Critical role of IL17 receptor signaling in acute TNBSinduced colitis.docx

Critical role of IL17 receptor signaling in acute TNBSinduced colitis.docx

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CriticalroleofIL17receptorsignalinginacuteTNBSinducedcolitis

CriticalroleofIL-17receptorsignalinginacuteTNBS-inducedcolitis

1.ZiliZhangMD,PhD1,

2.MingquanZhengMD2,

3.JulieBindasBS2,

4.PaulSchwarzenbergerMD3,

5.Dr.JayK.KollsMD1,*

Articlefirstpublishedonline:

14DEC2006

DOI:

 10.1097/01.MIB.0000218764.06959.91

Copyright©2006Crohn's&ColitisFoundationofAmerica,Inc.

Keywords:

CD4+lymphocytes;

colitis;

inflammation;

chemokines;

neutrophils

Abstract

Topofpage

Abstract

MATERIALSANDMETHODS

RESULTS

DISCUSSION

ACKNOWLEDGMENT

References

Background:

Inflammatoryboweldiseases（IBDs）suchasCrohn'sdiseaseandulcerativecolitisarecharacterizedbyrecurrentinflammationinthegastrointestinaltract.InfiltrationofCD4+lymphocytesandneutrophilsisoneofthepredominantfeaturesofIBD.MaterialsandMethods:

Recently,interleukin（IL）-23andthedownstreamTcell-derivedcytokineIL-17havebeenfoundtobeelevatedinintestinaltissueandserumofIBDpatients.However,theroleofIL-17andIL-17Rsignalingingutinflammationisunknown.Toexaminethisrole,weinvestigatedgutinflammationinwild-typeorIL-17Rknockoutmice.Results:

Usingamodelofacutetrinitrobenzenesulfonicacid（TNBS）-inducedcolitis,wefoundthatIL-17wasproducedincolontissueat24and48hoursandthatIL-17RknockoutmiceweresignificantlyprotectedagainstTNBS-inducedweightloss,IL-6production,colonicinflammation,andlocalmacrophageinflammatoryprotein-2induction.ThisprotectionoccurredinthepresenceofequivalentinductionoflocalIL-23andhigherlevelsofIL-12p70andinterferon-

inIL-17Rknockoutmicecomparedwithwild-typemice.Moreover,IL-17Rknockoutmiceshowedreducedtissuemyeloperoxidaseactivity.Furthermore,overexpressionofanIL-17RIgG1fusionproteinsignificantlyattenuatedcolonicinflammationafteracuteTNBS.Conclusions:

TheseresultsdemonstratethatIL-17RsignalingplaysacriticalroleinthedevelopmentofTNBS-inducedcolitisandmayrepresentatargetfortherapeuticinterventionforIBD.

Crohn'sdiseaseandulcerativecolitis,collectivelyreferredtoasinflammatoryboweldisease（IBD）,areuniquegastrointestinaldisorderscharacterizedbychronicrelapsinginflammation.1,2ThehistologyassociatedwithIBDincludesinflammationoftheintestinalmucosawithneutrophilandotherinflammatorycellinfiltration.3,4AlthoughtheoriginofIBDiscomplexandmutifactorial,itisbelievedthatdysregulatedhostimmunityinresponsetogutbacterialfloraisinvolvedinIBDpathogenesis.1,2RecentstudieshavedemonstratedthatCD4+lymphocytesplayanimportantroleinthedevelopmentofIBD.5,6CD4+lymphocytesareincreasedandactivatedinintestinallaminapropriaofIBDpatientsandinseveralcolitismodels.7Furthermore,reconstitutionofseverecombinedimmunodeficientmicewithfunctionalCD4+lymphocyteshasbeenshowntoleadtoalethalinflammatorycolitis.7ThedysregulatedTcellresponseinIBDisechoedbyalterationsinthemucosalcytokineexpression,5andCrohn'sdiseaseiswellcharacterizedforitsTH1cytokinepattern.8

