Clinic l viw in Allrgy &Immunology Clli nc c-cv wArAg y&I m& - PDF document

 Clinic l viw in Allrgy &Immunology Clli nc c-cv wArAg y&I m&uo CmmrC ncncc/sn2cn6-cnn- 2vn-x Clinc  ivw Arggvl yr&li&l& Clinnc nv Your article is protected by copyright andall rights are held exclusively by SpringerScience+Business Media, LLC. This e-offprintis for personal use only and shall not be self-archived in electronic repositories. If youwish to self-archive your work, please use theaccepted author’s version for posting to yourown website or your institution’s repository.You may further deposit the accepted author’sversion on a funder’s repository at a funder’srequest, provided it is not made publiclyavailable until 12 months after publication. LeakyGutandAutoimmuneDiseases AlessioFasano # SpringerScience+BusinessMedia,LLC2011 Abstract Autoimmunediseasesarecharacterizedbytissue damageandlossoffunctionduetoanimmuneresponsethatis directedagainstspecificorgans.Thisreviewisfocusedonthe roleofimpairedintestinalbarrierfunctiononautoimmune pathogenesis.Togetherwiththegut-associatedlymphoid tissueandtheneuroendocrinenetwork,theintestinalepithelial barrier,withitsintercellulartightjunctions,controlsthe antigens.Zonulinistheonlyphysiologicmodulatorof intercellulartightjunctionsdescribedsofarthatisinvolved intraffickingofmacromoleculesand,therefore,intolerance/ immuneresponsebalance.Whenthezonulinpathwayis deregulatedingeneticallysusceptibleindividuals,autoim- munedisorderscanoccur.Thisnewparadigmsubverts traditionaltheoriesunderlyingthedevelopmentofthese diseasesandsuggeststhattheseprocessescanbearrestedif theinterplaybetweengenesandenvironmentaltriggersis preventedbyre-establishingthezonulin-dependentintestinal evidencesupportthisnewparadigmandprovidetherationale forinnovativeapproachestopreventandtreatautoimmune diseases. Keywords Antigens . Autoimmunity . Gutpermeability . Immuneresponse . Tightjunctions . Zonulin Introduction Theintestinalepitheliumisthelargestmucosalsurface providinganinterfacebetweentheexternalenvironment andthemammalianhost.Itsexquisiteanatomicaland functionalarrangementsandthefinely-tunedcoordination ofdigestive,absorptive,motility,neuroendocrine,and ofthegastrointestinal(GI)system.Alsopivotalisthe regulationofmoleculartraffickingbetweentheintestinal lumenandthesubmucosaviatheparacellularspace.The dimensionsoftheparacellularspaceareestimatedtobe between10and15Å,suggestingthatunderphysiological circumstances,soluteswithamolecularradiusexceeding 15Å(~3.5kDa)willbeexcludedfromthisuptakeroute. Macromoleculetraffickingisdictatedmainlybyintestinal paracellularpermeability,whoseregulationdependsonthe modulationofintercellulartightjunctions(TJ).Afast growingnumberofdiseases,includingautoimmunedis- eases,arerecognizedtoinvolvealterationsinintestinal permeabilityrelatedtochangesinTJcompetency. ClassicalTheoriesonthePathogenesisofAutoimmune Soonafterautoimmunediseaseswerefirstrecognizedmore thanacenturyago,itwasbelievedthattheirdevelopment wasassociatedwithviralandbacterialinfections.The connectionbetweeninfectionandautoimmunediseaseis oftenexplainedbyamechanismknownas “ molecular mimicry, ” wherebymicrobialantigensarepostulatedto resembleself-antigens[ 1 ].Theinductionofanimmune responsetothemicrobialantigensresultsinacross-reaction withtheself-antigensandtheinductionofautoimmunity. A.Fasano( * ) MucosalBiologyResearchCenter, UniversityofMarylandSchoolofMedicine, Baltimore,MD21201,USA e-mail:afasano@mbrc.umaryland.edu ClinicRevAllergImmunol