsundergoingNETosisaroundtheworms.Surprisingly,NETdige stionordepletionofneutrophilswerenotsufficienttodiminishtheIF N-IsignatureonAF488^+DCs.Interestingly,expressionofIFN- Iintheskin,andtheIFN-I-

dependentupregulationofBST2ondLNDCs,requiredexpressionof TLR4.Consistentwiththisobserva-tion,addingGram-

negativebacteriatoAF488^+Nbbeforeinjec-

tionincreasedtheexpressionofBST2onAF488^+DCsindLN,andthe magnitudeoftheresultingTh2response.Thesefindingsstronglysug

gestthatmetazoanparasiteTLR4ligands,originat-

ingfromtheirassociatedmicroorganismsand/oralsofromtheircuticle glycans(7,30),inducethesecretionofIFN-

ItoenhanceDCmaturationandthedevelopmentofspecificTh2respon ses.

(4)

TLR4ControlsIFN-I- DependentTh2Responses Pellefiguesetal.

MaTerialsanDMeThODs MiceandTreatment

s

Seven-to1 0 - week-oldfemaleC 5 7 BL/6J,SiglecH-

DTR(31),TLR2KO(32),andTLR4KO(32)micewerebredandho usedinspecificpathogen-

freeconditionsattheMalaghanInstituteofMedicalResearchBiom edicalResearchUnit.Allexperimentalprotocolswereapprovedb ytheVictoriaUniversityofWellingtonAnimalEthicsCommittee(

Permit20 14 R 17 M) andperformedaccordingtoInstitutionalgu idelines.

NippostrongylusbrasiliensisinfectiveL3larvae(Nb)werecol- lected,washedinsterilePBS,killedbythreefreeze–

thawcycles,andinjectedintradermally(i.d.)intotheearpinnaofane sthetizedmiceaspreviouslydescribed(29).“LowEndotoxin”Nbp repara-tions(LE-

Nb)wereachievedbyaddingfiveextrawashingstepstothepreparat ion.EndotoxincontentwasquantifiedusingtheLALChromogeni cEndotoxinquantitationkit(Pierce)andwas

<5EU/mL.Nbsterilizationwasachievedbyantibiotictreatmentasp reviouslyd e scribed(27).Insomee x periments,Nbw aslabeledu singAlexaFluor488(AF488)succinimidylesterdyes(Molecular Probes)asdescribedpreviously(11).ToprepareNbsupernatant(S N),Nbsuspensionswerelefttosedimentfor5minatroomtemperatur e(RT),andSNcollected.

ToblockIFN-

Isignalinginvivo,miceweretreatedi.d.with250µ g M A R 1 - 5 A 3 ( blockinganti-mouseI F N -

a lphaandbetareceptor1 antibody,anti- IFNAR1)orisotypec ontrol( MOPC-

21)givenwithNbonday0.Thesameantibodydosewasgivenagain onday2byintraperitoneal(i.p.)injection.“InVivoPlus”MAR1- 5A3andMOPC-21werefromBioXCell(WestLebanon,NH,USA).

TodepleteplasmacytoidDCs(pDCs),SiglecH-

DTRmiceweregiven25ng/gdiphtheriatoxin(DT,Sigma)i.p.1day beforeNbinjection.Inallexperiments,flowcytometryanalysisofs pleencellsconfirmed>95%depletionofpDCs,identifiedasCD11 b^−CD11c+B 2 2 0 +Ly6C^+BST2^+cells,c omparedtoDT-

untreatedcontrols.Neutrophilswered e pletedbyi njecting0 . 5 m ganti-Ly6Gantibodyor2 0 0 µ g anti-

Gr1antibodyversust hesameamountoftheirisotypecontrol(IA8o rRB6-

8C5,respectively,InVivoPlus,BioXCell)i.p.1d aybeforeandont hedayofNbinjection.Depletionwasassessedinskinbyenumerati ngneutro-philinfiltrationasCD45+C D 1 1 b +Ly6C^intLy6B^+cells(50–

70%depletion)orC D 4 5^+CD11b+Ly6G^+cells( >95%d e pletion).T odigestNETs,micewereinjectedwith2,000UDNaseIi.d.

(Roche)togetherwithNb,followedby2,000Ui.p.every12huntil theendoftheexperiment.NETdigestionwasqualitativelyconfirm edbymicroscopyat2hafterDNaseIinjection(33,34).

QuantitativereverseTranscriptionPcr(rT- qPcr)

EarswerecollectedattheindicatedtimesandstoredinRNALater (Invitrogen)at4°C.Tissuewascutintosmallpieceswithscis- sorsandhomogenizedusingTissueLizerII(Qiagen)andRNAwas e x t ractedw ithTrizol( Invitrogen)followingt hesupplier’sinstru ctions.cDNAwassynthetizedusingtheHighcapacityRNA-to-

cDNAk it( AppliedBiosystems).RT-

qPCRw asp erformedusingSYBRGreenMasterMixandthefollowi ngprimers:Beta

(5)

Actin( F:5′C TAAGGCCAACCGTGAAAAG,R : 5 ′ACCAGA GGCATACAGGGACA),Ifnk(F:5′CCGCCCATCCAATCTCT GAA,R : 5 ′G G A A AGCCGGTCATGGTACT),Ifnb1( F:5′- GCACTGGGTGGAATGAGACT,R:5′-

AGTGGAGAGCAGTTGAGGACA),andIfna(F:5′- TCTGATGCAGCAGGTGGG,

R:5′-

AGGGCTCTCCAGACTTCTGCTCTG)toamplifyallIfnaspecie s(16),usingaQuantStudio7(AppliedBiosystems)andfollowingth emanufacturer’sguidelines.Transcriptlevelsareexpressedastherati oof2^−ΔCT(Transcriptofinterest)/2^−ΔCT(BetaActin)andnormalizedby comparisontopertinentexperimentalcontrols.

cellpreparationsandFlowcytometry

ForDCpreparations,auriculardLNswereharvestedanddigestedfor 3 0 m i n at3 7 ° C i n I M DM( Gibco)c ontaining1 0 0 µ g / m L DN aseIand100µg/mLLiberaseTL(Roche).ForTcellprepara-

tions,LNswerepassedthrougha70-

µmcellstrainer(Falcon).Forskincellpreparations,earsweresplitin tothedorsalandven-

trallayers,andthenmincedinAccutase(Stemcell)containing3U/m LDispaseII,100µg/mLDNAseI,and100ng/mLLiberaseTM(Roche )for30minat37°C.

