MC-12,an Annexin A1-Based Peptide, Is Effective in the

MC-12,anAnnexinA1-BasedPeptide,IsEffectiveintheTreatmentofExperimentalColitis

NengtaiOuyang1,CaihuaZhu1,DingyingZhou1,TingNie1,MaeF.Go2,RobertJ.Richards1,BasilRigas1*

1DepartmentofMedicine,StonyBrookUniversity,StonyBrook,NewYork,UnitedStatesofAmerica,2VASaltLakeCityHealthCareSystem,SaltLakeCity,Utah,UnitedStatesofAmerica

Abstract

AnnexinA1(ANXA1)inhibitsNF-kB,akeyregulatorofinflammation,thecommonpathophysiologicalmechanismofinflammatoryboweldiseases(IBD).MC-12,anANXA1-basedtripeptide,suppressesNF-kBactivation.Here,wedeterminedtheefficacyofMC-12inthecontrolofIBD.Micewithcolitisinducedbydextransodiumsulfate(DSS)or2,4,6-trinitrobenzenesulfonicacid(TNBS)weretreatedwithvariousdosesofMC-12administeredintraperitoneally,orallyorintrarectally.Wedeterminedcolonlengthandthehistologicalscoreofcolitis,andassayed:incolontissuethelevelsofTNF-a,IFN-c,IL-1b,IL-6andIL-10byRT-PCR;prostaglandinE2(PGE2),cytoplasmicphospholipaseA2(cPLA2)andmyeloperoxidasebyimmunoassay;andCOX-2andNF-kBbyimmunohistochemistry;andinserumthelevelsofvariouscytokinesbyimmunoassay.InbothmodelsMC-12:reverseddose-dependentlycolonicinflammation;inhibitedbyupto47%myeloperoxidaseactivity;hadaminimaleffectoncytoplasmicphospholipaseA2;reducedsignificantlytheinducedlevelsofTNF-a,IFN-c,IL-1b,IL-6andIL-10,returningthemtobaseline.DSSandTNBSmarkedlyactivatedNF-kBincolonicepithelialcellsandMC-12decreasedthiseffectby85.8%and72.5%,respectively.MC-12hadasimilareffectinculturedNCM460normalcolonepithelialcells.Finally,MC-12suppressedtheinductionofCOX-2expression,thelevelofPGE2inthecolonandPGE2metaboliteinserum.Inconclusion,MC-12,representinganovelclassofshortpeptideinhibitorsofNF-kB,hasastrongeffectagainstcolitisintwopreclinicalmodelsrecapitulatingfeaturesofhumanIBD.ItsmechanismofactioniscomplexandincludespronouncedinhibitionofNF-kB.MC-12meritsfurtherdevelopmentasanagentforthecontrolofIBD.

Citation:OuyangN,ZhuC,ZhouD,NieT,GoMF,etal.(2012)MC-12,anAnnexinA1-BasedPeptide,IsEffectiveintheTreatmentofExperimentalColitis.PLoSONE7(7):e41585.doi:10.1371/journal.pone.0041585Editor:Jan-HendrikNiess,UlmUniversity,Germany

ReceivedFebruary23,2012;AcceptedJune25,2012;PublishedJuly23,2012

Copyright:ß2012Ouyangetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

Funding:ThisworkwassupportedbyNationalInstitutesofHealthgrants:R01CA139454andR01CA09242308.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,http://wendang.chazidian.competingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:basil.rigas@stonybrookmedicine.edu