DOI10.1007/s12016-011-8291-x Accordingtothistheory,oncetheautoimmuneprocessisactivated,itbecomesindependentofcontinuousexposuretotheenvironmentaltriggerandisthereforeself-perpetuatingandirreversible.Epitope-specificcross-reactivitybetweenmicrobialantigensandself-antigenshasbeenshowninsomeanimalmodelstoinitiateautoimmunity[].Conversely,inmosthumanautoimmunediseases,molecularmimicryseemstobeafactorintheprogressionofapre-existingsubclinicalautoimmuneresponse,ratherthanintheinitiationofautoimmunity[].Anothertheorysuggeststhatmicroorgan-ismsexposeself-antigenstotheimmunesystembydirectlydamagingtissuesduringactiveinfection,andthatthisleadstothedevelopmentofautoimmunity.Thismechanismhasbeenreferredtoasthebystandereffect,anditoccursonlywhenthenewantigenispresentedwiththeorallyadminis-teredtriggeringantigen.Whetherpathogensmimicself-antigens,releasesequesteredself-antigens,orboth,however,remainstobeelucidated.NewProposedHypothesis:theLeakyGutasThirdElementinAutoimmunePathogenesisAcommondenominatorinautoimmunediseasesisthepresenceofseveralpre-existingconditionsthatleadtoanautoimmuneprocess[].Thefirstoftheseconditionsisthegeneticsusceptibilityofthehostimmunesystemtorecognize,andpotentiallymisinterpret,anenvironmentalantigenpresentedwithinthegastrointestinaltract.Thesecondisthatthehostmustbeexposedtotheantigen.Finally,theantigenmustbepresentedtothegastrointestinalmucosalimmunesystemfollowingitsparacellularpassagefromtheintestinallumentothegutsubmucosa;thisprocessisnormallypreventedbycompetentTJ[].Inmanycases,increasedintestinalpermeabilityseemstoprecedediseaseandcausesanabnormalityinantigendeliverythattriggersthemultiorganprocessleadingtotheautoimmuneresponsese3–5].Takingtheaboveinformationintoconsideration,weproposethatthepathogenesisofautoimmunediseasescanbedescribedbythreekeypoints[Autoimmunediseasesinvolveamiscommunicationbetweeninnateandadaptiveimmunity;Molecularmimicryorbystandereffectsalonemightnotexplainentirelythecomplexeventsinvolvedinthepathogenesisofautoimmunediseases.Rather,thecontin-uousstimulationbynonself-antigens(environmentaltriggers)seemstobenecessarytoperpetuatetheprocess.Contrarytogeneralbelief,thisconceptimpliesthattheautoimmuneresponsecantheoreticallybestoppedandperhapsreversediftheinterplaybetweengenespredis-posingindividualstothedevelopmentofautoimmunityandenvironmentaltriggersispreventedoreliminated;Inadditiontogeneticpredispositionandexposuretotriggeringnonself-antigens,thelossoftheprotectivefunctionofmucosalbarriersthatinteractwiththeenvironment(mainlythegastrointestinalandlungmucosa)isnecessaryforautoimmunitytodevelop.EvidenceSupportingThisNewTheoryCeliacdisease(CD)isthebesttestimonialofthevaliditytotheaccuracyofthenewparadigmforthepathogenesisofautoimmunityproposedabove.Celiacdiseaseisanauto-immuneconditiontriggeredbytheingestionofgluten-containinggrainsingeneticallysusceptibleindividuals(formoredetails,seeCDsectionbelow).Giventheundisputableroleofglutenincausinginflam-mationandimmune-mediatedtissuedamage,CDisauniquemodelofautoimmunityinwhich,incontrasttomostotherautoimmunediseases,aclosegeneticassociationwithHLAgenes,ahighlyspecifichumoralautoimmuneresponseagainsttissuetransglutaminaseauto-antigen,and,mostimportantly,thetriggeringenvironmentalfactor(gliadin),areallknown.Itistheinterplaybetweengenes(bothHLAandnon-HLAassociated)andenvironment(i.e.,gluten)thatleadstotheintestinaldamagetypicalofthedisease[].Underphysiologicalcircumstances,thisinterplayispreventedbycompetentintercellularTJ.EarlyinCD