Peripheralbloodleukocyteswereharvestedbycheekpuncturean dredbloodcellswerelysedinanammoniumchloride/TRISbuffer,as detailedelsewhere(35).Singlecellsuspensionswerefilteredon70- µmnylonmeshcellstrainers(Falcon)andblockedfor15minat4°CinF ACSBuffer(PBS1%bovineserumalbumin0.05%NaN³)containin ganti-

mouseCD16/CD32fromaffinitypurified2.4G2hybridomaSN.C ellswerethenstainedinFACSBufferfor2 0 m i n at4 ° C w ithanopti mizedc oncentrationoffluorophore-conjugatedantibodies.

Forintracellularcytokinestaining,cellswereculturedincomplet eIMDMcontaining10%FetalCalfSerum(FCS) andpenicillin/st reptomycin( allfromGibco)andstimulatedw ithPhorbol1 2 - Myristate1 3 -

Acetate( 5 0 ng/mL)andIonomycin(1µ g / m L ) for5 h at3 7 ° C i n t hepresenceofGolgiStop( BDBioscience).Aftersurfacestaining,c ellsweref i xedandper-

meabilizedwiththeCytofix/Cytopermkit(BDBioscience)andstai nedintracellularly.

ThefluorescentantibodiesusedwerespecificforCD11c(HL3),CD86 ( G L 1 ) , M HCII( M 5 / 1 1 4 ) , C D 3 2 6 ( G 8 . 8 ) , C D 4 ( R M 4 - 5 ) , CD3(145-

2C11),CD103(M290),IL4(11B11),IFNg(XMG1.2),andCD44 (IM7;allfromBD);IL10(JES5-16E3),CD8a(53-

6.7),CD11b(M1/70),CD45(30-F11),CD64(X54- 5/7.1FC),Ly6C

(HK1.4),Ly6G(IA8),CD317(BST2,clone927)fromBioLegend;IL17 A(eBio17B7)andB220(RA3-

6B2;bothfromeBioscience);Ly6B.2(7/4)fromThermofischer.IL 4-

AmCyanexpressionwasquantifiedwitha504/12filterafterexcita tionat445nm.Non-

viablecellsanddoubletswereidentifiedandexcludedusingDAPIorL I V E / DEADstaining( MolecularProbes).Compensationswerep erformedusingOneCompeBeads(Invitrogen)assinglestainedpos itivecontrolsandfluorescenceminusone(FMO)con-

trolswereusedtosetbackgroundexpression.Flowcytometrywasperf ormedonaBDLSRIIorLSRFortessaSORPflowcytometerwithFacs Diva6.1.1software(BectonDickinson).Analyseswereconductedusi ngFlowJovX(TreeStar)andtherepresentedvaluesofexpressioninten sityarethegeometricmeansoffluorescenceintensity(mfi).

(6)

cellcultur e

HumanE mbryonicKidney2 9 3 ( H E K ) c elll i nese ngineere dtoreportN F-

κBa ctivationw itht hesecretionofanoptimizedalkalinephosphata sewereusedtostudythesignalingpathwaysactivatedbyNb.HEK- BluecellsexpressingmurineTLR2,TLR4,TLR7,orT LR 9, andt hei rrespectivecontrolc ellli nes(Null1,Null2,Null1-v,Null2- k)wereculturedi n t riplicatei n 9 6 wellplatesat50,000/well,andsti mulatedO/Nat37°Cwitheither100Nbortheappropriatepositiveco ntrols.ReporterexpressionwasassessedusingQuantiBluemediu m(InvivoGen)followingmanu-

facturer’sinstructions.Absorbanceat640nmwasmeasuredonas pectrophotometer( HeliosG amma,T hermoScientific).Datafro meachreportercelllinewereexpressedasratioofthemeansofstimu latedversusunstimulatedcultures.

PrimaryBMcellswereharvestedbyflushingfemurs’contentusi ngmedium.O nem i llionc ellswereculturedi n R P M I and10%F CS(Gibco)for24hat37°C,inthepresenceof10µg/mLInVivoPlus mouseIgG1(MOPC1)oranti-IFNAR1(MAR-

5A3)fromBioXCell(Lebanon,NH,USA).Cultureswerestimulate dwithHDMwholebodies(Greer)at100µg/mL,or10–

200Nb,or10µg/mLlow-endotoxinPoly(I:C)

(Invitrogen).Escherichiacoli(E.coli)MG1655wasgrowninLuria Broth(Invitrogen)at37°C,quantifiedbyOD,harvestedatex ponen tialg rowthphase,andfixedin1%formalin.

Microscop y

OnehundredandfiftyAF488-

labeledNbwereinjectedintotheeardermisin30µlofsterilePBS.Aft erdifferenttimes,micewereeuthanizedandhairwasremovedwitha hairremovalcream(Veet)beforeharvestingtheears.Theeardorsal andventralpartsweresplitandfixedin4%formalin(Sigma)for30min ,washedinPBS,permeabilizedandblockedinPBSwith0.3%Triton X100(PBS-

T)5%Donkeyserumand2.4G2hybridomaSNatRTfor30min.Th esamebufferwasusedforantibodystaining:primaryantibodystain ingwasperformedusing5µg/mLofgoatantihuman/mouseMPO( R

& D,AF3667) , 1 / 50 0r abbitanti-

HistoneH 3 c itrulline(R2+R8+R17,ab5103)or1/100rabbitanti- mouseNeutrophilelastase(ab21595,bothfromAbcam)for2hatR Torovernightat4 ° C . S ampleswerew ashedf ivet imesi n PBS- Tandt henstainedfor1husing1/500donkeyanti-

goatAF594(ab140150)and/or1 / 5 0 0 d onkeyanti-

rabbitA F 6 4 7 ( ab181347,bothfromAbcam).Afterfivewashes,ti ssuewasmountedunderacoverslipinF luoromount( Sigma)andre cordedusinga ConfocalLaserScanningmicroscopeFV1200- IX83(Olympus).Imageanalysisandtri-

dimensionalreconstructionsweredonewiththeFijiver- sionofImageJandthehelpofa3Dviewerplugintojuxtaposez- stacks(36,37).

statisticalanalysi s

StatisticalanalyseswereperformedusingPrism7.0(GraphPad).T hedistributionofthedatagroupswasalwaysassessedusingaShapir o–

Wilktestfornormality.Datag roupswerec omparedusingoneway

ANOVAwithaTukey’smultiplecomparisontestfore x perimentsf ollowinga normald istribution,orusingt heMann–