Introduction

Inflammatoryboweldiseases(IBD)areasetofcomplex,life-longandforsomepatientsdevastatingdiseases,forwhichthereisnosatisfactorytreatment[1–3].Therearetwodistinctclinicalentities,ulcerativecolitisandCrohn’sdisease.Asharedclinicalmanifestationofthemisulcerationsintheintestinalmucosa;Crohn’sdiseasecanaffecttheentiredigestivetractwhileulcerativecolitisaffectsonlythecolon.ThetreatmentofIBDhasbeenbasedonanti-inflammatorymedications,withsteroidshavingbeenthemainstayoftreatmentforyears[4,5].Theprimelimitationsofanti-inflammatorymedicationsarevariableefficacyandsideeffects,which,inthecaseofsteroids,canlimitdosingordurationoftreatmentorforcephysicianstoaltogetherdiscontinuethem.Therecentlyintroducedbiologicalagentshavealsosignificantlimitations,and,inaddition,highcost[6].ItisclearthatthereisapressingneedfornewagentsforthecontroloftheclinicalmanifestationsofIBD.

InflammationistheunderlyingthemeinIBD(hencethewordinflammatoryintheirname).AkeyregulatorofinflammationisNF-kB,atranscriptionfactorthatisnormallysequesteredinthecytoplasm[7].Whenactivated,NF-kBtranslocatesintothenucleuswhereitregulatestheexpressionofamultitudeofgenesrelatedtoinflammation.Wehaverecentlyunraveledthe

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connectionbetweenglucocorticoidsandNF-kBandhaveproposedanovelmechanismbywhichtheyact[8].Briefly,wehavedemonstratedthatglucocorticoidsinducetheexpressionofannexinA1(ANXA1),whichthenbindstothep65subunitofNF-kB,inhibitingitsactivation.Thereisanearlyperfectcorrelationbetweentheanti-inflammatorypotencyofthevarioussteroidsandtheinductionofANXA1,ononehand,andthesuppressionofNF-kBontheother.Short(around20aminoacids)C-terminalfragmentsofANXA1areknowntohavemanyofitsbiologicalactivities[9,10].

WehavesynthesizedMC-12,atripeptidebasedonthestructureofANXA1thatisaseffectiveasANXA1insuppressingNF-kBactivation.ThispeptideisrepresentativeofseveralsuchANXA1peptidesthatinhibitNF-kB[8].WeassesseditspotentialefficacyincolitisusingtwomousemodelsofIBD,onebasedontheadministrationofdextransulfatesodium(DSS)andtheotherontrinitrobenzenesulfonicacid(TNBS)[11,12].Ourdatademon-stratethatadministrationofMC-12reversesinadose-dependentmannertheinflammatoryreactionofthecolonandpreventsthedevelopmentofulcerations.TherewerenoapparentsideeffectsfromtheadministrationofMC-12tomice.MC-12modulatesseveralinflammatorymediators,includingNF-kBandseveral

cytokines.

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Results

MC-12ReducesExperimentalColitisinMice

Byday8,micereceivingDSSlostonaverage8.4%oftheirbaselinebodyweight(Fig.1A).MC-12givenbyIPandPOatbothdosesandbyIRatthehigherdosepreventedsuchweightlossinadose-dependentmanner,withthosereceivingthehighestIPdoseofMC-12(25mg/kg),showing7.7%increaseintheirbodyweightcomparedtobaseline.Ofnote,http://wendang.chazidian.comparedtonormalcontrol,thecolonofDSS-treatedmicewasshorterby2.3cm(8.760.29vs.6.460.42cm;mean6SEM,forthisandallsubsequentvalues;p,0.01).AdministrationofMC-12preventeddose-dependentlymostofthisreductionincolonlength,withIP,POandIRadministrationproducingessentiallyidenticalresults;undertreatment,thelengthofthecolonrangedbetween7.360.29and7.760.20cm(p,0.05foralldifferencesfromvehiclecontrol).Macroscopically,shortenedcolonsshowedwalledemaandfewerfecesinthelumen.WealsostudiedcolitisinducedbyTNBSinSJL/Jmice.TheoptimaldoseofTNBSforourstudywas100mlofa2.5%ethanolicsolutioninstilledintracolonically.AsshowninFig.1C,TNBSreducedthebodyweightoftheanimalsonday3by18.2%andshortenedthelengthofthecolonby25%(Fig.1D),comparedtobaseline.MC-1225mg/kgfailedtopreventtheweightloss(84.761.1vs.81.861.3%invehicle-treatedcontrols,p.0.05)andtheshorteningofthecolon(6.460.20vs.6.460.07invehicle-treatedcontrols,p.0.05).