,TJsareopened[].Combined,thisinformationprovidestherationaleforthetreatmentofthediseasebasedoncompleteavoidanceofgluten-containinggrainsfromthepatientsdiet.Followingglutenwithdrawal,thesymptomsresolve,thebiomarkersoftheautoimmuneprocessreturnwithinnormallimits,andtheintestinalautoimmuneinsultheals.Theseoutcomessupportthenotionthattheautoimmuneprocesscanberevertedprovidedthattheinterplaybetweengenesandenvironmentaltrigger(s)canbeprevented.Besidesceliacdisease,severalotherautoimmunedis-eases,includingtype1diabetes[],multiplesclerosisosis15,16],andrheumatoidarthritis[],arecharacterizedbyincreasedintestinalpermeabilitysecondarytonon-competentTJsthatallowthepassageofantigensfromtheintestinalflora,challengingtheimmunesystemtoproduceanimmuneresponsethatcantargetanyorganortissueingeneticallypredisposedindividuals[IntestinalBarrierFunctionandItsRegulationAcenturyago,TJswereconceptualizedasasecretedextracellularcementforminganabsoluteandunregulatedbarrierwithintheparacellularspace[].BiologicalstudiesofthepastseveraldecadeshaveshownthatTJsaredynamicstructuressubjectedtostructuralchangesthat 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dictatetheirfunctionalstatusunderavarietyofdevelopmentalscenarios.Tomeetthemanydiversephysiologicalchallengestowhichtheepithelialandendothelialbarriersaresubjected,TJsmustbecapableofrapidandcoordinatedresponses.ThisrequiresthepresenceofacomplexregulatorysystemthatorchestratesthestateofassemblyoftheTJmultiproteinnetwork(Fig.).WhileourknowledgeonTJultrastructureandintracellularsignalingeventshavesignificantlypro-gressedduringthepastdecade,relativelylittleisknownabouttheirpathophysiologicalregulationsecondarytoextracellularstimuli.Therefore,theintimatepathogenicmechanismsofdiseasesinwhichTJsareaffectedhaveremainedunexploredowingtolimitedunderstandingoftheextracellularsignalinginvolvedinTJregulation.TheZonulinPathwayThediscoveryofzonulaoccludenstoxin(Zot),anenterotoxinelaboratedbyVibriocholeraethatreversiblyopensTJ[],increasedourunderstandingoftheintricatemechanismsthatregulatetheintestinalepithelialpara-cellularpathwayandledtothediscoveryofitseukaryoticcounterpartzonulin[].Thephysiologicalrole(s)ofthezonulinsystemremainstobeestablished.Thispathwayappearstobeinvolvedinseveralfunctions,includingTJregulationresponsibleforthemovementoffluid,macro-molecules,andleukocytesbetweenthebloodstreamandtheintestinallumenandviceversa.Anotherpossiblephysio-logicalroleofintestinalzonulinistheprotectionagainstmicroorganismcolonizationoftheproximalintestine(innateimmunity)[Sincezonulinisoverexpressedintissuesandseraofsubjectsaffectedbyautoimmunediseases,weelectedtouseserafromzonulin-positiveandzonulinnegativetype1diabetes(T1D)andCDsubjectstocharacterizefurtherthemolecularnatureofzonulin.Throughproteomicanalysisofhumansera,wehaverecentlyidentifiedzonulinaspre-haptoglobin(HP)2[],amoleculethat,todate,hasonlybeenregardedastheinactiveprecursorforHP2,oneofthetwogeneticvariants(togetherwithHP1)ofhumanHPs.MaturehumanHPsareheterodimericplasmaglycoproteins Fig.1Compositionofintercellulartightjunctions.Thestructuralcomponentsofintercellulartightjunctionscanbeclassifiedinintegralmembraneproteins(occludin,claudins,andJAM),junctionalcomplexproteins(ZO-1,ZO-2,p130orZO-3,7H6,symplekin,cingulin),andthecellcytoskeletonstructures(microtubules,intermediatefilaments,andmicrofilaments) ClinicRevAllergImmunol