Whitneytests,or,whenmoret hant hreei ndependentgroupswerec onsidered,a Kruskal–Wallisw itha D unn’smul-

tiplec omparisontestfore x perimentswhosed atap ointswere

(7)

notfollowinga normald istribution.Ina llc ases,a t wo-

tailedpvalue<0.05wasconsideredasthresholdforsignificance.Mean andSEMareshowninallgraphs.

resUlTs

TheMajorityofcellsTakingupnbMaterialint heskinat24hareneutrophilsandinflammat oryMonocytes

Stage3 ( L 3 ) l arvaefromt hehelminthN b c ani n fectrodents

byp enetratingt heirskinb arrier.Tostudyt hed evelopmentofantihe lminthTh2responsesinskinweusedasimplifiedmodelinvolvingin jectionofnon-

viableNbintotheeardermisofmice(29,38).Nbinjectioninducedaqu ickandtransientneutrophiliaint heblood( Figure1A)andana c cum ulationofC D 4 5^{+l e u}-

kocytesi n t heeard e rmisc omprisingmainlyC D 1 1 b^hiLy6G⁺ⁿeutr ophilsandCD11b^hiLy6G^−Ly6C^{hii n f l}ammatorymonocytesat24h(Fig ure1B).

ThereactiveaminesinthecuticleofNblarvaecanbestainedcoval entlywitht hesuccinimidylesterAF488, togenerateAF488^+Nbl ar vae(11,3 8).2 4 h afterA F 4 8 8^+Nbi njection,approximately90%

ofthecellsthathadtakenupAF488inthedermiswereneutrophilsan dinflammatorymonocytes.Indeed,themajorityofthesetwocelltyp esstainedpositivelyforAF488(Figures1C,D).Ofnote,AF488flu orescencewasconsistentlybrighterinmonocytesthanneutrophils, whichmightbeduetodifferentphagocytosisrates,dyestability,ora poptosisofthesecellpopulations.

Theseobservationsshowthatneutrophilsandinflammatorymo nocytesarerecruitedearlytotheeardermistointeractwithnon- viableNb.

injectionofnon-

viablenbinducesneTosisintheDer mis

Inordertovisualizetheearlyeventsintheinteractionof

non-viableN bw ithi n f i ltratingl e u kocytes,wec arriedouta

FigUre1|

Nippostrongylusbrasiliensis (Nb)inducestherecruitmentandactivationofneutrophilsintheskin.C57BL/6micewereinjectedintradermallyintotheearwith600 AF488⁺Nb

orPBS.Theresultinginflammatoryresponseinbloodandskinwasanalyzedbyflowcytometryattheindicatedtimepoints.Eachsymbol representsonemo use.(a)Proportionofneutrophils(CD11b^hiL

y6G⁺)inperipheralbloodleukocytes(PBL).Dataarepooledfromtwoindependentexperiments.

(B)Recruitmentofleukocytepopulationsintheearskinat24h.LeukocyteswereidentifiedasCD45⁺cells,

neutrophilsandmonocyteswereidentifiedaccordingtothegatinginpanelC.Dataarefromo neofthreeexperimentsthatgavesimilarresults.

(c)RepresentativedotplotsdepictingthegatingofearskinCD45⁺populations

expressingLy6G(neutrophils),highLy6C(monocytes),andAF488asameasureofNbuptake.

(D)PiechartshowingtherelativeproportionofAF488⁺populations

inskin,identifiedasin(c).Monocyte-

(8)

derivedDCs(moDCs)aredefinedasLy6G⁻L

y6C^hiCD11b_hiCD11c₊MHCII₊

,andCD11b⁺dendritic

cells(DCs)areLy6G⁻L

y6C⁻CD11c₊MHCII₊CD11b₊CD326₋

.Dataarefromone ofthreeexperimentsthatgavesimilarresults.Bargraphsshowmean±SEM.StatisticalanalysesusedtheMann–Whitneytest.NS:notsignificant;*p<0.05;**p<0.01;****p<0.0001.

(B)Symbolsclosetothelegendindicateacomparisonofthesamepopulationbetweenthetwogroups.

(9)

whole-

mountconfocalimagingoffixedeardermis.Anincreaseddensityof DAPI^+cellsclosetotheinjectedNbwasobservedasearlyas1hpost- injection(p.i.)

(Figure2A,left).Surprisingly,inadditiontot hec ellulari n f i ltrate,w ormswerea lsosurroundedbyl argeDAPI^+fiber-

shapedstructures( Figure2A,right).By2hafterNbinjection,mosto fthecellsassociatedwithNbwereLy6G⁺ⁿeutrophils( Figure2 B ,l e f t ) . Interestingly,t heLy6Gstainingwasoftenassociatedwiththef ormationofextracellularDAPI^+fiber-

shapedstructures,suggestinganeutrophilmediatedphenomenonin ducedlessthan1hafterNbinjection(Figure2B,right).

Immunohistochemistryexperimentstoinvestigatethenatureoft heDAPI^+structuressurroundingNbshowedthatextracellularDNAfi berswereoftenco-

localizedintheextracellularspacewiththeneutrophilgranuleenzym esneutrophilelastase(NE,Green)andmyeloperoxidase(MPO,Red) (Figure2C),commonlyfoundinN ETs.N ETformationhasbeensho wntobed e pendentonpeptidylargininedeiminase4activityanditsh istoneH3citrul-

lination.Peptidylargininedeiminase4facilitatesnuclearDNAdec ondensation,andt hereleasei ntot heext racellularspaceofDNAcoa tedbyneutrophilgranuleenzymesandhistones.Indeed,extracellular DNAfiberscouldbeobservedinthedermisforatleast48hafterNbinj ection,andstainedstronglyforhistoneH3citrullinesthusidentifying themasNETs(Figure2D)(39–42).

