Anti-Inflammatory Mechanisms of EntericHeligmosomoides polygyrus

Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.

Vol.76,No.10

Anti-InflammatoryMechanismsofEntericHeligmosomoidespolygyrusInfectionagainstTrinitrobenzeneSulfonicAcid-InducedColitisina

MurineModelᰔThomasL.Sutton,1AipingZhao,2KathleenB.Madden,3JustinE.Elfrey,2†BlaineA.Tuft,1‡

CarolynA.Sullivan,1JosephF.Urban,Jr.,4andTerezShea-Donohue2*

DepartmentofPediatrics,WalterReedArmyMedicalCenter,Washington,DC203071;DepartmentofMedicineandthe

MucosalBiologyResearchCenter,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland212012;

DepartmentofPediatrics,UniformedServicesUniversityoftheHealthSciences,Bethesda,Maryland208143;and

Diet,Genomics,&ImmunologyLaboratory,BeltsvilleHumanNutritionResearchCenter,

AgriculturalResearchService,U.S.DepartmentofAgriculture,Beltsville,Maryland207054Received1June2007/Returnedformodification17July2007/Accepted12July2008

Recentstudiesshowedthatenterichelminthinfectionimprovedsymptomsinpatientswithinflammatoryboweldiseaseaswellasinexperimentalmodelsofcolitis.Theaimofthisstudywastodeterminethemechanismoftheprotectiveeffectofhelminthinfectiononcolitis-inducedchangesinimmuneandepithelialcellfunction.BALB/cmicereceivedanoralinfectionofHeligmosomoidespolygyrusthird-stagelarvae,weregivenintrarectalsalineortrinitrobenzenesulfonicacid(TNBS)onday10postinfection,andwerestudied4dayslater.SeparategroupsofmicereceivedintrarectalsalineorTNBSonday10andwerestudiedonday14.Muscle-freecolonicmucosaeweremountedinUssingchamberstomeasuremucosalpermeabilityandsecre-tion.Expressionofcytokineswasassessedbyquantitativereal-timePCR,andmastcellswerevisualizedbyimmunohistochemistry.TNBS-inducedcolitisinducedmucosaldamage,upregulatedTh1cytokines,andde-pressedsecretoryresponses.HeligmosomoidespolygyruselevatedTh2cytokineexpression,increasedmastcellinfiltrationandmucosalresistance,andalsoreducedsomesecretoryresponses.PriorH.polygyrusinfectionpreventedTNBS-inducedupregulationofTh1cytokinesandnormalizedsecretoryresponsestospecificago-nists.TNBS-inducedcolitisdidnotalterH.polygyrus-inducedmastcellinfiltrationorupregulationofTh2cytokineexpression.TheresultsindicatethattheprotectivemechanismofentericnematodeinfectionagainstTNBS-inducedcolitisinvolvespreventionofTh1cytokineexpressionandimprovedcolonicfunctionbyamechanismthatmayinvolvemastcell-mediatedprotectionofneuralcontrolofsecretoryfunction.Similarresponsepatternscouldaccountfortheclinicalimprovementseenininflammatoryboweldiseasewithhelminthictherapy.

Thevertebrateimmunesystemhascoevolvedwithentericpathogens,leadingtotheelaborationofpolarizedTh1orTh2cytokineprofiles.Disturbanceofthebalancebetweentheseimmunepathwayshasdeleteriousconsequencesforthehost.Inflammatoryboweldisease(IBD),specificallyCrohn’sdis-ease,isdrivenbyaTh1-dominantimmuneresponse.Theini-tiatingfactoristhoughttobeadysregulatedimmuneresponsetonormalgutfloraingeneticallypredisposedindividuals,re-sultinginchronicgastrointestinal(GI)inflammationleadingtosignificanthostmorbidity.TheincidenceandprevalenceofIBDhaveincreasedsubstantiallyoverthepast50yearsinindustrializedcountries,buttheincidenceremainslowinun-derdevelopedareas.Thisobservationcouldnotbeexplainedcompletelybygeneticfactors,andthereforethe“hygienehy-pothesis”evolvedasaresultofthedramaticreductionin

exposureoftheguttopathogensasaconsequenceoflifestyleandenvironmentalchangesdesignedtoeliminateexposuretobacteriaandparasites,suchasnematodes(18).Inthispara-digm,theabsenceofhelminthinfectionseliminatesthenormalupregulationoftheTh2and/orTregulatoryimmuneresponseinchildhood,culminatinginamoreTh1-proneimmunere-sponsecharacteristicofautoimmuneandinflammatorydis-eases(18).

Theinteractionbetweenimmuneandstructuralcells,suchassmoothmuscleandepithelialcells,iscriticaltoTh2cyto-kine-mediatedimmunity.Infection-inducedalterationsingutfunctionplayamajorroleinhostresistanceagainstentericnematodeinfection.ItiswellestablishedthatinductionofTh2cytokines,includinginterleukin-4(IL-4)andIL-13,isapre-requisiteforexpulsion.Incontrast,theTh1profile,featuringhighlevelsofIL-12,tumornecrosisfactoralpha(TNF-␣),orgammainterferon(IFN-␥),promoteswormsurvivalintheGItract.BecauseoftheabilityofTh2cytokinestomodulateTh1cytokines,thereisconsiderableinterestintheircontribution,aswellasthecontributionsoftheirindividualreceptors,tobothTh1-andTh2-predominantpathologies.

Theinfectiousthird-stagelarvae(L3)ofHeligmosomoidespolygyrusinvadethesubmucosaoftheduodenumforapprox-imately8daysbeforetheadultstageemergesandpersistsin

4772

*Correspondingauthor.Mailingaddress:MucosalBiologyRe-searchCenter,UniversityofMarylandSchoolofMedicine,20PennStreet,Baltimore,MD21201.Phone:(410)706-5503.Fax:(410)706-5508.E-mail:tdonohue@mbrc.umaryland.edu.

†Presentaddress:NewYorkCollegeofOsteopathicMedicine,OldWestbury,NY11568.

‡Presentaddress:DepartmentofPediatrics,NavalMedicalCenter,Portsmouth,VA23708.ᰔPublishedaheadofprinton21July2008.

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theproximalsmallintestineforweekstomonths.PrimaryexposureresultsinachronicinfectionwithanupregulationoftheTh2cytokineresponseandminimaldamage(45).PreviousreportsdemonstratedthatseveraldifferententericpathogensamelioratedexperimentalTh1-drivencolitisinmice(16,26,37),andH.polygyrussignificantlyreducedtheinflammationseeninIL-10-deficientmice(17).Morerecently,inhumans,administrationofTrichurissuiseggswasshowntoimprovetheclinicalseverityofCrohn’sdiseaseandulcerativecolitis(49,50).Theprotectiveabilityofnematodeinfectionisthoughttobeimmunomediated;however,therearefewdatashowingchangesinlocalcolonicexpressionofcytokines.

ItisknownthatTh1-driveninflammationisassociatedwithalterationsinmucosalfunctioninboththesmallintestineandcolonthatcontributetothedevelopmentofdiarrhea,amajorsymptomofIBD.Oneofthemajoractionsofthemucosalepitheliumistosecreteafluidthatfacilitatesremovalofnox-iousmaterialsandagentsanddeliversimmunoglobulinAintothegutlumen.Reflexcircuitsthatcontrolsecretionareacti-vatedbyanumberofmechanicalandchemicalstimulithatoriginateinthelumenandinvolveserotonin(5-HT)andace-tylcholine(13).Mastcellsalsoaltersecretionbyincreasingthesensitivityofnervestostimulationthatcanleadtohypersecre-toryresponsescharacteristicofallergy(3).Itwasofinterest,therefore,thatexperimentalmodelsofIBDinduceastereo-typichyposecretioninresponsetoanumberofstimuli(41,42,61).Thisparadoxicaleffectonsecretionsuggeststhatthedi-arrheaobservedinIBDisattributedtodecreasedsodiumabsorptionduetolossordisruptionofsurfaceepithelialcells.Mastcellsarekeymediatorsofallergicandothermodelsofinflammation,includingintestinalhelminthinfection(29).Mastcellmediators,includingtryptase,5-HT,andhistamine,canmodulateepithelialcellsecretion(20).Tryptasebindstooneoftheprotease-activatedreceptors(PARs)amongtheclassofuniqueGprotein-coupledreceptors.AlthoughatleastfourPARshavebeenidentified,activationofPAR-1orPAR-2hasimportantimplicationsinthegut(62,).Thesereceptorsareautoactivatedthroughtheirown“tetheredligand”uponenzymaticcleavagebythrombin(PAR-1),trypsin(PAR-2),ormastcelltryptase(PAR-2)(39,56).BothPAR-1andPAR-2activationincreasesmucosalsecretionandpermeabilityandexertsproinflammatoryeffectsintheGItract(6–9,11,28,55).Histamineisanothermajormastcellmessengerthathasnu-merousphysiologicandpathophysiologiceffectsmediatedthroughatleastfourdistinctGprotein-coupledreceptors,representedbyhistaminereceptorsthatincludeH1R,H2R,H3R,andH4R(25).Inthegut,H1Risexpressedonepithelialcellsandisinvolvedinchloridesecretion(2).Incontrast,themorerecentlydiscoveredH4R(33,38,40,65),preferentiallyexpressedonimmunecellsofhematopoieticorigin,ispro-posedtoplayaroleintheregulationofimmuneandinflam-matoryprocesses(59).