composedof-and-polypeptidechains.Whilethechain(36kDa)isconstant,thechainexistsintwoforms,1(~9kDa)and2(~18kDa).ThepresenceofoneorbothofthechainsresultsinthethreehumanHPphenotypes,i.e.,HP1-1homozygote,HP2-1heterozygote,andHP2-2homozygote.ThezonulinpathwaymodulatingTJpermeabilityisdescribedinFig.Zonulin-DependentImpairedIntestinalBarrierFunctionandAutoimmuneDiseasesCeliacDiseaseCDisanimmune-mediatedchronicenteropathywithawiderangeofpresentingmanifestationsofvariableseverity.Itistriggeredbytheingestionofgliadinfractionofwheatglutenandsimilaralcohol-solubleproteins(prolamines)ofbarleyandryeingeneticallysusceptiblesubjectswithsubsequentimmunereactionleadingtosmallbowelinflammationandnormalizationofthevillousarchitectureinresponsetoagluten-freediet[].CDnotonlyaffectsthegut,butitisasystemicdiseasethatmaycauseinjurytoanyorgan.Itisacomplexgeneticdisorder,andHLAstatusappearstobethestrongestgeneticdeterminantofriskforceliacautoimmunity.Glutenisacomplexmoleculemadeofgliadinandglutenins,bothtoxicforCDpatients.Therepertoireofglutenpeptidesinvolvedinthediseasepathogenesisisgreaterthanappreciatedpreviously,withatleast50toxicepitopesinglutenpeptidesexertingcytotoxic,immunomodulatory,andgutpermeatingactivities.Theseactivitieshavebeenpartiallymappedtospecificdomainsin-gliadin:thecytotoxicpeptide3143,theimmunomodulatorypeptide5789(33-mer),theCXCR3binding,zonulinreleasing(gutpermeating)peptides111130and151170,andtheIL8-releasingpeptide261277[].TheeffectofthepermeatinggliadinpeptidesinvivowasconfirmedbytheanalysisofintestinaltissuesfrompatientswithactiveCDandnon-CDcontrolsprobedforzonulinexpression[].QuantitativeimmunoblottingofintestinaltissuelysatesfromactiveCDpatientsconfirmedtheincreaseinzonulinproteincomparedtocontroltissuess8].ZonulinupregulationduringtheacutephaseofCDwasconfirmedbymeasuringzonulinconcentrationinseraof189CDpatientsusingasandwichELISA.Comparedtohealthycontrols,CDsubjectshadhigherzonulinserumconcen-trations(0.000001)duringtheacutephaseofthediseasethatdecreasedfollowingagluten-freediet[Currentdatasuggestthatalteredprocessingbyintra-luminalenzymes,changesinintestinalpermeability,andactivationofinnateimmunitymechanismsseemtoprecedetheactivationoftheadaptiveimmuneresponse[].Basedonthesedataandonthegliadinepitopemappingdescribedabove,itisconceivabletohypothesizethefollowingsequenceofevents:afteroralingestion,gliadininteracts Fig.2ProposedzonulinintracellularsignalingleadingtotheopeningofintestinalTJ.Zonulininteractswithaspecificsurfacereceptor[]whosedistributionwithintheintestinevaries.TheproteinthenactivatesphospholipaseC[]thathydrolyzesphosphatidylinositol[]toreleaseinositol1,4,5-trisphosphate(PPI-3)anddiacylglycerol(DAG)[PKCisthenactivated[],eitherdirectly(viaDAG)[]orthroughthereleaseofintracellularCa(viaPPI-3)().Membrane-associated,activatedPKCKC6]catalyzesthephosphorylationoftargetprotein(s),withsubsequentpolymerizationofsolubleG-actininF-actin[].Thispolymerizationcausestherearrangementofthefilamentsofactinandthesubsequentdisplacementofproteins(includingZO-1)fromthejunctionalcomplex[].Asaresult,intestinalTJbecomeslooser(seefreezefractureelectronmicroscopy).Oncethezonulinsignalingisover,theTJsresumetheirbaselinesteadystate ClinicRevAllergImmunol withthesmallintestinalmucosacausinginterleukin(IL)-8releasefromenterocytes(peptide261277),soleadingtoimmediaterecruitmentofneutrophilsinthelaminapropria.Atthesametime,gliadinpermeatingpeptides111130and170initiateintestinalpermeabilitythroughaMyD88-dependentreleaseofzonulin(aswehaverecentlycon-firmedbyidentifyingCXCR3asthereceptorthatreleaseszonulininaMyD88-dependentmanner,seeref.