Asexpected,DSSinducedcolitisinthesemice.ThehistologicalsectionsshowninFig.2A–FdemonstratechangesinthecolonicmucosabyDSS,includingsignificantinflammation,accumulationofmucusanddevelopmentofulcers.Thegranulocytespresentinthemucosaestablishthedevelopmentofacuteinflammation.TreatmentwithMC-12reducedthedegreeofinflammation,essentiallyrestoringtheintegrityofthemucosa.AftertreatmentwithDSSthehistologicalcolitisscorebecame25.862.01from0innormalcontrols(Fig.2I).MC-12reducedthehistologicalcolitisscoredose-dependently,http://wendang.chazidian.comparedtovehicle-treatedcontrols,MC-12givenIPreducedthehistologicalscoreby48.9%at5mg/kg(13.261.62vs.25.862.01,p,0.01)and66.8%at25mg/kg(8.661.43vs.25.862.01,p,0.01).Thedoseof25mg/kgshowedalowerhistologicalscorethanthedoseof5mg/kg(8.661.43vs.13.261.62;p,0.05).Givenorally,MC-12wasslightlylesseffective,reducingthisscoreby50.2%at5mg/kg(12.961.01vs.25.862.01,p,0.01)and48.4%at25mg/kg(13.361.05vs.25.862.01,p,0.01).Similarly,theIRadministrationofMC-12decreasedthehistologicalscoreby33.3%(p,0.05)atthedoseof5mg/kgand58.1%(p,0.01)atthedoseof25mg/kgcomparedtoDSScontrolgroup.RegardlessofitslackingeffectonbodyweightandcolonlengthintheTNBSmodel,MC-12hadasignificantanti-inflammatoryeffectonthecolonicmucosaasshowninFig.2G–H,reducingthehistologicalscoreby39.1%,comparedtoTNBScontrolasshowninFig.2J(38.361.7vs.23.363.3;p,0.01).

TheConstituentAminoAcidsofMC-12HaveNoEffectonColitis

Sincepeptidesaresubjecttohydrolyticcleavageoftheirpeptidebonds,especiallywhenadministeredorally,weexaminedwhetherMC-12actsagainstcolitisafteritspotentialdegradationtoitsthreeconstituentaminoacids.Tothisend,wetreatedmicewithDSS-inducedcolitisusingasolutioncontainingthethreeaminoacidsofMC-12(Ac-Gln,AlaandTrp)atequimolarconcentra-tions.WetreatedmicewithDSS-colitisfollowingthesameprotocolasinthepreviousstudies.Theaminoacidsolutionwasgivenipatadoseequivalentto25mg/kgofintactMC-12.Theaminoacidsolutionhadnosignificanteffectonanyofthethreeparametersthatweevaluated:bodyweight(98.8%vs.96.7%),colonlength(6.560.13vs.6.260.17)andhistologicalscore(19.962.0vs.24.861.9);alldifferenceswerestatisticallynotsignificant.

MC-12ReducesDSS-andTNBS-inducedInflammationinColonicMucosa:EffectsonMPO,cPLA2andCytokines

ToassesstheeffectofMC-12ontheinflammatorychangesassociatedwithexperimentalcolitis,wedeterminedincolontissuesamplestheactivityofmyeloperoxidaseandcytosolicphospholi-paseA2(cPLA2)aswellastheresponseoffiveinflammatorycytokines.MPOactivityisanindicatorofthedegreeofacuteinflammationinagiventissue[13].cPLA2,aphospholipaserecognizingthesn-2acylbondofphospholipids,releaseslysopho-spholipidandarachidonicacid,whichcanthenbeconvertedtoprostaglandinsandleukotrienes,bothinflammatorymediators[14–16].Finally,thepro-inflammatorycytokinesTNF-a,IFN-c,IL-1bandIL-6,andanti-inflammatorycytokineIL-10havebeenimplicatedinexperimentalandhumancolitis[17].Indeed,cytokinesarethoughttoorchestratethedevelopment,recurrenceandexacerbationofIBD[18].