Theprecisemechanismforthebeneficialeffectsofnema-todeinfectiononTh1-driveninflammationremainsunclearbutmayincludealterationsinthelocalimmuneresponseand/orepithelialcellfunction.Theaimsofthisstudyweretodetermine(i)iftheprotectiveeffectsofintestinalnematodeinfectiononcolitis-inducedalterationsareduetolocalim-munemodulation,includingcolonicTh1orTh2cytokineex-pression,H4Rexpression,andmastcellactivity;and(ii)if

intestinalhelminthinfectionaffectscolitis-inducedalterationsinepithelialcellfunction.Ourresultssuggestthattheprotec-tiveeffectsofparasiticnematodeinfectionareduetolocalimmunemodulationoftrinitrobenzenesulfonicacid(TNBS)-inducedinflammatorychangesleadingtoattenuationorpre-ventionofinflammation-inducedchangesincolonicmucosalfunction.

MATERIALSANDMETHODS

Animals.FemaleBALB/cmice(aged8to10weeks)wereobtainedfromCharlesRiver,Inc.(NCI,Frederick,MD).Themicewereagematchedwithcontrolsinallfourexperimentalgroups.Thesestudieswereconductedinaccor-dancewithprinciplessetforthintheGuidefortheCareandUseofLaboratoryAnimals(38a)andbytheUniversityofMarylandSchoolofMedicineInstitu-tionalCareandUseCommittee.

Treatmentgroups.ThetimingofTNBSintrarectalinjectionandH.polygyrusinoculationwascoordinatedtoensurethatmicefromeachtreatmentgroupwerestudiedonthesameday.

(i)TNBS-inducedcolitis.Micewerefastedfor2hpriortotreatmentbutwereallowedfreeaccesstowater.AnesthetizedmicereceivedanintrarectalinjectionofTNBS(2mg/mouse)in40%ethanolorsaline(totalvolume,0.1ml)andwereheldbythetailfor3mintoensurecontinuedexposuretotheagent.Micewerestudied4dayslater.

(ii)Heligmosomoidespolygyrusinfection.Infective,ensheathedL3ofH.polygy-rus(specimensonfileattheU.S.NationalHelminthologicalCollection,USDA,Beltsville,MD)werepropagatedandmaintainedasdescribedpreviously(53)andwerestoredat4°Cuntiluse.BALB/cmicewereinoculatedorally,viaan18-gaugeball-tippedfeedingtube,with200L3onday0andwerestudiedat14dayspostinfection.AprimaryH.polygyrusinfectionpersistsforanumberofweeks.

(iii)HeligmosomoidespolygyrusinfectionplusTNBStreatment.At10dayspostinfection,one-halfoftheH.polygyrus-infectedBALB/cmicereceivedintrar-ectalTNBSandtheotherhalfreceivedsaline,asdescribedabove.

Histology.Sections(1.5cm)ofdistalcolonwereopenedalongthemesentericborder,rinsedpromptlyinsaline,andplacedin4%paraformaldehyde.Thetissuewasembeddedinparaffin,sectionedtransversely(5␮m),andstainedwithGiemsastaininordertovisualizegranulocytes.Thedegreeofinflammationandtissuedamageonmicroscopiccrosssectionsofthecolonwasgradedsemiquan-titativelyfrom0to10,usingastandardizedscoringsysteminwhichtissuewasgiven1pointforthepresenceofanyofthefollowingfeatures:disruptionofepithelialcells(lossofepithelialcells,mucosallifting,mucosalulceration,ordistortionofepithelialcrypts),lossofgobletcellsorgobletcellmucus,mucosaledema,thepresenceofneutrophilicorlymphocyticinfiltrateorgranulomas,hemorrhage,ormucosalorsmoothmusclehypertrophy.Twoinvestigatorswhowereunawareofthegiventreatmentperformedthegrading;thescoreswereaveragedforeachslidetoobtainafinalslidescoreforeachanimal.Thesescoreswerethenaveragedforeachtreatmentgroup.

Immunofluorescencestaining.FrozenblocksofcolonwerepreparedbyusingtheSwissrolltechniqueandwerestoredatϪ80°Casdescribedpreviously().Tissuesections(5␮m)werecutfromfrozenblocksbyuseofanHM505Ecryostat(RichardAllanScientific).SlideswerekeptondryiceandthenstoredatϪ80°C.Forimmunofluorescencestaining,tissueslideswerefixedincoldacetonefor30minandsubsequentlyblockedwith10%normalrabbitseruminphosphate-bufferedsalinefor1hatroomtemperature.Aftertheadditionof1:100dilutedprimarygoatantibodiesformastcellprotease(MCP;SantaCruzBiotechnology),slideswereincubatedovernightinahumidifiedchamberat4°C.Slideswererinsedfor30mininphosphate-bufferedsaline,followedbyincuba-tionwithfluoresceinisothiocyanate-conjugatedrabbitanti-goatantibody(Jack-sonImmunoresearchLaboratories)for1h,usinga1:200dilution(fromtheoriginalstocksolution).TheslideswerethencoverslippedwithVectorshield(Vec-torLaboratories)anddigitallyphotographedwithaNikonEclipse80imicroscopeusingImage-ProPlus5.1software.Theintensityofstainingwasdeterminedbyestablishingsettingsforthesamplesfromthevehiclegroupandusingthesameconditionstoevaluatethesamplesfromtheinfectedortreatedgroups.Comparisonsweremadeonlyamongtheslidespreparedonthesameday.

Ussingchambers.One-centimetersegmentsofmucosawerestrippedofmus-cleandmountedinUssingchambersthatexposed0.126cm2oftissueto5mlKrebsbuffer.Agar-saltbridgesandelectrodeswereusedtomeasurepotentialdifference.Every50s,thetissueswereshortcircuitedat1V(DVC1000voltageclamp;WorldPrecisionInstruments,Sarasota,FL),andtheshortcircuitcurrent

4774SUTTONETAL.TABLE1.Primersequencesforreal-timequantitativePCR

Gene

Primerorientation

Primersequence(5Јto3Ј)

PAR-1ForwardGCTGGAGGGTAGGGCAGTCTReverseGTACACGGAGGGCATGAAGAGPAR-2ForwardCTGCATCTGTCCTCACTGGAReverseACAGAGAGGAGGTCAGCCAA

IFN␥ForwardGCATAGATGTGGAAGAAAAGAGTCTCTReverseTGGCTCTGCAGGATTTTCATG

TNF-␣ForwardCATCTTCTCAAAATTCGAGTGACAAReverseCCAGCTGCTCCTCCACTTGIL-4ForwardCGGAGATGGATGTGCCAAACReverseGCACCTTGGAAGCCCTACAGIL-13ForwardGACCAGACTCCCCTGTGCAAReverseTGGGTCCTGTAGATGGCATTGH1RForwardTCCGAAGACAAGATGTGTGAGCReverseCACTGTGACCAGGAGATACTACH4RForwardGGCTCCATACTGTCTGTTCAC

ReverseCAGAAAGGGATTAACAAACGAATTGMCP-1

ForwardTGGGAAGTTCCACAAAGTTAAAAACReverseGCCACACCAGCACACAGAAG

(Isc)wascontinuouslymonitored.Inaddition,every50s,theclampvoltagewasadjustedto1Vfor10stoallowforcalculationoftissueresistanceusingOhm’slaw.

Aftera15-minperiod,concentration-dependentchangesinIscinseparatetissuesweredeterminedfollowingthecumulativeadditionofacetylcholine,his-tamine,or5-HT(allat1nMto1␮M)totheserosalside.ChangesinIscwerealsodeterminedinresponsetoPAR-1(TFLLR;100␮M)andPAR-2(SLIGRL;100␮M)agonists.Afterthepeakresponsetothefinalconcentrationofeachsecretagoguewasrecorded,theKrebsbufferoneachsideofthechamberwasreplaced.Todeterminethecontributionofentericnervestotheresponse,tissueswereincubatedwiththesodiumchannelblockertetrodotoxin(TTX;1␮M)for15minpriortotheadditionofsecretagogues.