NippostrongylusbrasiliensisinducesaniFnar 1-Dependenta n d T lr4-

DependentexpressionofBsT2onBMcells

Werecentlyshowedthattheinjectionofnon-viableNbintheear dermisi nducesa strongI F N -

I t ranscriptionals i g natureont hemigratoryDCsinfiltratingtheskin dLN,andthatIFN-

IsignalingisimportantforanoptimalTh2immuneresponseinthism odel(11).InordertostudytheNbsensingmechanismsthatleadtoIF N-

Isecretion,weusedprimaryBMcellsastheycontainhighproportion sofneutrophilsandmonocytes,whicharethemaincellt ypesrecruite dtot heeard e rmisearlyafterN bi njection.Ananalysisofthesurface markersexpressedbythesecellpopulationsshowedthat,comparedt oAF488^−/lowcells,AF488^+inflammatorymonocytes,andtoalessere xtentneutrophils,overexpressedtheIFN-I-

inducedmarkerBST2(11,43)

(Figure3 A ).Treatmentw ithI F NAR1-

blockingantibodiesreversedBST2upregulation,confirmingthatit wasdependentonIFNAR1signaling(Figures3B,C).Coculturewit hSNfromNbpreparations,todetermineiftheIFN-

IinducingfactorpresentinNbpreparationssedimentedwiththeNbbo dyorwasasolublefac-

tor,andcoculturewithhousedustmitewholebodies(HDM)alsoindu cedanIFN-I-

dependentupregulationofBST2(Figure3C).Thislatterobservatio nisconsistentwithaveryrecentandelegantreportshowingthatSchis tosomamansoniandHDMinduceanIFN-

IdependentTh2response(12).Thus,severalTh2stimulicaninduceth esecretionofIFN-IbyprimaryBMcells.

IFN-

Iisknowntobeproducedinhighamountsduringantiviralandcellulari mmuneresponses,butmuchlessisknownaboutitssecretionduringme tazoanparasiteinfectionsorotherTh2responses(12,44).Themechan ismsleadingtothesecretionofIFN-

Ihavebeenmostlyassociatedwiththedetectionofviral

(10)

nucleicacidsorbacterialLPS(45–47).Weusedcommercialtoll- likereceptor(TLR)reportercelllinestoinvestigatewhetherNbpre parationswereabletotriggeractivesignalingthroughtheserecept ors.Invitro,non-viableNblarvaeinducedtheactivationofNF- κBonlyinreportercelllinesexpressingTLR2orTLR4,butnotint hoseexpressingTLR7orTLR9,ortheirrespectivecontrols(Fig ure3D).ThissuggeststhatNb-inducedIFN-Isecre-

tionisunlikelytobemediatedbynucleicacidsensing,andthatNbca ninsteadbedetectedbyTLR2andTLR4.

Cocultureofnon-

viableNbwithfreshBMcellsshowedthatNbcouldinduceanIFNA R1-

dependentexpressionofBST2onWTandTLR2KO,butnotTLR4K O,BMmonocytes(Figure3E;FigureS 1 A i n SupplementaryMa terial),whichisc onsistentwiththereporteddifferentialcapacityof thesereceptorstotriggerI F N - I secretion(47).Anti-

IFNAR1antibodyt reatmentdecreasedB ST2e x pressionbelowc ontroll evels,suggestinga constitutiveIFN-

Is ig nalingin B M cultures.Of note,IF NAR1expressiononmon ocytesw asd e creaseduponN bstimulationofWTandTLR2KOB M,butwasunaffectedintheTLR4KOBMcultures( F i g u reS 1 B i n SupplementaryMaterial).T h isisconsistentw itha l i g and- inducede ndocytosisofI F NAR1,andanabsenceofIFN-

IsecretiononlyintheTLR4KObackground.Importantly,IFNAR1 expressionandNb-

AF488dyeuptakewerenotinfluencedbyTLR2orTLR4expression, revealingthattheseprimaryc ellswerenotde fectivei n IFN- Isecretion,s ig naling,orphagocytosisintheseconditions(Figur e3E;FigureS1CinSupplementaryMaterial).

BST2e x pressiononmonocytesandneutrophilsw asd o se- dependentandsaturableafterinductionbyNborbyformalin- fixedGram-

negativeE . colib acteria( d atanotshown).T heseexperimentsc o nfirmedt hatt hesecretionofI F N - I i nducedbyGram-

negativebacteria,knowntobedependentonTLR4signal- ing(47),couldalsobeobservedinBMcultures.

Theseresultsshowt hatN bpreparationsareabletos i g nalthrou ghTLR2andTLR4inBMcells,andelicittheupregula-

tionofB ST2ex pressionviaaT L R 4 - de pendentandI F NAR1- dependentpathway.However,theprecisenatureoftheseTLR4lig andsremainstobedefined.

Tlr4M e d i a t e s n b-

inducedI f n b expression,butnotneTosis, intheskinW

ew ishedtoassesst hee x pressionofI F N - I i n t heskinafterNbi njection.Non-

viableA F 4 8 8^+Nbl arvaei nducede x pres-sionoftheIFN-I- inducedmarkerBST2onAF488^+monocytes,monocyte- derivedDCsandCD11b^+dermalDCsintheskinat24hp.i.

(Figures4A,B),implyingthatIFN-

Iwassecretedintheskinbefore24h.ThemaincomponentsoftheIF N-

Ifamilyaretranscribedfrom14Ifna,1Ifnb,1Ifnk,and1Ifnegenes(

48).UsingRT-

qPCR,wedetectedatransientexpressionofIfnb1inearskin,peaki ngat2hp.i.anddisappearingquicklyatlatertimepoints(FigureS2 AinSupplementaryMaterial).OthercommonIFN-

IspeciesincludingIfna2andIfna4werenotdetectedinsig- nificantamountsduringthefirst6hp.i.(datanotshown).WhenNb-

inducedIfnexpressionwasassessedinC57BL/6andTLR4KOmice, noIfnb1transcriptscouldbedetectedinTLR4KOmice.Ofnote,wecou ldalsoobserveatrendtowardalowexpression

(11)

FigUre2|

Nippostrongylusbrasiliensis(Nb)inducestheformationofneutrophilextracellulartrapsinskin.C57BL/6micewereinjectedintradermallyintotheearwith150 AF488⁺Nb,

andz-stacksofwhole-

mounteardermiswereanalyzedbyimmunofluorescenceandconfocalmicroscopyatdifferenttimepoints.Imagesarerepresentativeofatleasttwoindependentex periments.(a)Dermisat1hafterAF488⁺Nb

injectionshowingDAPI⁺nuclei

(blue)andAF488⁺Nb

(magenta).

Bar=200µm.(B)Dermisat2hafterAF488⁺Nb

(blue),AF488⁺Nb

(magenta),andLy6G⁺neut

rophils(Green).Bar=50µm.

(12)

(c)Dermisat24hafterAF488⁺Nb

(blue),AF488⁺Nb

(magenta),myeloperoxidase(Red),andneutrophilelastase(Green).Bar=50µm .

(D)Dermisat48hafterAF488⁺Nb

(blue),autofluorescentNb(magenta),histoneH3citrullination(Green),andneutrophilelastase(

Red).Bar=200µm.