Fig.3summarizesourfindings.DSSandTNBSincreasedMPOactivity2.4-and3.5-foldcomparedtonormalcontrols.Inbothmodels,MC-12inhibitedtheenhancedMPOactivitysignificantlyandinadose-dependentmanner.Thisreductionwas30%(p,0.05)and47%(p,0.01)at5mg/kgand25mg/kg,respectively,intheDSSmodeland39%(p,0.01)intheTNBSmodel.

SimilartoMPO,theactivityofcPLA2incolonmucosawasincreased2.9-foldbyDSSand4.5-foldbyTNBScomparedtonormalmice.TheeffectofMC-12oncPLA2activitywasmodestandstatisticallynotsignificant;inthethreeMC-12treatmentgroups,thereductionincPLA2activityrangedbetween18%and24%.

WealsodeterminedtheresponseofthecytokinesTNF-a,IFN-c,IL-1b,IL-6andIL-10inthecolonbymeasuringtheircorrespondingmRNAlevelsusingreal-timePCR.AsshowninFig.4E–F,comparedtocontrols,DSSincreasedthemRNAlevelsofallthesecytokinesby9-to17-foldandTNBSby4-to10-fold.TreatmentwithMC-12ateitherdosesignificantlyreducedthemRNAlevelsofTNF-a,IL-1b,IFN-c,IL-6andIL-10,

Next,wemeasuredthelevelsofthesecytokinesintheserumoftheexperimentalanimals.AsshowninFig.4,DSSorTNBSinducedtheTNF-a,IFN-c,IL-1b,IL-6,andIL-10by2.4-to19-fold.TreatmentwithMC-12administeredPO,IPorIRsignificantlyreducedby1.7-to6.4-foldthelevelsofallthesecytokines.

MC-12isnotCytotoxictoNormalColonicEpithelialCells

MC-12failedtoinducecytotoxicitytotheNCM460normalcolonicepithelialcellline.The24-hIC50valueofMC-12washigherthan5mM;higherconcentrationswerenotstudied.ThisconcentrationfarexceedsconcentrationsofMC-12thatinhibited,forexample,theactivationofNF-kB(lowmMrange;shownbelow).

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MC-12InhibitstheActivationofNF-kBinvivoandinvitro

ThechronicmucosalinflammationinIBDischaracterizedbyhyperactivationofeffectorimmunecells,whichproducehigh

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Figure1.TheeffectofMC-12onDSS-andTNBS-inducedcolitisinmice.Mice(7/group)received2%DSSindrinkingwateror2.5%TNBSintra-colonicallytoinducecolitisandweretreatedwithvehicleorMC-12givenIP,POorIR.A:ThebodyweightofmiceofDSSmodelduringtreatmentbyIP,POorIR,expressedaspercentageofbaseline(day0).B:ThecolonlengthofDSSmodelwithMC-12treatmentsbyIP,POorIR.C:ThebodyweightofmiceofTNBSmodelduringtreatment,expressedaspercentageofbaseline(day0).D:ThecoloniclengthofmiceofTNBSmodelinthreetreatmentgroups.Thesestudieswererepeatedatleastoncegivingsimilarresults.Valuesaremean6SEM.*,statisticallysignificantdifferencefromthevehicle-treatedgroup.