RNAextraction,cDNAsynthesis,andreal-timequantitativePCR.TotalRNAswereextractedfromsamplesofmid-colonwithTRIzolreagent(Invitro-gen,GrandIsland,NY)perthemanufacturer’sinstructions.RNAintegrityandquantityandgenomicDNAcontaminationwereassessedusinganAgilentBio-analyzer2100machineandanRNA6000Labchipkit(AgilentTechnologies,PaloAlto,CA)asdescribedpreviously(63).OnlythoseRNAsampleswith28S/18Sratiosbetween1.5and2andnoDNAcontaminationwerestudied.RNAsamples(2␮g)werereversetranscribedtocDNAsbyuseofafirst-strandcDNAsynthesiskit(MBIFermentas,Hanover,MD)witharandomhexamerprimer.Real-timequantitativePCRwasperformedonaniCyclerdetectionsystem.PrimersequencesweredesignedbyusingBeaconDesigner4.0(PremierBiosoftInternational,CA)andweresynthesizedbytheBiopolymerLaboratoryoftheUniversityofMaryland.TheprimersequencesforPAR-1,PAR-2,IFN-␥,TNF-␣,IL-4,IL-13,H1R,H4R,andMCP-1arelistedinTable1.PCRwasperformedina25-␮lvolume,usingSYBRgreenSupermix(Bio-Rad,CA).Amplificationconditionswereasfollows:95°Cfor3minand50cyclesof95°Cfor15s,60°Cfor15s,and72°Cfor20s.ThechangesinmRNAexpressionforPAR-1,PAR-2,IFN-␥,TNF-␣,MCP-1,IL-4,IL-13,H1R,andH4Rwerede-terminedrelativetotherespectivevehiclegroupsofmiceafternormalizationtothe18SrRNAhousekeepinggene.

Solutionsanddrugs.Krebsbuffercontained4.74mMKCl,2.54mMCaCl2,118.5mMNaCl,1.19mMNaH2PO4,1.19mMMgSO4,25.0mMNaHCO3,and12mMglucose.Thetissueswereallowedtoequilibratefor15mininKrebsbuffer.AlldrugswereobtainedfromSigma(St.Louis,MO)unlessstatedoth-erwise.Stocksolutionswerepreparedasfollows.ThePAR-1peptideagonistTFLLRandthePAR-2peptideagonistSLIGRL(10mM;synthesizedbytheUniversityBiomedicalInstrumentationCenter,UniformedServicesUniversityoftheHealthSciences)weredissolvedin20%dimethylsulfoxideandstoredatϪ70°Cinaliquots.Onthedayoftheexperiment,PAR-1,PAR-2,5-HT,andhistamineweredissolvedinwater,andappropriatedilutionsof5-HTandhista-mineweremadeusingdistilledwater.

Dataanalysis.Foreachmouse,responsesforeachsecretagogueweredeter-minedforasingletissue,andthereforethenforeachgroupreflectsthenumberofmice.Resistancewascalculatedforalltissuesegmentsfromeachmouseandwasaveragedtoyieldonemeanperanimal.Statisticalanalysiswasperformed

INFECT.IMMUN.

usingone-wayanalysisofvariancetocompareinflammatoryscores,resistances,andmaximalIscresponsesamonggroups.Cumulativedoseresponseswerecomparedusingmultipleanalysisofvariancewithposthocanalysisformultiplecomparisons.PvaluesofϽ0.05wereconsideredsignificant.

RESULTS

PriorH.polygyrusinfectionattenuatedTNBS-inducedcolonicinjury.Incontrolmice,thecolonicmucosaexhibitedintactepithelia,anevendistributionofgobletcells,minimalinfiltrateinthelaminapropriaandsubmucosa,andnormalsmoothmusclethickness(Fig.1A).Heligmosomoidespolygyrusinfectiondidnotcausemucosaldamage(Fig.1B)butinducedgobletcellhyperplasia,lymphocyticinfiltration,andsmoothmusclethickeninginthecolon.AdministrationofTNBStomicesignificantlyincreasedtissuedamageinthecolon(Fig.1C),includingdisruptionofepithelialcells,dilationofcrypts,wideningofthesubmucosalspace,andthickeningofthesmoothmuscle.Separatesectionsofcolonalsoshowedin-creasedlymphocyticinfiltrate,lossofnormalmucosalarchi-tecture,andgranulomas(Fig.1D).Interestingly,priorH.po-lygyrusinfectionmarkedlyattenuatedTNBS-inducedcolonicdamageandinflammation,withanabsenceofgranulomafor-mationandsubmucosaledema(Fig.1E).Therewasareten-tionofsmoothmusclehypertrophyandoftheelevatednumberofgobletcellscharacteristicofinfectionalone(Fig.1B),andthusthemicroscopicdamagescoreremainedelevatedabovethatofcontrols.Themeandamagescore(Fig.1F)demon-stratestheprotectiveeffectofpriornematodeinfectiononTNBS-inducedcolitis.

TheprotectiveeffectofpriorH.polygyrusinfectiononcolitiswasassociatedwithadecreaseinmRNAexpressionofTh1cytokines.ItiswellestablishedthatincreasedproductionofTh1cytokinesplaysanimportantroleinthepathogenesisofcolitis.Consistentwithpreviousstudies,micetreatedwithTNBSexhibitedanupregulationoftheTh1cytokinesIFN-␥andTNF-␣(Fig.2A),butnotoftheTh2cytokinesIL-13andIL-4(Fig.2B),inthecolon.Heligmosomoidespolygyruspref-erentiallycolonizesthesmallintestine,andthereforetheabil-ityoftheinfectiontoupregulateexpressionofIL-4andIL-13(17.4-and356.0-fold,respectively)wasconfirmedinthisre-gion.Heligmosomoidespolygyrusinfectionalsoinducedasig-nificantincreaseintheexpressionofTh2cytokinesIL-4andIL-13inthecolon(Fig.2B)buthadnoeffectonexpressionoftheTh1cytokines(Fig.2A).PriorH.polygyrusinfectionpre-ventedtheTNBS-inducedupregulationofTh1cytokines(Fig.2A).Incontrast,theH.polygyrusinfection-inducedupregula-tionofTh2cytokinesinthecolonwasunalteredbyadminis-trationofTNBS(Fig.2B).

PriorH.polygyrusinfectionimprovedcolitis-inducedchangesinepithelialcellfunction.Abnormalcolonicepithelialcellfunctionisimplicatedinthepathogenesisofcolitis.TodetermineiftheprotectiveeffectofpriorH.polygyrusinfectiononTNBS-inducedcolitisisassociatedwiththealterationofcolonicepithelialcellfunction,muscle-freesectionsofmuco-saeweremountedinUssingchambers.Colonicmucosalresis-tance,anindexofpermeability,wasincreasedsignificantlybyH.polygyrusinfection(Fig.3),indicatingthebeneficialeffectofinfectiononbarrierfunction.AlthoughTNBStreatmentalonedidnotaltermucosalresistance,thepriorH.polygyrusinfec-

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FIG.1.Samplesofdistalcolonwerefixed,paraffinembedded,sectioned(5␮m),andstainedwithGiemsastaintovisualizeinflammatoryinfiltrate.Representativesamplesfromcontrols(A)andthetreatmentgroupsareincluded.(B)H.polygyrusinfectionshowedfewchanges,withthemostnotablebeingagobletcellhyperplasiaandthickeningsmoothmuscle.(C)Incontrast,TNBSadministrationinducednumerousinflammatorychanges,includingcryptexpansion(asterisk),neutrophilinfiltration(arrowheads),anddisruptionofsurfaceepithelialcells(arrow).(D)Inaddition,therewereareasofgraulomatousinfiltration.(E)InfectionwithH.polygyruspriortoTNBSexposuremarkedlyattenuatedTNBS-inducedinflammation,includingprotectionagainstmucosaldamageandneutrophilinfiltration.(F)ThemeanscoresforallgroupsdemonstratethatpriorH.polygyrusinfectionprotectsagainstthedamageandinflammationinducedbyTNBS(*,PϽ0.05versuscontrol;**,PϽ0.01versuscontrol;␾,PϽ0.05versusTNBSgroup).

tion-inducedincreaseinmucosalresistance(decreaseinper-meability)remainedevenafterthesubsequentadministrationofTNBS(Fig.3).