InlightofthecriticalroleofTH1CD4+lymphocytesinIBD,furtherelucidationofCD4+cell-derivedcytokineregulatorynetworkswillgreatlyfacilitatetheunderstandingofthediseaseandthedevelopmentofnoveltherapeuticstrategies.7,9Recently,ithasbeenshownthatinfusionofmonoclonalantibodyagainstinterleukin（IL）-12,acriticalregulatorofTH1celldevelopment,isefficaciousinCrohn'sdisease.9However,thisantibodyisagainsttheIL-12p40subunit,whichissharedbyIL-23.10Thus,neutralizationofIL-23couldinpartberesponsibleforthetherapeuticeffectofthisreagent.Infact,theothersubunitofIL-23,IL-23p19,hasbeenshowntobeelevatedinintestinaltissueofpatientswithIBD.11OurlaboratoryandothershaveshownthatIL-23isproducedafterToll-likereceptor4stimulationandiscriticalforCD4+TcellIL-17production12,13;andrecentlyitwasdemonstratedthatIL-23specificallyexpandsapopulationofCD4+TcellscalledThIL-17cells,whichproduceIL-17A,IL-17F,IL-6,andtumornecrosisfactor-

.14–17TheseIL-23-drivenCD4+TcellsarehighlypathogenicandelicitIL-17-dependentinflammation,includingexperimentalautoimmuneencephalitisandcollagen-inducedarthritis.14

IL-17isproducedmainlybyactivatedCD4+Tcells18althoughCD8+TcellsalsohavebeenreportedtoproduceIL-17.12IL-17inducesneutrophilia19andupregulatesnumerousinflammatorymediators,includinginduciblenitricoxidesynthase,IL-6,andtheCXCchemokinesIL-8andGCP-2,20andthemousehomologuesmacrophageinflammatoryprotein-2（MIP-2）21,22andLIX.23Recently,IL-17expressionhasbeenfoundtobeincreasedintheserumandintestinalmucosaofIBDpatients.24Fromthesedata,wehypothesizedthatIL-17andIL-17RsignalingplaysakeyproinflammatoryroleintheearlyphaseofIBDpathogenesis,leadingtotheobservedleukocyteinfiltration.Totestthis,weinvestigatedtheroleofIL-17Rsignalinginanacutetrinitrobenzenesulfonicacid（TNBS）-inducedmodelofcolitis.

MATERIALSANDMETHODS

Topofpage

Abstract

MATERIALSANDMETHODS

RESULTS

DISCUSSION

ACKNOWLEDGMENT

References

Animals

AllmicewerehousedinanAAALAC-approvedspecificpathogen-freevivariumatLouisianaStateUniversityHealthSciencesCenterorChildren'sHospitalofPittsburgh.ColoniesofIL-17receptorknockoutmice（IL-17RKO）withaC57BL/6geneticbackgroundwereestablishedfrombreederpairsdevelopedatImmunexCorp（Seattle,Wash）.22ControlC57BL/6micewerepurchasedfromtheJacksonLaboratory（BarHarbor,Me）.Six-week-oldmalemicewererandomizedintocontrolandexperimentalgroups（n=6–10）.

InductionofAcuteColitis

Micewereanesthetizedwithintraperitonealinjectionofketamine（100mg/kg）.Arubbercatheterwasinsertedintotherectumofmice,and2mgofTNBSin50%ethanol（vol/vol）orvehiclealonewasintroducedintothecolonthroughthecatheterinserted5cmdistaltotheanus.Then,24or48hoursafterthechallenge,themicewerekilled,andthecolonswereremovedformacroscopicscoringofinflammationaccordingtothefollowingsemiquantitativecriteria:

0=novisibleedemaorhemorrhage;1=focalhyperemiaorbowelwalledemaandnohemorrhage;2=focalhyperemiaorbowelwalledemaandhemorrhageat1site;3=extendedhyperemiaorbowelwalledemaandhemorrhageat>1site;and4=extendedhyperemiaorbowelwalledema,hemorrhageat>1site,andperforation.Afterscoring,thetissuewasprocessedforhistology,tissuemyeloperoxidase（MPO）assay,andcytokineanalysis.