doi:10.1371/journal.pone.0041585.g001

levelsofpro-inflammatorycytokines,resultingincolonictissuedamage.NF-kB,themasterregulatorofallinflammation[19,20],hasbeenidentifiedasakeyregulatorinthisimmunologicalsetting.Itsactivation,markedlyinducedinIBDpatients,stronglyinfluencesthecourseofmucosalinflammation[21,22].NF-kBisalsothemoleculartargetoftheactivityofMC-12[8].Therefore,wedeterminedbyimmunohistochemis-trythelevelofNF-kBactivationinthecolonicmucosaofourmice,usinganantibodyrecognizingthephosphorylationofser276ofNF-kB’sp65subunit.Asamethodologicalcontrol,wealsoshowedmarkedactivationofNF-kBincolonsamplesfrompatientswithulcerativecolitisusingthesameprimaryantibodyandmethod(FigureS1)[23].

AsshowninFig.5A–E,therewasminimalbaselineactivationofNF-kBinthecolonofnormalanimals.DSSactivatedNF-kBincolonicepithelialcells(0.760.20vs.8.861.45,p,0.01),inagreementwithpreviousreports[24].MC-12reversedthiseffectnearlycompletely(Fig.5I),regardlessofitsrouteofadministration:atthehighestdose(25mg/kg)MC-12decreasedthiseffectofDSSby85.8%(8.861.45vs.1.260.32;p,0.01).Fig.5F–Hdemon-stratesthatTNBSactivatedNF-kBinthecolonicepithelium;as

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showninFig.5J,thepercentageofcellswithactivatedNF-kBis8.461.32inTNBS-treatedmicevs.1.160.25incontrols(p,0.01).TreatmentwithMC-12inhibitedtheTNBS-inducedNF-kBactivationby72.5%(2.360.70vs.8.461.32,p,0.01).

TheinhibitoryeffectofMC-12onNF-kBwasalsodocumentedinculturedNCM460cells(normalcolonepithelialcells).Asexpected[25–27],DSSsignificantlyincreasedNF-kB-DNAbindingactivityintheNCM460cells.MC-12atconcentrationsof30mMand300mMessentiallyeliminatedthisNF-kBactivation(Fig.5K).

MC-12InhibitstheInductionofCOX-2andDecreasesPGE2Levelsinvivo

Theeicosanoidcascadeseemstobeinvolvedinthepathogen-esisofIBDbutthereissomecontroversyregardingitsspecificrole.COX-2isinducedinexperimentalcolitis[28–30]whereasNSAIDsarethoughttoexacerbatecolitisinhumans[31].ThusweevaluatedtheeffectofMC-12onCOX-2expressionandPGE2levelsincolonictissueanditsmetabolite13,14-dihydro-15-ketoprostaglandinE2intheserumof

mice.

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Figure2.MC-12amelioratescolitisinducedbybothDSSandTNBS.Paraffinsectionsofcolonictissueswerestainedwithhematoxylin&eosinandtheirhistologicalscoresweredeterminedasinMethods.A:NormalmucosaoftheC57BL/6mouse.B:Severeinflammationincludinginfiltrationbyinflammatorycells,edema,lossofcryptsandulcerationsareseeninaDSS+vehicle-treatedmouse.C,D:MC-12,5or25mg/kg,IP,significantlydecreasedDSS-inducedcolonicinflammation.E:TreatmentwithMC-12,25mg/kgPO,decreasedDSS-inducedcolonicinflammation.F:TreatmentwithMC-12,25mg/kg,IR,markedlyreducedDSS-inducedcolitis.G:ColonicmucosafromaTNBSvehicle-treatedmouse,showingseverecryptlossandinflammatorycellinfiltration(arrow).Inset:normalmucosaofahealthySJL/Jmouse.H:TreatmentwithMC-1225mg/kgfor2daysdecreasedtheinflammatorycellinfiltrationandcryptloss(arrowindicatesremainingcrypts).I,J:ThehistologicalscoreofthevariousstudygroupsofbothDSS-andTNBS-inducedcolitis.Valuesaremean6SEM.H&Estaining;magnification100x.doi:10.1371/journal.pone.0041585.g002

AsshowninFig.6,inbothmodelsofexperimentalcolitisMC-12markedlyreducedtheexpressionofCOX-2inthecolonicmucosa(Fig.6AandB).InadditionitdecreasedthelevelsofPGE2incolonicmucosa(87.9615.8or127.0612.5vs.241.4673.7,p,0.05,Fig.6C)andPGE2metaboliteinserum(4.360.9or4.262.4vs.16.864.9,p,0.01,Fig.6D).