Wefurtherassessedcolonicepithelialcellsecretaryre-sponsestovarioussecretagogues.5-HT,releasedfromentero-chromaffin-likecells,bindstoreceptorsonsensorynervesformingtheafferentarmofreflex-mediatedsecretion.Secre-toryresponsesto5-HTweredecreasedsignificantlybyTNBSbutnotbyH.polygyrus(Fig.4A).PriorentericinfectionwithH.polygyruspreventedtheTNBS-inducedinhibitionofsecre-tioninresponseto5-HT.Acetylcholine,uponreleasefromentericnervesaspartofthereflexivesecretoryarc,bindstomuscarinicreceptorsonepithelialcells.Colonicsecretoryre-sponsestoacetylcholineweredecreasedbyTNBSadministra-tion(Fig.4B).AlthoughH.polygyrusinfectionalonedidnotaffecttheresponse,itdidpreventthehyposecretioninre-

4776SUTTONETAL.FIG.2.Samplesofcolonweretakenfromeachgroup.SignatureTh1andTh2cytokineswerequantitatedusingreal-timequantitativePCRonwholecolonictissue.(A)TNBSinducedasignificantincreaseinTh1cytokineexpression,whileH.polygyrussignificantlyupregulatedTh2cytokineexpression.PriorexposuretoH.polygyruspreventedupregulationofTh1cytokines.(B)TNBShadnoeffectontheupregu-lationofTh2cytokinesbyH.polygyrus(*,PϽ0.05versuscontrol).

sponsetoacetylcholineinducedbyTNBS(Fig.4B).TTX,aneurotoxin,significantlyreducedtheresponsetoacetylcholine,by38%,incontrols,indicatingasignificantcontributionbyentericnerves(Fig.4B).Thisneuralcontributionwasen-hancedinH.polygyrus-infectedmice,asTTXdecreasedtheresponsetoacetylcholineby73%.Incontrast,TTXdidnotalterresponsestoacetylcholineinTNBS-treatedmice,dem-onstratingalossofthecontributionofnervestoinflammation.PriorinfectionwithH.polygyrusrestoredtheTTXsensitivitytoacetylcholineandrenderedtheresponsesnearly100%depen-dentonentericnerves(Fig.4B).

WeshowedpreviouslythataprimaryH.polygyrusinfectioninducesasignificantmastocytosisinBALB/cmice(45).Mastcellsproduceanumberofproteases,andmastcellmediatorscanalterintestinalsecretion,inpartbyincreasingthesensi-tivityorresponsestoneuralinput(45).EndogenousligandsofPAR-1(thrombin)andPAR-2(tryptaseandtrypsin)haveanumberofeffectsonepithelialcellfunction.Colonicexpres-sionofPAR-1andPAR-2wasunchangedbycolonicinflam-

INFECT.IMMUN.

FIG.3.Resistance,anindexofmucosalpermeability,wasdeter-minedwithUssingchambers,usingmuscle-freecolonicmucosa.TNBSdidnotalterpermeability,whileH.polygyrussignificantlyincreasedresistance(Hp1).ThisincreasedresistancepersistedafterinjectionwithTNBS(Hp1ϩTNBS)(**,PϽ0.01versuscontrol).

mationorintestinalinfection(Table2).ColonicsecretioninresponsetothePAR-1agonistTFLLRwasdecreasedinallgroups(Fig.4C).Incontrast,therewasaselectiveinhibitionoftheresponsetothePAR-2agonistSLIGRLbyTNBSthatwascompletelypreventedbypriorinfectionwithH.polygyrus(Fig.4C).

PriorH.polygyrusinfectionalteredTNBS-inducedchangesinhistaminereceptorexpression.Histamineisalsosecretedbymucosalmastcellsandhasdirectprosecretoryeffectsbybind-ingtoreceptorsonepithelialcellsaswellasindirecteffectsbyincreasingthesensitivityofentericnervestostimulation.Inthepresentstudy,responsestohistaminewereinhibitedbyalltreatments(Fig.5A).HistamineincreasessecretionbybindingtoH1Rlocatedonepithelialcells.ThedecreaseinH1Rex-pressionintheTNBS-treated,H.polygyrus-infected,andH.polygyrus-plus-TNBS-treatedgroups(Fig.5B)isconsistentwiththehyposecretoryresponsetohistamineseeninthesegroups(Fig.5A).H4Risanimportantproinflammatoryme-diatorthatisfoundonimmunecellsofhematopoieticlineage.TNBSsignificantlyupregulatedH4Rexpression(Fig.5C);thiseffectwaspreventedcompletelybypreviousH.polygyrusinfec-tion.

MastcellshavearoleintheprotectiveeffectofH.polygyrusonTNBS-inducedinflammation.ToexplorethecontributionofmastcellstotheprotectiveeffectofH.polygyrusonTh1-driveninflammatorychanges,weassessedexpressionofMCP-1byreal-timePCRandimmunohistochemistry.MCP-1geneexpressionwasincreasedsignificantlybyH.polygyrusbutwasunchangedbyTNBS(Fig.6A).Inaddition,theinfection-inducedincreaseinMCP-1wasnotalteredbyTNBSadmin-istration.ControlsshowedmodestimmunostainingforMCP-1(Fig.6B)thatwasmarkedlyincreasedinH.polygyrusinfection(Fig.6C).Incontrast,immunostainingforMCP-1wascom-pletelyabsentintheTNBSgroup(Fig.6D),andTNBSde-creasedstaininginmicewithpriorH.polygyrusinfection(Fig.6E).TheeffectofTNBStreatmentindecreasingstaininginuninfectedmice(versusvehicle-treatedcontrols)andinfected

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FIG.4.Secretioninresponseto5-HT(A),acetylcholine(B),andthePAR-1agonistTFLLRandPAR-2agonistSLIGRL(C)wasde-terminedinmuscle-freecolonicmucosaebyuseofUssingchambers.TNBSuniformlyinducedahyposecretioninresponsetoallsecreta-goguestested(AtoC).PriorentericinfectionwithH.polygyrus(Hp1ϩTNBS)preventedtheTNBS-inducedhyposecretioninre-sponsetoacetylcholine,5-HT,andthePAR2,butnotPAR-1,agonist.SecretioninresponsetoacetylcholinewasreducedbyTTXincontrolandH.polygyrus(Hp1)-infectedmice(B).Thissensitivityofacetylcho-line-inducedresponsestoTTXwaslostinTNBS-treatedmicebutwasrestoredinmicetreatedwithTNBSafterinfectionwithH.polygyrus.Ͻ0.05versusrespectivecontrol;**,PϽ0.01versusrespective*,Pcontrol;␾,PϽ0.05versusrespectivevehicle;␾␾,PϽ0.01versusrespectivevehicle.

TABLE2.EffectsofH.polygyrusinfectiononTNBS-inducedalterationsinPARexpressionandinsecretoryresponses

toacetylcholine

FoldchangeinexpressionResponseGroup

(IPAR-1

PAR-2

toscacetylcholine͓␮A/cm2͔)aControl1.0Ϯ0.041.0Ϯ0.163Ϯ13TNBS

1.2Ϯ0.20.7Ϯ0.124Ϯ8*H.polygyrusinfection0.9Ϯ0.21.0Ϯ0.153Ϯ13H.polygyrusinfectionϩ1.2Ϯ0.21.0Ϯ0.170Ϯ24

TNBS

a*,PϽ0.05versuscontrolgroup.The͓acetylcholine͔was100␮M(nϭ6to8).

mice(versusinfectedcontrols)isconsistentwithaninflamma-tion-inducedlossand/ordegranulationofmastcells.

DISCUSSION

Itiswellknownthatthebalanceofimmunoderivedcyto-kinesinvivoiscriticaltothecontrolofimmunefunctionandthatpolarizationtowardtheTh1orTh2cytokineprofilecanhaveconsequencesthatareinappropriatetohostdefense.TheincreasedTh1responsetoTNBSisassociatedwithmoderatetoseveregranulomatous,andsometimestransmural,inflam-mationsimilartothatobservedinCrohn’sdisease.Theabilityofinfection-inducedupregulationofTh2cytokinestoattenu-ateTh1-mediatedinflammationintheGItractisclear(16,26,37,49,50),butpreviousstudiesdidnotaddressimmunome-diatedchangeslocalizedtotheaffectedareaorassesspossiblebeneficialeffectstogutfunction.Inthepresentstudy,main-tenanceoftheTh2-polarizedresponseinducedbyH.polygyrusinthesmallintestineandcoloneffectivelypreventedTNBS-inducedcolonicupregulationofgeneexpressionfortheTh1cytokinesIFN-␥andTNF-␣,pathologicalinjurytothemucosa,andalterationsinepithelialcellfunction.Thesedataarecon-sistentwiththehypothesisthatnematodeinfectionpreventsanexacerbatedresponsetoproinflammatorystimuliinthecolon.Thus,thebeneficialeffectsofnematodeinfectioninthesmallintestineorchestrateimmunomediatedchangesandprotectionfrominflammationatadistantsiteinthecolon.