(13)

FigUre3|Toll-likereceptor4(TLR4)expressionisnecessaryforNippostrongylusbrasiliensis(Nb)-

dependentBST2expressioninvitro.Bonemarrow(BM)cellswereharvestedfromC57BL/6miceortheindicatedstrains,stimulatedovernightinthedescribedco nditions,andanalyzedbyflowcytometry.

(a)Identificationofneutrophils(CD11b^hiL

y6G⁺)andmonocytes(CD11b^hiL y6G⁻L

y6C⁺)andtheiruptakeofAF488andexpressionofBST2afterstimulationwithAF48 8⁺Nb

(red)ormedium(black).(B)BST2expressiononmonocytesfromBMculturesthatwerestimulatedwithAF488⁺Nb

inthepresenceofIFNAR1- blockingantibodiesorisotypecontrol.

(c)BST2expressiononmonocytesfromBMculturesstimulatedwithNb,Nbsupernatant(SN),housedustmite(HDM)orPoly(I:C)inthepresenceofIFNAR1- blockingantibodiesormIgG1.Dataa refromoneofatleasttwoindependentexperimentsthatgavesimilarresults;eachdotcorrespondstoaBMculturefromasepara temouse.

(D)StimulationofTLRreporteractivitybyNb.CelllinesexpressingtheindicatedTLRandtheirrespectivecontrolswerecoculturedovernightwithNbornostimulus,andT LRreporteractivitywasquantifiedusingacolorimetricassay.Reporteractivityforeachcelllineisexpressedasfold-

change(FC)ofthereadingsforstimulatedversusunstimulatedcultures.Eachdotcorrespondstoanindependentexperimentandistheaverageoftriplicatecultures.

(e)BMcellsfromC57BL/6(WT),TLR4KOorTLR2KOmicewereculturedwithNborPoly(I:C)ornostimulus,inthepresenceofIFNAR1-

blockingantibodiesorisotypecontrol.BST2expressiononmonocyteswasassessedafterovernightculture.Dataarefromoneofatleasttwoindependentexperime ntsthatgavesimilarresults;eachdotcorrespondstoaBMculturefromaseparatemouse.StatisticalanalysesusedtheANOVAwithTukey’smultiplecomparisonstes t.NS:notsignificant;**p<0.01, ***p<0.001, ****p<0.0001. Symbolsaboveindividualbarsrefertothepvalueoftheindicatedgroupversusitscontrolcondition,w hichwaseitherunstimulated (c)orWT(e).

ofIfna(allspecies)

(16)andIfnkinC57BL/6recipients,butagainthiswasnotobservedin TLR4KOmice(Figure4C).

PlasmacytoidDCsarek nowntobet hemainproducerofIFN- Ionapercellbasisinantiviralresponsesandsomeauto- immuned iseases.Int hesec onditions,p DCI F N -

I secretionismainlyinducedbythedetectionofnucleicacidsthroug hTLR7andTLR9,whereastheTLR4pathwayisnotinvolved(49).W e,therefore,usedSiglecH-

DTRmicetoassessthecontributionofpDCstoIFN-

IproductionafterNbinjection.DepletionofpDCsbyi.p.treatmentwit hDThadnoeffectontheexpressionofIfnb1inearskin2hafterNbinjecti on(FigureS2BinSupplementary

(14)

Material).TranscriptsforIfnaorIfnk,whichcouldbedetectedatlow levelsinC57BL/6mice,wereundetectableinSiglecH-

DTRmiceona Balb/cb ackground( F i g u reS 2 B i n Supplement aryMaterial).Thesediscrepanciesmightbeexplainedbycelltype andstrain-specificdifferencesintheexpressionofIFN-

Ifamilymembers(50).Together,theseresultssuggestthatpDCsar enotinvolvedintheIFN-IresponsetoNb.

NETosisisknowntobeinducedbyvarioussignalingpath- ways,includingTLR2andTLR4activation(51,52).AsNETosishas beenassociatedwiththesecretionofIFN-

Ibyvariouscelltypes(22–24,53),weinvestigatedwhetherNb- inducedNETosis

(15)

FigUre4|Nippostrongylusbrasiliensis(Nb)inducesatoll-likereceptor4(TLR4)-dependentexpressionofIFN- Iintheskin.C57BL/6(WT)orTLR4KOmicewereinjectedintradermallyintotheearwith600AF488⁺Nb.

ExpressionofIFN-ItranscriptsandoftheIFN-I- dependentmarkerBST2wereexaminedineartissueasindicated.Eachdotcorrespondstoonemouse.

(a)RepresentativeflowcytometrycontourplotsdepictingAF488uptakeandexpressionofBST2onCD45⁺skin

populations24hafterPBSinjection,orco- injectionofAF488⁺Nb

togetherwithIFNAR1-blockingantibodiesorisotypecontrol.CellpopulationswereidentifiedasinFigure1.FluorescenceMinusOne(FMO)forBST2isshownasacontrol.

(B)ExpressionofBST2onAF488⁻and

AF488⁺skin

populationsdefinedasin(a).Bargraphsshowmean±SEMfromoneoftwoindependentexperimentsthatgavesimilarresults.

(c)QuantitativeReverseTranscriptionPCRforIfnb1,Ifna(allspecies)andIfnkineartissue2hafterNbinjection.Bargraphsshowmean±SEMfromtwoindependentexperimentseachwiththreemice/gr oup.StatisticalanalysesusedtheANOVAwithTukey’smultiplecomparisonstestin(B),orKruskal–

WallistestwithDunn’smultiplecomparisonin(c)asthedistributionofthedatacouldnotbeconsiderednormal(Shapiro–Wilknormalitytest).Ns:notsignificant;*p<0.05;**p<0.01.

requiredt hee x pressionofT L R 2 orT L R 4 . Wewereabletoobse rveNETosisintheskinofmutantmiceasearlyas1hafternon- viableNbinjection,rulingoutakeyrequirementforeitherTLR2orT LR4inmediatingNb-

inducedNETosis(FigureS3inSupplementaryMaterial).

expressionofaniFn-

isignatureonmigratoryDcrequiresh ostTlr4expressionbutnotneTformatio n

InjectionofAF488^+NbisfollowedbythemigrationofAF488⁺ skinDCsfromtheskintothedLN.AF488^+DCsindLNpeakinnumb eratd ay2 p.i.andmostlyc ompriset heC D 1 1 b^+andCD326⁻CD103

−CD11b⁻,orTriplenegative( T N ) , DCsubsets.Workfromourgrou phasshownthatthesetwosubsetsofmigra-toryDCsexpressanIFN- IsignaturethatcanberevealedbytheirexpressionofBST2(11).Wec omparedexpressionofBST2onCD11b^+andTNDCsfromWTorTLR 4KOmice,andfoundthat,despitecomparableAF488uptake,BST2w asnotupregulatedinAF488^+DCsfromTLR4KOmice(Figure5A).Thi

sobservationisconsistentwiththelackofdetectableIFN- ItranscriptsinTLR4KOmice(Figure4C).