[])thatenablesparacellulartranslocationofgliadinanditssubse-quentinteractionwithmacrophages(through33-merandotherimmunomodulatorypeptides)withintheintestinalsubmucosa[].ThisinteractioninitiatessignallingthroughaMyD88-dependent,butTLR4andTLR2-independentpathway,resultingintheestablishmentofaproinflammatory(Th1-type)cytokinemilieu[]thatresultsinmononuclearcellinfiltrationintothesubmucosa.Thepersistentpresenceofinflammatorymediatorssuchastumornecrosisfactor(TNF)-andinterferon(IFN)-causesfurtherincreaseinpermeabilityacrosstheendothe-lialandepitheliallayers[],suggestingthattheinitialbreachoftheintestinalbarrierfunctioncausedbyzonulincanbeperpetuatedbytheinflammatoryprocessaftertheaccessofgliadintothesubmucosa.Ingeneticallypredis-posedindividualsthis,inturn,maypermittheinteractionofTcellswithantigenpresentingcells,includingmacro-phages,leadingultimatelytotheantigen-specificadaptiveimmuneresponsecausingtheautoimmuneinsultoftheintestinalmucosaseeninpatientswithCD[Onceglutenisremovedfromthediet,serumzonulinlevelsdecrease,theintestineresumesitsbaselinebarrierfunction,theautoantibodytitersarenormalized,theautoimmuneprocessshutsoffand,consequently,theintestinaldamage(thatrepresentsthebiologicaloutcomeoftheautoimmuneprocess)healscompletely.Type1DiabetesType1diabetes(T1D)isanautoimmunecondition,sometimesassociatedtodiseasesthatarecharacterizedbymarkedimmunologicfeatures,suchasCDandthyroiditistis33,34].GIsymptomsindiabetesmellitushavebeengenerallyascribedtoalteredintestinalmotilitysecondarytoautonomicneuropathy[].However,otherstudiessuggestthatanincreasedpermeabilityofintestinalTJisresponsibleforboththeonsetofthediseaseandtheGIsymptomsthatthesepatientsoftenexperience[].Thishypothesisissupportedbyastudyperformedonaspontaneouslydiabeticanimalmodel[].TheauthorsofthisstudyshowedanincreasedpermeabilityofthesmallintestineofBioBreedingDiabetesProne(BBDP)/Wordiabetic-proneratsthatprecedesatleastamonththeonsetofdiabetes.Further,histologicalevidenceofpancreaticisletdestructionwasabsentatthetimeofincreasedpermeabilitybutclearlypresentatalatertime[].Therefore,theauthorspresentedevidencethatincreasedpermeabilityoccurredbeforeeitherhistologicalorovertmanifestationofdiabetesinthisanimalmodel.Weconfirmedthesedatabyreportinginthesameratmodelthatzonulin-dependentincreaseinintestinalpermeabilityprecedestheonsetofT1Dby23weekss38].Oraladministrationofthezonulininhibitor,AT1001(nowcalledLarazotideacetate),toBBDPratsblockedautoantibodyformationandzonulin-inducedincreasesinintestinalpermeability,soreducingtheincidenceofdiabetes[].Thesestudiessuggestthatthezonulin-dependentlossofintestinalbarrierfunctionisoneoftheinitialstepsinthepathogenesisofT1DintheBBDPanimalmodelofthedisease.TheinvolvementofzonulininT1Dpathogenesiswascorroboratedbyourstudiesinhumansshowingthat~50%ofT1Dpatientshaselevatedserumzonulinlevelsthatcorrelatedwithincreasedintestinalpermeability[].Wealsoprovidedpreliminaryevidencesuggestingthat,asintheBBDPratmodelofthedisease,zonulinupregulationprecedestheonsetofdiabetesinT1Dpatients[].Interestingly,asmallerpercentage(~25%)ofunaffectedfamilymembersofprobandswithT1Dhavealsobeenfoundtohaveincreasedserumzonulinlevelsandincreasedgutpermeability[],suggestingthatlossofintestinalbarrierfunctionisnecessarybutnotsufficientfortheonsetoftheautoimmuneprocess.Severalreportshavelinkedgliadin(theenvironmentaltriggerofCDautoimmunitythatalsocauseszonulinreleasefromthegut,seerefs.