TheresultsofthepresentstudyareincontrasttothoseshowingthathelminthinfectionworsenedcolitiscausedbyinfectionwithCitrobacterrodentium,anenteropathogensimi-lartoEscherichiacoli(10).ItiswellestablishedthatinductionoftheTh1profile,featuringhighlevelsofIL-12,TNF-␣,orIFN-␥,isaprerequisiteforexpulsionofanumberofentericpathogens,includingC.rodentium.Incontrast,upregulationofTh2cytokines,includingIL-4andIL-13,impairsitsclearance.Itisnotsurprising,therefore,thattheabilityofH.polygyrustopreventupregulationoftherequisiteTh1responsewouldworsenC.rodentium-inducedinflammationbyprolongingex-posuretounrestrictedpathogenproliferation.Itshouldbenoted,however,thatwhilethecommensalbacteriaplayanimportantroleinthepathogenesisofIBD(22)aswellasotherspontaneousanimalmodelsofinflammation(30),Crohn’sdis-easeandulcerativecolitisareconsideredtobeimmunomedi-atedratherthaninfectious.TheetiologyoftheTNBS-inducedmodelofcolitismaythereforebeclosertoclinicalautoimmunediseaseinthisregard.

4778SUTTONETAL.FIG.5.Secretoryresponsestohistamine(changesinIsc)weredeterminedinmuscle-freecolonicmucosaebyuseofUssingcham-bers,andsecretionwasdecreasedsignificantlyinalltreatmentgroups.Expressionofhistaminereceptorswasdeterminedbyquan-titativereal-timePCRwithcolonictissue.(A)TNBSinhibitedsecretioninresponsetohistamine,andthiswasnotpreventedbypriorentericinfectionwithH.polygyrus.(B)H1Rexpressionwasdownregulatedsignificantlyinallgroups.(C)H4RexpressionwasincreasedsignificantlyonlyintheTNBSgroup.PriorH.polygyrusinfectioncompletelypreventedtheTNBS-inducedupregulation.Ͻ0.05versuscontrolorvehicle;**,PϽ0.01versuscontrolor*,Pvehicle.

INFECT.IMMUN.

TheeffectsofTh1-dominantinflammatoryprocessesarewelldocumentedandincludereducedepithelialsecretioninIBDpatientsaswellasanimalmodelsofcolitis(44,60).Thismaybeduetoadirecteffectofimmuneand/orinflammatorymediatorsonepithelialfunctionoranindirecteffectduetothelossofepithelialcellsininflammation.Secretionisanimpor-tantpartofmucosaldefensebecauseitfacilitatesremovalofharmfulsubstancesandbacteriainthelumenawayfromtheepithelialsurfaceandreducesbacterialtranslocation(1).Inthepresentstudy,TNBSuniformlyinhibitedsecretion(Iresponsestoallsecretagoguestested,althoughitdidnotaffectsc)inresistance,indicatingalargelyintactbarrierfunction.

5-HTiscriticallyinvolvedintheafferentarmofthemucosalsecretoryreflexarcbytransducingsignalsfromthelumentotheafferentnerves(Fig.7).Recentstudiesdemonstratedal-terationsin5-HTsignalinginfunctionalGIdisorders,suchasirritablebowelsyndrome,chronicconstipation,diarrhea,andfunctionaldyspepsia(46).Thegutisthelargestsourceof5-HT,whoseeffectsaremediatedthroughnumerous5-HTreceptors.Inresponsetothesestimuli,enterochromaffincellsrelease5-HT,whichbindstospecificreceptorsonprimaryafferentnervesorepithelialcells(13).Inthecurrentstudy,thedecreasein5-HT-stimulatedsecretioninducedbyTNBSisconsistentwitheitherlossordamagetoepithelialcellsand/ornerves.

Inthesecretoryreflex,5-HT-orsubstanceP-mediatedac-tivationofmotorneuronsresultsinreleaseofneurotransmit-ters,suchasacetylcholine(14,15).Secretioninresponsetoexogenousacetylcholineisacombinationofeffectsmediatedbybindingtonicotinicreceptorsonentericneuronsorthroughmuscarinicreceptorsonepithelialcells(Fig.7).SecretioninresponsetoacetylcholinewasreducedbybothH.polygyrusinfectionandTNBS.ItisinterestingthatthecontributionofentericnervestoacetylcholinewaslostafterTNBSbutwasprotectedbypriorinfectionwithH.polygyrus,indicatingthatentericinfectionprotectsagainstdamagetothereflexsecre-torycircuits.Mastcellsapproximateentericnerves,andthemastocytosisofentericinfectionenhancesthisinteraction(45).Thesepathwaysplayanimportantroleinmucosalprotectionaspartofthe“weepandsweep”responsetopotentialnoxiousstimuli(3).TheincreasedmRNAexpressionandantibodystainingforMCP-1afterH.polygyrusinfectionandH.polygyrusinfectionplusTNBS-inducedcolitiscomparedtothoseafterTNBSinductionaloneindicatethataninfluxofmastcellsmayserveaprotectiverolebyincreasingthesensitivityofsensorynervestostimulation,effectivelyrestoringsecretiontocontrollevels.ThisisconsistentwiththeobservationthatresponsestoacetylcholineintheH.polygyrusinfectionandH.polygyrusinfection-plus-TNBSgroupswerenearlyabolishedinthepres-enceofTTX,indicatingahighlevelofdependenceonentericnervesafterinfection(Fig.7).AlthoughMCP-1mRNAexpres-sionwassimilarinH.polygyrusinfectionandH.polygyrusinfectionwithTNBSinduction,therewasareductioninantibodystainingforMCP-1forH.polygyrusinfectionwithTNBSinductionversusH.polygyrusinfection.TherewasalsoareductioninstainingforbaselinelevelsofmastcellsinmiceaftertreatmentwithTNBSalonecomparedtothatforvehicle-treatedcontrolmice,suggest-ingthatTNBSmaydegranulatemastcellsevenwhentheyareactivatedbyinfectionwithH.polygyrus.

Colitis-inducedabnormalitiesinentericnervesmayalsoal-

VOL.76,2008ANTI-INFLAMMATORYEFFECTSOFH.POLYGYRUSONCOLITIS4779

FIG.6.(A)MCP-1mRNAexpressionwasdeterminedbyreal-timePCR.ExpressionwaselevatedsignificantlybyH.polygyrusinfection.**,PϽ0.01versuscontrol.(B)MastcellinfiltrationisindicatedbyimmunofluorescentstainingofMCP-1.Vehicle-treatedcontrolmicehadsomestainingofmastcells(arrows).(C)Heligmosomoidespolygyrusinfectionincreasedthemarkedmastcellinfiltration.(D)Incontrast,TNBStreatmentcausedadecreaseinbackgroundmastcellstaining.(E)PriorH.polygyrusinfectionpartiallystabilizedthemastcellstainingthatwasdecreasedbyTNBS.

terresponsestoPARagonists.PAR-2isexpressedalongtheentirelengthoftheGItractandhasbeenidentifiedinanumberofcells,includingenterocytes(48).TheroleofPAR-2ininflammationisbetterdefinedthanthatofPAR-1(23,31).PAR-2isalsolinkedtosensitizationofvisceralafferentslead-ingtohyperalgesia(12),andPAR-2agonistsincreaseepithe-lialsecretioninboththesmallintestineandcolon(4,28,36).Inthepresentstudy,weshowedthattheTNBS-inducedinhi-bitionofresponsestoPAR-2activationwasnormalizedwithpriorexposuretoH.polygyrus.MCPs(e.g.,tryptaseinhumans)areimportantmediatorsofPAR-2activation,suggestingthattherestorationofsecretiontoPAR-2maybemediatedbyaneffectofH.polygyrusonmucosalmastcellsandtheireffectsonentericnerves.

4780SUTTONETAL.FIG.7.SchematicshowingtheinteractionbetweenimmunecellsandepithelialcellsduringTNBS-inducedcolitis.(A)Inthisparadigm,secretioniselicitedbyluminaleventsthatstimulateenterochromaffincells(ECcells)torelease5-HT,whichactsonreceptorsonsensoryafferentnervesthatmaytransmitinformationtothecentralnervoussystem(CNS)oractlocallytoreleasesubstanceP(SP)atneuronswithintheentericnervoussystemwithinthewallofthegut.Thisispartofasecretoryreflexculminatinginthereleaseofneurotransmitters,suchasacetylcholine(ACH),thatbindtomuscarinicreceptorsonepithelialcellstoreleasechlorideions(ClϪ)andfluid.TNBSupregu-latesTh1cytokines,increasesneutrophilinfiltration,inducesalossofand/ordegranulationofmastcells,andcausesmucosaldamage,in-cludinginjurytoentericnerves(dashedlines).(B)InH.polygyrusinfection,thereisanincreasedproductionofTh2cytokinesthatpre-ventsTNBS-inducedupregulationofTh1cytokines.Thisisassociatedwithanenhancednumberofmastcellsclosetoentericnervesthatincreasethesensitivityofthesenervestostimulationandrendertheresponsesmoredependentonentericnerves.PriorinfectionwithH.polygyruspreventsthedevelopmentoftheTh1response,limitsmuco-saldamageandinflammationandneutrophilinfiltration,andenhancesthecontributionofnervestosecretoryresponsesinvolvingmastcells.