(16)

InfectionwithliveNbalsoinvolvesexposuretothemicroor- ganismsnaturallyassociatedwiththesehelminths.IndeedGr1^+cell s( i ncluding,butnotl i m itedto,neutrophils,i n f l ammatorymon ocytes,andp DCs),havebeenshowntobed e terminantinc ontrolli ngb acterialproliferationandt hesurvivalofm i c e infectedwithN b,therebyenablingthedevelopmentofacanoni-

calTh2responseinsteadofaTh1response(27).

Wei nvestigatedwhetherc ontaminationbyGram- negativefecalbacteriawastriggeringtheIFN-

IresponsetoNb.Antibiotic-sterilizedNbpreparations(sNb) (27)inducedsimilarexpressionofBST2andofthematurationmark erCD86onAF488+C D 1 1 b +andTNDCsinfiltratingtheeardLNatday2 p.i.(FigureS4AinSupplementaryMaterial).

Injectingl i m itingnumbersofanN bpreparationt hathadbeen e x tensivelyw ashedtol owere ndotoxinc ontent( L E - N b,

<5EU/mLversusNb,>40EU/mL)stillinducedsignificantBST2upre gulationonAF488^+DCsindLN(Figure5B).Co-

injectionof10,000E.colitogetherwithLE-NbenhancedtheIFN- Isigna-

tureofAF488^+DCs(Figure5B).TheseresultsshowthatTLR4ligan dsfromhelminthsand/ortheirassociatedmicroorganismsallowth edevelopmentofanIFN-IsignatureonmigratoryDCsinskindLN.

(17)

FigUre5|Toll-likereceptor4(TLR4),notextracellularDNA,drivesthedevelopmentofanIFN-

IsignatureonLNdendriticcells(DCs)afterNippostrongylusbrasiliensis(Nb)injection.C57BL/6orTLR4KOmicewereinjectedintradermallyintotheearwithAF488⁺ Nb.UptakeofAF488andexpressionofCD86andtheIFN-I-

dependentmarkerBST2wereexaminedineardraininglymphnode(dLN)DCsbyflowcytometry48hafterNbinjection.CD11b⁺DCs

wereCD11c⁺MHCII_hiCD11b₊CD 326₋CD103₋

.TripleNegative(TN)DCswereCD11c⁺MHCII_hiCD11b₋CD326₋CD103₋

.Eachdotcorrespondstoonemouse.Dataarefromoneofatleasttworepeate xperimentsthatgavesimilarresults.

(a)AF488uptakeandBST2expressiononsubsetsofmigratoryDCsindLNofC57BL/6(WT)orTLR4KOmiceafterinjectionof600AF488⁺Nb.

(B)BST2expressiononsubsetsofmigratoryDCsinthedLNofC57BL/6miceafterco-injectionof300Low-Endotoxin(LE,

<5EndotoxinUnits/mL)AF488⁺Nb

withorwithout10,000formalin-fixedEcoli.

(c)AF488uptakeandBST2orCD86expressiononsubsetsofmigratoryDCsinthedLNofC57BL/6miceafterinjectionof600AF488⁺Nb

withorwithoutDNaseItreatment.Bargraphsshowmea n±SEM.StatisticalanalysesusedtheANOVAwithTukey’smultiplecomparisonstest.Ns:notsignificant;*p<0.05;**p<0.01;***p<0.001.

AsN bi njectioni nducedN ETosisandI F N -

I secretion,weinvestigatedwhetherN ETsc ouldmediateN b-

inducedI F N -

I expression.R egulara d m i n istrationsofh i ghd o sesofDNaseI arer

(18)

eportedtodigestNETsinvivo(33).Here,i.d.injectionof2,000U DN

aseI togetherw ithN bi n hibitedt heformationof NETsat2hp.i.,asassessedbymicroscopy(datanotshown).Howev er,regularinjectionsofDNaseI(i.d.andtheni.p.every12h)wasuna bletoreduceBST2expressiononAF488^+DCsindLNat24hand48ha fterNbinjection(Figure5Canddatanotshown).Neutrophild e pleti onusinganti-Ly6Goranti-Gr1

(19)

antibodytreatmentwasalsounabletopreventNb-

inducedBST2e x pressiononA F 4 8 8^+DCsi n d L N ( F i g u reS 5 A , B i n SupplementaryMaterial).TheseresultsshowthattheIF N-IsignatureondLNDCsfromNb-

injectedmicewasnotuniquelydependentonneutrophilsorextracell ularDNA.

Together,thesedatasuggestthatNbanditsassociatedTLR4ligan dsinducetheexpressionofIFN-Iintheskin(7,54–56),andanIFN- Isignatureonantigen-bearingmigratoryDCsinthedLN(11).

Tlr4signalingenhancestheDevelop mentofTh2responsestonb

AsshowninFigure5,TLR4signalingisnecessaryforNb-induced BST2expressiononAF488^+DCsindLN.Wehavepreviouslyshow nthatTh2immuneresponsestoNbarereducedbyblock-ingIFN- Isig naling(11).We,therefore,assessedtheabilityofNbpreparatio nswithdifferentendotoxincontenttoinduceTcellresponses.Assho wninFigure6A,reducingtheendotoxin

contentofNbpreparationsdecreaseddLNcellularityandIL4^+andI F N γ^+Tc ellresponses.Adding1 0 , 0 0 0 E . colito“Low-

Endotoxin”Nbpreparationswasabletorescuethepercentagesandn umbersofbothIL4^+andIFNγ^{+C D 4 +T}cells(Figure6A).IL17A^+CD4^+T cellswerenotdetectedinsignificantnumbersafterNbinjectionthus theeffectofTLR4signalingcouldnotbeassessedinthispopulation.