Discussion

Ourdatademonstratethestronganti-inflammatoryeffectofthenoveltripeptideMC-12intwomodelsofexperimentalcolitis.MC-12,designedasaninhibitorofNF-kBbasedonarecentlyunraveledmechanismofNF-kBcontrol,hadaprofoundinhibitoryeffectonNF-kBactivationandonacircuitryofdependentinflammatorymediators.

Experimentalmodelsofcolitisenableustonotonlystudyitspathogeneticcomponentsduringthevariousphasesofcolitisbutalsotoassessthetherapeuticefficacyofexperimentalagents[12].Thetwomurinemodelsofcolitisthatweemployedinthesestudiesrepresenttwodifferententities.TheDSSmodel,technicallyamodelofchemicalinjurytothecolon,recapitulatesfeaturesofulcerativecolitis,includingsevereleukocyteinfiltration,cryptdamage,andtissueedemaoftenaccompaniedbysevereulcera-tion.TheTNBSmodel,ontheotherhand,hasmanyofthecharacteristicfeaturesofCrohn’sdiseaseinhumanssuchasseveretransmuralinflammation.Bothareassociatedwithweightlossandgastrointestinalmanifestations.

Asexpected,inbothmodelsweobservedsignificantweightlossanddecreasedcolonlength,bothofwhichwerereversedbyMC-12inDSS-treatedmice,eithertotallyormostly,dependingonMC-12’sdose.Interestingly,MC-12failedtoreverse

these

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Figure3.TheeffectofMC-12onparametersofinflammation.MPO(A,B),cPLA2(C,D)activityandthemRNAlevelsofproinflammatoryandanti-inflammatorycytokines(E,F)weremeasuredincolontissuesamplesofbothDSS-andTNBS-inducedcolitismousemodels.InbothmodelsMC-12significantlyinhibitedMPOactivityandthemRNAlevelsofallcytokinesbutnotcPLA2activity.Valuesaremean6SEM.*p,0.05comparedtovehicle-treatedgroup,**p,0.01comparedtovehicle-treatedgroup.doi:10.1371/journal.pone.0041585.g003

changesintheTNBSmodel.Regardless,however,ofitseffectsonbodyweightandcolonlength,MC-12displayedastronganti-inflammatoryeffectinbothmodels.Theinhibitionofinflamma-tionreachednearly70%inDDS-treatedmiceandalmost40%inTNBS-treatedmice.ThedoseofMC-12wasrelevantaswasitsrouteofadministration.IntheDSSmodelinwhichtwodrugdosesandtworoutesofadministrationwereevaluated,thedoseeffectwasunmistakableandtheiproutewasmodestlybetterthantheoralroute.ThelattermaywellreflectagreaterchancetohydrolyzeMC-12asittravelsdownthealimentarycanal.Ourdataconfirmedthatfullyhydrolyzed,MC-12isrenderedineffective;inareconstitutionexperimentthethreeaminoacidsofMC-12wereadministeredorallybuthadnoeffectoncolitis.Indirectasthisresultmaybe,onecannotfailtosurmisethatproperlyformulatedtheMC-12tripeptidemayhaveevenhigherefficacyincolitis.

EvensimpleinspectionofthetissuesectionsofthecolonmakesitclearthatMC-12actsasananti-inflammatoryagent;ourdatahaveobjectivelyandquantitativelyconfirmedthis.Inadditiontohistologicalscoring,inbothmodelsMC-12significantly

reduced

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