PAR-1isexpressedthroughoutthegut,havingbeenidenti-fiedinthesmoothmuscle,epithelial,nerve,andlaminapropriacells(48).PAR-1isoverexpressedinthecolonsofIBDpa-tientsand7daysafterTNBS-inducedcolitisinmiceduetotheincreasednumbersofinfiltratinginflammatorycells(55).Ac-tivationofPAR-1leadstoincreasedintestinalsecretion,per-meability,andleukocyteinfiltration(6,7,11,55).Inthepresentstudy,however,PAR-1expressionwasunchanged4

INFECT.IMMUN.

daysafterTNBSadministration.Thismaybeduetothetiming(4versus7days)ormethodofanalysis(real-timeversusquan-titativereversetranscription-PCR).PreviousstudiesshowedthatPAR-1agonistshadnoeffectoncolonicsecretionintheunstrippedmurinecolon(6)buthadincreasedsecretioninhumanepithelialcelllines(7).Theuniforminhibitionofre-sponsestoPAR-1inalltreatmentgroupsindicatesthattheeffectisnotspecifictoinflammationbutmaybeageneralizedresponsetoCD4ϩT-cell-mediatedimmuneactivation.

Histamineisoneofmanymastcellproducts,andincreasedlevelsofhistamineanditsmetabolitesarereportedinactiveIBD(27,43,57,58).Muchoftheeffectofhistamineonepi-thelialcellsecretionismediatedbyH1R.Inthecurrentstudy,weobservedaninhibitionofhistaminesecretioninthecolonforalltreatmentgroupsthatwasassociatedwithasignificantreductioninH1Rexpression.WeshowedpreviouslythatH.polygyrushadnoeffectonhistamineresponsesinthesmallintestine(44),indicatingaregionalspecificitygovernedbychangesinH1Rexpressionafterinfection.

H4Rispreferentiallyexpressedonhematopoieticcells,in-cludingmastcells,dendriticcells,Tcells,eosinophils,andpossiblyneutrophils(33,38,40,65).Itexhibitsnumerousproinflammatoryeffects,includingneutrophil,mastcell,andeosinophilchemotaxis(5,21,24,32,51,52).IncreasedH4RexpressionwasobservedintheTNBSgroup,coincidentwithsignificantinfiltrationofimmune/inflammatorycellsandmu-cosaldamage.TheabilityofH.polygyrustoblocktheincreaseinH4RexpressionsuggeststhatitsprotectiveeffectisrelatedtolimitinginfiltrationoractivationofcellsspecifictoTh1-dominantinflammation.Neutrophilsarethemostlikelycan-didate,whichissupportedbyaveryrecentstudyshowingthatanH4RantagonistsignificantlyinhibitedTNBS-inducedinjuryandmyeloperoxidaseactivity,anindexofneutrophilinfiltra-tion,intheratcolon(54).ThesedataindicatethatH4Rmaybeanoveltherapeutictargetininflammatorypathologies.Mucosalbarrierfunctionplaysacriticalroleinthepatho-genesisofanumberofdiseases,includingIBD(19);however,mucosalpermeabilitywasunaltered4daysafterTNBStreat-ment.Previousstudiesusingwaterfluxand/orclearanceofradioisotopestomeasurechangesincolonicpermeability4hto2weeksafterTNBS-inducedcolitisinratsshowedincreasedpermeabilityafterTNBStreatment(47,60).Itshouldbenoted,however,thatthesestudiesreflectpermeabilitychangesalongtheentiregut,andthus,increasedpermeabilitymaybeattributedtoalteredresistanceinthesmallintestineratherthanthecolon.TheabilityofH.polygyrusinfectiontoimprovecolonicresistance,evenafteradministrationofTNBS,isan-otherbenefitoftheupregulationofTh2cytokines.Incontrast,weshowedpreviouslythatIL-4andIL-13bothdecreaseresis-tanceinthesmallintestine(34,35,45).Thenotedincreaseincolonicresistancemayservetolimitmovementofantigensortoxinsacrossthecolon,anareaofslowtransitandhighbac-terialloadrelativetothesmallintestine.

Inconclusion,thesedatademonstratethatH.polygyrusin-fection-inducedupregulationofTh2cytokinesattenuatedTNBS-inducedchangesincolonicepithelialcellfunctionbyattenuatingtheTh1response,restoringnormalsecretoryfunc-tionto5-HT,acetylcholine,andPAR2,andenhancingcolonicresistance.Partofthiseffectmaybemediatedbyaprotectiveeffectofinfectiononentericnervesand/orfacilitationofen-

VOL.76,2008ANTI-INFLAMMATORYEFFECTSOFH.POLYGYRUSONCOLITIS4781

tericnerveandmastcellinteractionstomaintainsecretion.TheupregulationofH4Rinthelocaltissuemayunderscoreanimportanthistaminergiccontributiontochroniccolonicin-flammationandmayserveasapotentialtherapeutictarget.UpregulationofTh2cytokinesthroughnematodeinfectionpreventsthedevelopmentoftheTh1response,suggestingthateliminationofhelminthinfectioninindustrializedcountriesexacerbatestheresponsetoproinflammatorystimuliandfacilitatesthedevelopmentofimmunogenicpathologiessuchasIBD.

REFERENCES

1.Asfaha,S.,C.J.Bell,J.L.Wallace,andW.K.MacNaughton.1999.Pro-longedcolonicepithelialhyporesponsivenessaftercolitis:roleofinduciblenitricoxidesynthase.Am.J.Physiol.Gastrointest.LiverPhysiol.276:G703–G710.

2.Ash,A.S.,andH.O.Schild.1966.Receptorsmediatingsomeactionsofhistamine.Br.J.Pharmacol.Chemother.27:427–439.

3.Berin,M.C.,A.J.Kiliaan,P.C.Yang,J.A.Groot,Y.Kitamura,andM.H.Perdue.1998.Theinfluenceofmastcellsonpathwaysoftransepithelialantigentransportinratintestine.J.Immunol.161:2561–2566.

4.Bohm,S.K.,W.Kong,D.Bromme,S.P.Smeekens,D.C.Anderson,A.Connolly,M.Kahn,N.A.Nelken,S.R.Coughlin,D.G.Payan,andN.W.Bunnett.1996.Molecularcloning,expressionandpotentialfunctionsofthehumanproteinase-activatedreceptor-2.Biochem.J.314:1009–1016.

5.Buckland,K.F.,T.J.Williams,andD.M.Conroy.2003.HistamineinducescytoskeletalchangesinhumaneosinophilsviatheH(4)receptor.Br.J.Pharmacol.140:1117–1127.

6.Buresi,M.C.,A.G.Buret,M.D.Hollenberg,andW.K.MacNaughton.2002.Activationofproteinase-activatedreceptor1stimulatesepithelialchlo-ridesecretionthroughauniqueMAPkinase-andcyclo-oxygenase-depen-dentpathway.FASEBJ.16:1515–1525.

7.Buresi,M.C.,E.Schleihauf,N.Vergnolle,A.Buret,J.L.Wallace,M.D.Hollenberg,andW.K.MacNaughton.2001.Protease-activatedreceptor-1stimulatesCa(2ϩ)-dependentCl(Ϫ)secretioninhumanintestinalepithelialcells.Am.J.Physiol.Gastrointest.LiverPhysiol.281:G323–G332.

8.Cenac,N.,A.M.Coelho,C.Nguyen,S.Compton,P.Andrade-Gordon,W.K.MacNaughton,J.L.Wallace,M.D.Hollenberg,N.W.Bunnett,R.Garcia-Villar,L.Bueno,andN.Vergnolle.2002.Inductionofintestinalinflamma-tioninmousebyactivationofproteinase-activatedreceptor-2.Am.J.Pathol.161:1903–1915.

9.Cenac,N.,R.Garcia-Villar,L.Ferrier,M.Larauche,N.Vergnolle,N.W.Bunnett,A.M.Coelho,J.Fioramonti,andL.Bueno.2003.Proteinase-activatedreceptor-2-inducedcolonicinflammationinmice:possibleinvolve-mentofafferentneurons,nitricoxide,andparacellularpermeability.J.Im-munol.170:4296–4300.