[]and[])toT1Dautoimmunitybothinanimalmodelsandinhumanstudies[].Morerecently,wereportedadirectlinkbetweenantibodiestoGlo-3a(awheat-relatedprotein),zonulinupregulation,andisletautoimmunityinchildrenatincreasedriskforT1DT1D43].Glo-3Aantibodylevelswereinverselyassociatedwithbreast-feedingdurationanddirectlyassociatedwithcurrentintakeoffoodscontaininggluteninisletautoimmunitycasesbutnotincontrols[].Further,zonulinwasdirectlyassociatedwithGlo-3Aantibodylevelsincasesbutnotincontrols,suggestingthatthepresenceofGlo-3Aantibodiesandzonulinupregulationinisletautoimmunitycasesarerelatedtoanunderlyingdifferenceinmucosalimmuneresponseascomparedtocontrols.AsthmaAsthmaisacomplexclinicalsyndromecharacterizedbyairflowobstruction,airwayhyperresponsiveness,andin-flammation.Themechanismsbywhichairwayinflamma-tionandalterationsinairwayfunctionaremaintainedareincompletelyunderstood.Becausewheezingcanalsobetriggeredbyfoodchallengesinsomeasthmaticchildren,increasedintestinalpermeabilityofasthmatics[]mayplayaroleinsusceptibilitytoenvironmentalallergens.We 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havegeneratedpreliminarydatasuggestingthatserumzonulinlevelsarehighinasubsetofsubjectsaffectedbyasthmaandthatapproximately40%ofasthmaticpatientshaveanincreasedintestinalpermeability[].Thisprelim-inaryobservationsuggeststhat,besidesinhalation,analternativerouteforthepresentationofspecificantigensorirritantsmayoccurthroughtheGImucosalimmunesystemfollowingtheirparacellularpassage(normallypreventedbytheintercellularTJ).MultipleSclerosisBesidesanincreaseinbloodbrainbarrierpermeability,multiplesclerosis(MS)patientsmayalsoexperienceanincreasedpermeabilityofintestinalTJ.Yacyshynandcoworkershavedemonstratedthat25%ofMSpatientsstudiedhadanincreasedintestinalpermeability[].ThefactthatpatientswithMS[]andCrohnsdisease[bothpresentanincreasednumberofperipheralBcellsexhibitingCD45RO,amarkerofantigenexposure,furthersupporttheconceptofpreexisting,geneticallydeterminedsmallintestinalpermeabilityabnormalitieswithsubsequentalteredantigenexposureasapathogenicfactorcommontothesediseases.Tochallengethishypothesis,wemeasuredserumlevelsofzonulininMSpatientswithdifferentsubtypesremitting[RRMS]vs.secondaryecondary—andactivitiestoascertainwhetherexpressionofzonulinintoperipheralcirculationcandifferentiatethesetwogroups.Approximately29%ofpatientswitheitherRRMSorSPMShadelevatedserumzonulinlevels(apercentagesimilartoincreasedintestinalpermeabilityinMSpatientsreportedbyYacyshynetal.,seeref.[]),withoverallaverageserumlevels~2.0-foldhigherthanincontrols.Interestingly,patientswithRRMSinremissionshowedserumzonulinlevelscomparabletocontrols[InflammatoryBowelDiseasessdiseaseandulcerativecolitisareinflammatorydiseasesinvolvingtheGItractinwhichabnormalpara-cellularpermeabilitydefectsprecedethedevelopmentofbothsyndromesand,therefore,appeartoplayanimportantroleindiseasepathogenesis[].Thepathogenesisofinflammatoryboweldisease(IBD)remainsunknown,althoughinrecentyearsthereisconvincingevidencetoimplicategenetic,immunological,andenvironmentalfac-torsininitiatingtheautoimmuneprocess.Severallinesofevidence,however,suggestthatanincreasedintestinalpermeabilityplaysacentralroleinthepathogenesisofIBD.InclinicallyasymptomaticCrohnsdiseasepatients,increasedintestinalepithelialpermeabilityprecedesclinicalrelapsebyasmuchas1year,suggestingthatapermeabilitydefectisanearlyeventindiseaseexacerbation[].ThehypothesisthatabnormalintestinalbarrierfunctionisagenetictraitinvolvedinthepathogenesisofIBDisfurthersupportedbytheobservationthatclinicallyasymptomaticfirst-degreerelativesofCrohnsdiseasepatientsmayhaveincreasedintestinalpermeability