WethenassessedtheimpactofTLR4deficiencyonthecon- tributionofIFN-

IsignalingtotheTcellresponse.AsshowninFigure6B,TLR4KOmi ceshowedlowerdLNcellularityandlowercytokineresponsestoNbi njection.Inaddition,treatmentwithIFNAR1-

blockingantibodieswasunabletodecreaseIL4^+TcellresponsesinTL R4KOhosts,whichisconsistentwiththelackofIFN-

Iproductioninthesemice.AsimilareffectwasseenalsoonIL10^+CD4+Tc ells.IFNγ^+CD4+TcellresponseswereessentiallyablatedbyblockingIF N-

IsignalingandbyTLR4deficiency.Ofnote,asmallpopulationofIL4⁺

I L 10^{+C D}4^+Tcellswasinduced

FigUre6|Toll-

likereceptor4(TLR4)enhancesthedevelopmentofCD4⁺IL4⁺T

cellsafterNippostrongylusbrasiliensis(Nb)injection.C57BL/6orTLR4KOmicewereinjectedintradermallyintotheearwith150Nbfromdif ferentpreparations.Tcellresponsesweremeasuredinthedraininglymphnodebyintracellularcytokinestainingandflowcytometry7daysafterNbinjection.Eachdotcorrespondstoonemouse.Da taarefromoneofatleasttworepeatexperimentsthatgavesimilarresults.

(a)TcellcytokineresponseinC57BL/6miceinjectedwithNb,orLowEndotoxin(LE,<5EndotoxinU/mL)Nb,orLowEndotoxinNbpreparations(LE-Nb)plus10,000formalin-fixedEcoli.

(B)TcellcytokineresponseinC57BL/6(WT)andTLR4KOmiceinjectedwithNbtogetherwithIFNAR1-

blockingantibodiesorisotypecontrol.Bargraphsshowmean±SEM.StatisticalanalysesusedtheANOVAwithTukey’smultiplecomparisonstest.Ns:notsignificant;*p<0.05;

(20)

**p<0.01;***p<0.001;****p<0.0001.

(21)

afterNbimmunization,butwasindependentofIFN-

IsignalingorTLR4expression(13.47±2.47%ofIL4^+CD4+Tcells,d atanotshown).

TheseresultsshowthatTLR4ligandscanamplifyNb- inducedTc ellresponsest hroughanI FN - I -

de pendentmechanism.T heroleofT L R 4 andI F N -

I s i g nalingappearstobei n e n hancingthemagnitudeoftheTcellim muneresponsetoNb,ratherthanspecificallyskewingtheresponseto wardTh2.

DiscUssiOn

Inthispaper,weshowthatthedetectionofNband/oritsassociatedmic roorganismsthroughTLR4allowsatransientsecretionofIFN- Iintheskin.PreventingTLR4signalingablatedtheupregulationofIF N-I-

inducedmarkersonDCsanddampenedthedevelopmentofIFNγ,IL1 0,andIL4-

secretingTcells,suggestingthatTLR4andIFNARsignalingareimp ortantbutnon-

specificamplifiersofadaptiveimmuneresponsestoNb.Wealsosho wthatNbinducesapotentrecruitmentandactivationofneutrophilsin thedermisandtheformationofNETs.However,wewereunabletode monstrateaneffectofneutrophilsorNETsonthenumberofNb^+DCsin dLN,orontheirexpressionoftheIFN-I-

inducedmarkerBST2andtheactivationmarkerCD86,suggestingth atNETformationdoesnotdriveimmuneactivationinresponsetoNb.

Neutrophilsarethemaincelltyperecruitedearlytothesiteofnon- viableNbinjection.WeshowthatneutrophilsinteractwithAF488^+N bintheskin,takingupasubstantialquantityofthefluorescentdyesas sociatedwithit(Figure1).NETshavebeenassociatedwiththesecre tionofIFN-

Iindifferentmodels,andhavebeenrecentlyi mplicatedi n mediati ngt hee x a c e r b ationofanasthmaticT h 2 responsei nducedbyrh inovirusi n fection(23,24,34,53).Unexpectedly,wecouldnotfinda nyimpactofneutrophild e pletionorN ETd i gestionont hed evelop mentoftheIFN-Isignatureonantigen-

bearingdLNDCsinourmodel.Indeed,Gr1^+cells,includingneutrop hils,havenotbeenreportedtoc ontributetot hed evelopmentofprima rya d aptivei m muneresponsestosterilepreparationsofliveNb(27).

However,neutrophilshavebeenshowntoi mprovememoryprotecti veresponsestoN bt hrought heirc ontrolofa lternativea ctivationof macrophagesi n t helung(28).AsN ETosisispreferentiallyinduced bythesensingoflargepathogens(39),itcouldrepresentamechanism ofhelminthtrappingandkilling(57).Asantibodiesandcomplement areimportantforneutrophilactivationduringNETosis(40),itappea rstobeworthwhileinvestigatingwhethertheNETosisprocesscontri butestoantihelminthicprotectiveimmunityduringsecondaryrespo nses.

Weshowt hate ndotoxinsore ndotoxin-

containingb acteria,whicharebothcommonlypresentinthehelmin thnaturalenvironment,canincreasetheTh2responsetoNbbyinduc ingaquickandtransientexpressionofIFN-

Iintheskin.AntibioticsterilizationofNbpreparationsdidnotaffect BST2expressiononA F 4 8 8^+DCsi n d L N , revealingt hatb acteria lproliferationorsecretionsinvivowerenoti mportantfort hismecha nism(FigureS4AinSupplementaryMaterial).Addingextrawashin gstepsduringN bpreparationtod i m i n isht heirp otentiale ndo-

toxincontentwassufficienttodecreasethedevelopmentofanIFN- IsignatureonAF488^+dLNDCsandtheTh2responseto

(22)

theworms.Theseeffectscouldbereversedbytheadditionofasmall numberofE.coliatthetimeofNbinjection.Inaddition,theSNfrom NbpreparationscouldenhancetheIFN-

IsignatureonprimaryB Mc ellsasmuchasNbt hemselves(Figure 3C).Therefore,Gram-

negativebacteriaorendotoxinshaveapoten-

tialroleinshapingDCactivationafterhelminthinjection,andthed evelopmentofantihelminthicTh2 responses(Figures5Band6A ).Inthisregard,itisnonethelessimportanttonotethatoureffortstoc ompletelyeliminatetheTLR4-

dependentactivityfromNbpreparationswereoverallunsuccessful ,suggestingthepossibilitythatselectedNbcomponentsmaybeabl etodirectlyengageTLR4andinitiatesignalingaswasdescribedf orHDMDerP1(58).Indeed,v arioushelminthsarereportedtoe x p ressglycans,glycolipids,orproteinaseswhoseproductsareabletoa ctivateTLR4(7,54,56,59).