10.Chen,C.C.,S.Louie,B.McCormick,W.A.Walker,andH.N.Shi.2005.

Concurrentinfectionwithanintestinalhelminthparasiteimpairshostresis-tancetoentericCitrobacterrodentiumandenhancesCitrobacter-inducedcolitisinmice.Infect.Immun.73:5468–5481.

11.Chin,A.C.,N.Vergnolle,W.K.MacNaughton,J.L.Wallace,M.D.

Hollenberg,andA.G.Buret.2003.Proteinase-activatedreceptor1activationinducesepithelialapoptosisandincreasesintestinalpermeability.Proc.Natl.Acad.Sci.USA100:11104–11109.

12.Coelho,A.M.,N.Vergnolle,B.Guiard,J.Fioramonti,andL.Bueno.2002.

Proteinasesandproteinase-activatedreceptor2:apossibleroletopromotevisceralhyperalgesiainrats.Gastroenterology122:1035–1047.

13.Cooke,H.J.1998.“Enterictears”:chloridesecretionanditsneuralregula-tion.NewsPhysiol.Sci.13:269–274.

14.Cooke,H.J.,M.Sidhu,P.Fox,Y.Z.Wang,andE.M.Zimmermann.1997.

SubstancePasamediatorofcolonicsecretoryreflexes.Am.J.Physiol.272:G238–G245.

15.Cooke,H.J.,M.Sidhu,andY.Z.Wang.1997.5-HTactivatesneuralreflexes

regulatingsecretionintheguinea-pigcolon.Neurogastroenterol.Motil.9:181–186.

16.Elliott,D.E.,J.Li,A.Blum,A.Metwali,K.Qadir,J.F.Urban,Jr.,andJ.V.

Weinstock.2003.ExposuretoschistosomeeggsprotectsmicefromTNBS-inducedcolitis.Am.J.Physiol.Gastrointest.LiverPhysiol.284:G385–G391.17.Elliott,D.E.,T.Setiawan,A.Metwali,A.Blum,J.F.Urban,Jr.,andJ.V.

Weinstock.2004.HeligmosomoidespolygyrusinhibitsestablishedcolitisinIL-10-deficientmice.Eur.J.Immunol.34:2690–2698.

18.Elliott,D.E.,J.F.Urban,Jr.,C.K.Argo,andJ.V.Weinstock.2000.Does

thefailuretoacquirehelminthicparasitespredisposetoCrohn’sdisease?FASEBJ.14:1848–1855.

19.Fasano,A.,andT.Shea-Donohue.2005.Mechanismsofdisease:theroleof

intestinalbarrierfunctioninthepathogenesisofgastrointestinalautoim-munediseases.Nat.Clin.Pract.Gastroenterol.Hepatol.2:416–422.

20.Frieling,T.,J.M.Palmer,H.J.Cooke,andJ.D.Wood.1994.NeuroimmunecommunicationinthesubmucousplexusofguineapigcolonafterinfectionwithTrichinellaspiralis.Gastroenterology107:1602–1609.

21.Gantner,F.,K.Sakai,M.W.Tusche,W.W.Cruikshank,D.M.Center,and

K.B.Bacon.2002.Histamineh(4)andh(2)receptorscontrolhistamine-inducedinterleukin-16releasefromhumanCD8(ϩ)Tcells.J.Pharmacol.Exp.Ther.303:300–307.

22.Hanauer,S.B.2006.Inflammatoryboweldisease:epidemiology,pathogenesis,

andtherapeuticopportunities.Inflamm.Bowel.Dis.12(Suppl.1):S3–S9.

23.Hansen,K.K.,P.M.Sherman,L.Cellars,P.Andrade-Gordon,Z.Pan,A.

Baruch,J.L.Wallace,M.D.Hollenberg,andN.Vergnolle.2005.Amajorroleforproteolyticactivityandproteinase-activatedreceptor-2inthepatho-genesisofinfectiouscolitis.Proc.Natl.Acad.Sci.USA102:8363–8368.24.Hofstra,C.L.,P.J.Desai,R.L.Thurmond,andW.P.Fung-Leung.2003.

HistamineH4receptormediateschemotaxisandcalciummobilizationofmastcells.J.Pharmacol.Exp.Ther.305:1212–1221.

25.Hough,L.B.2001.Genomicsmeetshistaminereceptors:newsubtypes,new

receptors.Mol.Pharmacol.59:415–419.

26.Khan,W.I.,P.A.Blennerhasset,A.K.Varghese,S.K.Chowdhury,P.

Omsted,Y.Deng,andS.M.Collins.2002.Intestinalnematodeinfectionamelioratesexperimentalcolitisinmice.Infect.Immun.70:5931–5937.27.Knutson,L.,O.Ahrenstedt,B.Odlind,andR.Hallgren.1990.Thejejunal

secretionofhistamineisincreasedinactiveCrohn’sdisease.Gastroenterol-ogy98:849–854.

28.Kong,W.,K.McConalogue,L.M.Khitin,M.D.Hollenberg,D.G.Payan,

S.K.Bohm,andN.W.Bunnett.1997.Luminaltrypsinmayregulateentero-cytesthroughproteinase-activatedreceptor2.Proc.Natl.Acad.Sci.USA94:8884–88.

29.Krishnaswamy,G.,J.Kelley,D.Johnson,G.Youngberg,W.Stone,S.K.

Huang,J.Bieber,andD.S.Chi.2001.Thehumanmastcell:functionsinphysiologyanddisease.Front.Biosci.6:D1109–D1127.

30.Kuhn,R.,J.Lohler,D.Rennick,K.Rajewsky,andW.Muller.1993.Inter-leukin-10-deficientmicedevelopchronicenterocolitis.Cell75:263–274.31.Lindner,J.R.,M.L.Kahn,S.R.Coughlin,G.R.Sambrano,E.Schauble,D.

Bernstein,D.Foy,A.Hafezi-Moghadam,andK.Ley.2000.Delayedonsetofinflammationinprotease-activatedreceptor-2-deficientmice.J.Immunol.165:6504–6510.

32.Ling,P.,K.Ngo,S.Nguyen,R.L.Thurmond,J.P.Edwards,L.Karlsson,

andW.P.Fung-Leung.2004.HistamineH4receptormediateseosinophilchemotaxiswithcellshapechangeandadhesionmoleculeupregulation.Br.J.Pharmacol.142:161–171.

33.Liu,C.,X.Ma,X.Jiang,S.J.Wilson,C.L.Hofstra,J.Blevitt,J.Pyati,X.

Li,W.Chai,N.Carruthers,andT.W.Lovenberg.2001.Cloningandphar-macologicalcharacterizationofafourthhistaminereceptor(H(4))ex-pressedinbonemarrow.Mol.Pharmacol.59:420–426.

34.Madden,K.B.,L.Whitman,C.Sullivan,W.C.Gause,J.F.Urban,Jr.,I.M.

Katona,F.D.Finkelman,andT.Shea-Donohue.2002.RoleofSTAT6andmastcellsinIL-4-andIL-13-inducedalterationsinmurineintestinalepi-thelialcellfunction.J.Immunol.169:4417–4422.

35.Madden,K.B.,K.A.Yeung,A.Zhao,W.C.Gause,F.D.Finkelman,I.M.

Katona,J.F.Urban,Jr.,andT.Shea-Donohue.2004.EntericnematodesinducestereotypicSTAT6-dependentalterationsinintestinalepithelialcellfunction.J.Immunol.172:5616–5621.

36.Mall,M.,T.Gonska,J.Thomas,S.Hirtz,R.Schreiber,andK.Kunzelmann.

2002.Activationofionsecretionviaproteinase-activatedreceptor-2inhu-mancolon.Am.J.Physiol.Gastrointest.LiverPhysiol.282:G200–G210.37.Moreels,T.G.,R.J.Nieuwendijk,J.G.DeMan,B.Y.DeWinter,A.G.

Herman,E.A.VanMarck,andP.A.Pelckmans.2004.ConcurrentinfectionwithSchistosomamansoniattenuatesinflammationinducedchangesinco-lonicmorphology,cytokinelevels,andsmoothmusclecontractilityoftrini-trobenzenesulphonicacidinducedcolitisinrats.Gut53:99–107.

38.Morse,K.L.,J.Behan,T.M.Laz,R.E.West,Jr.,S.A.Greenfeder,J.C.

Anthes,S.Umland,Y.Wan,R.W.Hipkin,W.Gonsiorek,N.Shin,E.L.Gustafson,X.Qiao,S.Wang,J.A.Hedrick,J.Greene,M.Bayne,andF.J.Monsma,Jr.2001.Cloningandcharacterizationofanovelhumanhistaminereceptor.J.Pharmacol.Exp.Ther.296:1058–1066.