AdenovirusVectors

ToantagonizeIL-17RsignalinginestablishedTNBScolitis,weadministeredacontroladenovirusencodingfireflyluciferase（AdLuc）oravectorencodingasolublemurineIL-17F:

Fcfusionprotein（AdIL-17R:

Fc）thatneutralizesIL-17bioactivity2212hoursafterTNBSadministration,andmicewerekilledat48hoursfortissueandcytokineanalyses.Theconstructionofthesevectorshasbeendescribed.22

HistologyandRibonucleaseProtectionAssays

Harvestedcolonswerefixedin10%neutralformalin,dehydrated,embeddedinparaffin,andsectioned.Thesectionswerestainedwithhematoxylinandeosinandevaluatedunderlightmicroscopy.TotalRNAwasextractedfromcolonictissuewithTri-Zol（Invitrogen）andhybridizedto32p-labeledribonucleaseprotectionassayprobesusingtheMCK-5RiboQunatkit（BDBioSciences,SanJose,Calif）.

Enzyme-linkedImmunosorbentAssayofIL-17,IL-6,andMIP-2

IL-17,IL-6,andMIP-2weremeasuredwithanenzyme-linkedimmunosorbentassay（ELISA）accordingtothemanufacturer'sprotocol（R&DSystems,Minneapolis,Minn）.MurineIL-23wasmeasuredbyELISA（E-Bioscience,SanDiego,Calif）.ToprepareELISAsamplesfromcolontissues,thecolonsineachtreatmentgroupwerecollectedandhomogenizedwith0.5mLofphosphate-bufferedsalinecontaining1%TritonX-100withproteinaseinhibitorcocktail（Amersham,Piscataway,NJ）.Thehomogenateswerecentrifugedat12,000gfor15min,andthesupernatantswerecollectedforELISA.IL-6wasassayedinserum.

MPOAssay

MPOisanenzymelocalizedinazurophilicgranulesofneutrophils.MPOactivitycanbeusedasanindexofneutrophilinfiltration.TomeasureMPO,thetissuewascollectedandhomogenizedasdescribedabove,andthesupernatantswereassayedforMPOusingthepublishedprotocolsaspreviouslydescribed.22

StatisticalAnalysis

Statisticalcomparisonsweremadebetweenexperimentalgroups,andwhendifferenceswerenotedamongtreatmentresponses,multiple-comparisontestswereconductedusingtheANOVAftestatthe0.05significancelevel.

RESULTS

Topofpage

Abstract

MATERIALSANDMETHODS

RESULTS

DISCUSSION

ACKNOWLEDGMENT

References

InductionofIL-17ProductioninTNBS-treatedColon

ToinvestigatetheroleofIL-17ininflammatorycolitis,wefirstexaminedwhetherTNBStreatmentinducedthelocalproductionofIL-17.Atboth24and48hoursaftertreatment,thecolonswereharvested,andIL-17inthetissuehomogenatesandserumwasmeasuredbyELISA.Comparedwithcontrolmicereceivingvehiclealone（Fig.1）,TNBStreatmentincreaseddetectableIL-17by≈3-fold.TheproductionofIL-17wascompartmentalizedtothecolonbecauseIL-17wasnotdetectedintheserumofeithergroup（datanotshown）.Tissueexaminationat48hoursafteradministrationofTNBSrevealedincreasednumbersoftissueneutrophils（Fig.2）.Moreover,usingaribonucleaseprotectionassay,weobservedtheinductionoftranscriptsforanumberofchemokinesinvolvedinneutrophilrecruitment（MIP-1

MIP-1β,MIP-2）,Tcells,eosinophils（RANTES）,andmacrophages（MCP-1）.

Figure FIGURE1.