WepreviouslyshowedthatTLR4deficiencyhadnoeffectonthe inductionofTh2responsestoNbinIL4-

reporterG4mice(29).TheTh2immuneresponsetoNbisdose- dependent,andusinga“saturating”doseofNb,suchas600L3larva e,preventedusfromobservingsignificantdifferencesatthattime.I nthiscontext,ifthedevelopmentofaTh2immuneresponseistheres ultoftheintegrationofdiverseredundantsignals,onemightnotobs ervethecontributionofeachofthesesignalsifthesystemissaturate dbyanoptimaldoseofstimulus.Indeed,IL25,IL33,andTSLPcane achcontributeredundantlyt otheoptimalTh2i m muneresponsein variousorgansandcontexts,possiblycompensatingfortheeffects ofTLR4-dependentIFN-

Isecretion(5).Here,wewereabletoobserveaTLR4dependencyon thedevelopmentofaTh2immuneresponsetoasuboptimaldoseofN b(Figure6B).

OurdatasuggestthatTLR4,whichisnotexpressedonconventio nalDC,enablesthesensingofNbanditsassociatedmicroorganism sasad angersig naltoi nduceIFN-

Iex pressionandindirectlyenhanceDCactivation,maturation,an d/orfunc-tionalabilities(16,60–62).ThesecretionofIFN- IinducedbyNbwascompletelyabrogatedinTLR4KOmice.Asaresu lt,wecouldnotobserveanyeffectofIFNARblockadeontheTh2resp onsetoNbinTLR4KOmice.Moreimportantly,TLR4anditscorece p-

torCD14areknowntobemainlyexpressedbymonocyteandmacr ophagep opulations.Asneutrophilsandmonocyteswerefoundtob ethemaincelltypesinteractingwithNbearlyintheskin,andasneut rophilswerenotfoundtobecriticalinmediatingNb-inducedIFN- Isecretion,wecanspeculatethatNbsensingbymonocyteand/orm acrophagepopulationsisinvolvedinthisprocess.Indeed,LPSrec ognitionthroughTLR4 expressionbymacrophagesisawell- knownstimulusofTRIF-

dependentIfnbexpression(47).However,wecannotexcludethepo ssibilitythatothercellssuchaskeratinocytes(63),orTLR4- mediatedIFN-

Iindependenteffects,mightalsocontributetothedevelopmentofT h2responses.Indeed,inHDM-

mediatedasthmamodels,TLR4expressionbyairwayepithelialcell shasbeenshowntocontributeina MyD88-dependentbutT R I F- independentf ashiontot hedevelopmentofanIL1-, alarmin-,and GM-CSF-

mediatedDCactivationtoallowthedevelopmentofapathogenicTh 2response(64–

66).Interestingly,theexpressionofIRF3byDCswasfoundtobecrit

icalfortheiroptimalmaturation,andforthedevelop-

mentofasthmainsimilarmodels.However,norolewasfoundforTR IForIFN-IsignalingthroughIFNAR2blockadeinthose

(23)

studies(67).Veryrecently,WebbetalreportedthatIFN-Isignal- ingw asi ndeedi mportantforDCa ctivation,maturation,andtheirin ductionofTh2responsesbybothHDMandSchistosomamansoni(12 ).AsHDMandS.mansoniarebothknowntoactivateTLR4,theTLR 4–IFN-

IaxismightbeanimportantmechanismenhancingDCmaturationan dTh2responsesinvariouspatholo-

gies,fromhelminthiasestoallergicdiseases.

Thel i fecycleofmanyparasitichelminthsi nvolvesseveraldistin ctphaseswherehelminthsfeedonb acteriaduringt heirfree- livingphaseinthesoil,andthenspendmostoftheirparasiticphaseint hegutwhileinfectingtheirhost.Itisdifficulttocon-

ceivehowanaturalhelminthinfectioncouldtakeplacewithoutinvol vingbarrierdisruptionofthehostandasmall-

scaleinvasionbyitssurroundingmicroorganisms.Gram-

negativebacteriasuchasWolbachiacanbeassociatedwithhelminth s,includingmosthuman-

infectingfilarialnematodes,insymbiotic(orobligatorysymbiotic) relationships.Wolbachia-

derivedendotoxinshavebeenshowntostronglyaffectt hei n f l am matoryresponsetoBrugiamalayinematodesthroughTLR4(68).O urresultsheresuggestthat,ifDCsintegratesignalsfromhelminthsa ndfromepithelialb arrierd isruptiontoshapea T h 2 i m munerespon se,thedetectionoftheendotoxinsoriginatingfromhelminth- associatedm i c roflorac oulda lsop articipateviaa T L R 4 andIF N-I-dependentmechanism.

Ourworkstronglysuggestst hat,i f a l i m iteda ctivationofantib acterials i g nalingp athwaysisl i kelytoo ccuri n a naturalhelminthi n fection,itm i ghtc ontributeto,andnoti n hibit,t hedevelopmentof antihelminthicTh2responses.

eThicss TaTeMenT

Alle x perimentalprotocolswereapprovedbyt heVictoriaUniversit yofWellingtonAnimalEthicsCommittee(Permit2014R17M)and performedaccordingtoInstitutionalguidelines.

aUThOrcOnTriBUTiOns

CPcarriedoutexperiments,analyzedthedataandwrotethemanusc ript.S-

CTgeneratedNbstocksandcarriedo utexperiments.ASandJDdes igned,o ptimized,andanalyzedconfocalmicroscopyexperiments .RWandOLcarriedo utRT-

qPCRtimecourses.CRp rovidedessentialr eagentsandexperime ntaladvice.LCprovidedconceptualinsightsanddataonthemodel.

GLGandFRprovidedconceptualinsightsandFRsupervisedthepr oject.AllAuthorsprovidedfeedbackonthemanuscript.

acKnOWleDgMenTs

Theauthorswishtothanktheex pertsupportoftheMalaghanInstitut eofMedicalResearchHughGreenCytometryCore,ResearchInfor mationTechnologiesandBiomedicalR esearchUnitstaff.Weareex tremelygratefultoProfessorShizuoAkira,OsakaUniversity,fordo natingTLR2KOandTLR4KOmice.

FUnDing

ThisresearchwasfundedbyanIndependentResearchOrgani- zationsgrantfromtheHealthResearchCouncilofNewZealandtothe MIMR,andinpartbyaPolishNationalCenterforSciencegrant2013 /11/B/NZ3/00189toJD.Thisworkwasmadepossiblebyt heg rac iousg iftofa F V 1 2 0 0 c onfocalm i c roscopebyt heInfinityFounda tion,NewZealand.

sUPPleMenTarYMaTerial

TheSupplementaryMaterialforthisarticlecanbefoundonlineathttp ://www.frontiersin.org/article/10.3389/fimmu.2017.01575/full

#supplementary-material.

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