38a.NationalResearchCouncil.1996.Guideforthecareanduseoflaboratory

animals.NationalAcademyPress,Washington,DC.

39.Nystedt,S.,A.K.Larsson,H.Aberg,andJ.Sundelin.1995.Themouse

proteinase-activatedreceptor-2cDNAandgene.Molecularcloningandfunctionalexpression.J.Biol.Chem.270:5950–5955.

40.Oda,T.,N.Morikawa,Y.Saito,Y.Masuho,andS.Matsumoto.2000.

Molecularcloningandcharacterizationofanoveltypeofhistaminereceptorpreferentiallyexpressedinleukocytes.J.Biol.Chem.275:36781–36786.41.Perez-Navarro,R.,O.Martinez-Augustin,I.Ballester,A.Zarzuelo,and

D.M.Sanchez.2005.Experimentalinflammationoftheratdistalcoloninhibitsionsecretionintheproximalcolonbyaffectingtheentericnervoussystem.NaunynSchmiedebergsArch.Pharmacol.371:114–121.

42.Perez-Navarro,R.,I.Ballester,A.Zarzuelo,andF.SanchezdeMedina.

2005.Disturbancesinepithelialionicsecretionindifferentexperimentalmodelsofcolitis.LifeSci.76:14–1501.

43.Raithel,M.,M.Matek,H.W.Baenkler,W.Jorde,andE.G.Hahn.1995.

MucosalhistaminecontentandhistaminesecretioninCrohn’sdisease,ul-

4782SUTTONETAL.cerativecolitisandallergicenteropathy.Int.Arch.AllergyImmunol.108:127–133.

44.Sandle,G.I.2005.Pathogenesisofdiarrheainulcerativecolitis:newviewsonanoldproblem.J.Clin.Gastroenterol.39:S49–S52.

45.

Shea-Donohue,T.,C.Sullivan,F.D.Finkelman,K.B.Madden,S.C.Morris,J.Goldhill,V.Pineiro-Carrero,andJ.F.Urban,Jr.2001.TheroleofIL-4inHeligmosomoidespolygyrus-inducedalterationsinmurineintes-tinalepithelialcellfunction.J.Immunol.167:2234–2239.

46.

Spiller,R.C.,D.Jenkins,J.P.Thornley,J.M.Hebden,T.Wright,M.Skinner,andK.R.Neal.2000.Increasedrectalmucosalenteroendocrinecells,Tlymphocytes,andincreasedgutpermeabilityfollowingacuteCampy-lobacterenteritisandinpost-dysentericirritablebowelsyndrome.Gut47:804–811.

47.Stein,J.,J.Ries,andK.E.Barrett.1998.Disruptionofintestinalbarrierfunctionassociatedwithexperimentalcolitis:possibleroleofmastcells.Am.J.Physiol.274:G203–G209.

48.

Steinhoff,M.,J.Buddenkotte,V.Shpacovitch,A.Rattenholl,C.Moormann,N.Vergnolle,T.A.Luger,andM.D.Hollenberg.2005.Proteinase-activatedreceptors:transducersofproteinase-mediatedsignalingininflammationandimmuneresponse.Endocr.Rev.26:1–43.

49.Summers,R.W.,D.E.Elliott,J.F.Urban,Jr.,R.Thompson,andJ.V.Weinstock.2005.TrichurissuistherapyinCrohn’sdisease.Gut54:87–90.50.Summers,R.W.,D.E.Elliott,J.F.Urban,Jr.,R.A.Thompson,andJ.V.Weinstock.2005.Trichurissuistherapyforactiveulcerativecolitis:aran-domizedcontrolledtrial.Gastroenterology128:825–832.

51.

Takeshita,K.,K.Sakai,K.B.Bacon,andF.Gantner.2003.CriticalroleofhistamineH4receptorinleukotrieneB4productionandmastcell-depen-dentneutrophilrecruitmentinducedbyzymosaninvivo.J.Pharmacol.Exp.Ther.307:1072–1078.

52.

Thurmond,R.L.,P.J.Desai,P.J.Dunford,W.P.Fung-Leung,C.L.Hofstra,W.Jiang,S.Nguyen,J.P.Riley,S.Sun,K.N.Williams,J.P.Edwards,andL.Karlsson.2004.ApotentandselectivehistamineH4re-ceptorantagonistwithanti-inflammatoryproperties.J.Pharmacol.Exp.Ther.309:404–413.

53.Urban,J.F.,Jr.,I.M.Katona,andF.D.Finkelman.1991.Heligmosomoidespolygyrus:CD4ϩbutnotCD8ϩTcellsregulatetheIgEresponseandprotectiveimmunityinmice.Exp.Parasitol.73:500–511.

54.

Varga,C.,K.Horvath,A.Berko,R.L.Thurmond,P.J.Dunford,andB.J.R.Whittle.2005.InhibitoryeffectsofhistamineH4receptorantagonistsonexperimentalcolitisintherat.Eur.J.Pharmacol.522:130–138.

Editor:W.A.Petri,Jr.

INFECT.IMMUN.

55.Vergnolle,N.,L.Cellars,A.Mencarelli,G.Rizzo,S.Swaminathan,P.Beck,

M.Steinhoff,P.Andrade-Gordon,N.W.Bunnett,M.D.Hollenberg,J.L.Wallace,G.Cirino,andS.Fiorucci.2004.Aroleforproteinase-activatedreceptor-1ininflammatoryboweldiseases.J.Clin.Investig.114:1444–1456.56.Vu,T.K.,D.T.Hung,V.I.Wheaton,andS.R.Coughlin.1991.Molecular

cloningofafunctionalthrombinreceptorrevealsanovelproteolyticmech-anismofreceptoractivation.Cell:1057–1068.

57.Weidenhiller,M.,M.Raithel,S.Winterkamp,P.Otte,J.Stolper,andE.G.

Hahn.2000.MethylhistamineinCrohn’sdisease(CD):increasedproductionandelevatedurineexcretioncorrelateswithdiseaseactivity.Inflamm.Res.49(Suppl.1):S35–S36.

58.Winterkamp,S.,M.Weidenhiller,P.Otte,J.Stolper,D.Schwab,E.G.

Hahn,andM.Raithel.2002.UrinaryexcretionofN-methylhistamineasamarkerofdiseaseactivityininflammatoryboweldisease.Am.J.Gastroen-terol.97:3071–3077.

59.Xie,H.,andS.H.He.2005.Rolesofhistamineanditsreceptorsinallergic

andinflammatoryboweldiseases.WorldJ.Gastroenterol.11:2851–2857.60.Yamada,Y.,S.Marshall,R.D.Specian,andM.B.Grisham.1992.A

comparativeanalysisoftwomodelsofcolitisinrats.Gastroenterology102:1524–1534.

61.Zamuner,S.R.,N.Warrier,A.G.Buret,W.K.MacNaughton,andJ.L.

Wallace.2003.Cyclooxygenase2mediatespost-inflammatorycolonicsecre-toryandbarrierdysfunction.Gut52:1714–1720.

62.Zhao,A.,andT.Shea-Donohue.2003.PAR-2agonistsinducecontractionof

murinesmallintestinethroughneurokininreceptors.Am.J.Physiol.Gas-trointest.LiverPhysiol.285:G696–G703.

63.Zhao,A.,J.F.Urban,Jr.,M.Morimoto,J.E.Elfrey,K.B.Madden,F.D.

Finkelman,andT.Shea-Donohue.2006.Contributionof5-HT2Areceptorinnematodeinfection-inducedmurineintestinalsmoothmusclehypercon-tractility.Gastroenterology131:568–578.

.Zhao,A.,M.Morimoto,H.Dawson,J.E.Elfrey,K.B.Madden,W.C.Gause,

B.Min,F.D.Finkelman,J.F.Urban,Jr.,andT.Shea-Donohue.2005.Immuneregulationofprotease-activatedreceptor-1expressioninmurinesmallintestineduringNippostrongylusbrasiliensisinfection.J.Immunol.175:2563–2569.

65.Zhu,Y.,D.Michalovich,H.Wu,K.B.Tan,G.M.Dytko,I.J.Mannan,R.

Boyce,J.Alston,L.A.Tierney,X.Li,N.C.Herrity,L.Vawter,H.M.Sarau,R.S.Ames,C.M.Davenport,J.P.Hieble,S.Wilson,D.J.Bergsma,andL.R.Fitzgerald.2001.Cloning,expression,andpharmacologicalcharacter-izationofanovelhumanhistaminereceptor.Mol.Pharmacol.59:434–441.