[].WehaverecentlygeneratedevidencesuggestingthatzonulinupregulationisdetectableintheacutephaseofIBDandthatitsserumlevelsdecrease(butstillarehigherthannormal)oncetheinflammatoryprocesssubsidesfollowingspecifictreatmentment21].Whileaprimarydefectoftheintestinalbarrierfunction(possiblysecondarytoactivationofthezonulinpathway)maybeinvolvedintheearlystepsofthepathogenesisofIBD,theproductionofcytokines,includingandTNFsecondarytotheinflammatoryprocessservetoperpetuatetheincreasedintestinalpermeabilitybyreorganizingTJproteinsZO-1,junctionaladhesionmole-cule1,occludin,claudin-1,andclaudin-4[].Inthismanner,aviciouscycleiscreatedinwhichbarrierdysfunctionallowsfurtherleakageofluminalcontents,therebytriggeringanimmuneresponsethatinturnpromotesfurtherleakiness.AnkylosingSpondylitisAnkylosingspondylitis(AS)isacommonandhighlyfamilialrheumaticdisorderthattypicallyaffectsyoungandmiddle-agedadultsandischaracterizedbystiffnessandpainintheback.ThelinkbetweenincreasedintestinalpermeabilityandAShasbeenclearlyestablished[UsingdifferentmarkersofTJpermeability,twoindepen-dentstudies[]foundanincreasedintestinalperme-abilityinbothASpatientsandtheirrelatives.Thesechangesprecedetheclinicalmanifestationsofthedisease,suggestingapathogenicroleofTJdysfunctioninAS.Usingaproteomicapproach,Liuetal.haveidentifiedHPasanASbiomarker[].TheauthorsinvestigatedtheserumproteinprofilesofASpatientsandhealthycontrolsfromalargeChineseASfamilyusingtwo-dimensionalelectrophoresisanalysis.AgroupoffourhighlyexpressedproteinspotswasobservedinallankylosingspondylitispatientsprofilesandsubsequentlyidentifiedasisoformsofHPbyESI-Q-TOFMS/MS[ProofofPathogenicRoleofZonulin-MediatedIntestinalBarrierDefectinAutoimmunity:theCeliacDiseaseandType1DiabetesParadigmsCDandtype1diabetesautoimmunemodelssuggestthat,whenthefinelytunedtraffickingofmacromoleculesisderegulatedduetoaleakygut,autoimmunedisorderscanoccuringeneticallysusceptibleindividuals[].Thistheoryimpliesthatremovinganyofthethreekeyelements 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(genes,environmentaltrigger(s),orimpairedbarrierfunc-tion)shouldblocktheautoimmuneprocess.Tochallengethishypothesis,zonulininhibitorLarazotideacetatewasusedwithencouragingresultsintheBBDPratmodelofautoimmunity[].Besidespreventingthelossofintestinalbarrierfunction,theappearanceofautoantibodies,andtheonsetofdisease,pretreatmentwithLarazotideacetateprotectedagainsttheinsultofpancreaticisletsand,therefore,oftheinsulitisresponsiblefortheonsetoftype1diabetes[Thisproof-of-conceptinananimalmodelofautoimmunityprovidedtherationaletodesignhumanclinicaltrialsinwhichLarazotideacetatewasinitiallytestedinaninpatient,double-blind,randomizedplacebocontrolledtrialtodetermineitssafety,tolerability,andpreliminaryefficacy[].NoincreaseinadverseeventswasrecordedamongpatientsexposedtoLarazotideascomparedtoplacebo.Followingacuteglutenexposure,a70%increaseinintestinalpermeabilitywasdetectedintheplacebogroup,whilenochangeswereseenintheLarazotideacetategroup[].GastrointestinalsymptomsweresignificantlymorefrequentamongpatientsoftheplacebogroupascomparedtotheLarazotideacetategroupp54].Larazotideacetatehasnowbeentestedinapproximately500subjectswithexcellentsafetyprofileandpromisingefficacyasconcernprotectionagainstsymptomscausedbyglutenexposureinCDpatients[ConclusionsTheclassicalparadigmofautoimmunepathogenesisinvolv-ingspecificgenemakeupandexposuretoenvironmentaltriggershasbeenrecentlychallengedbytheadditionofathirdelement,thelossofintestinalbarrierfunction.Geneticpredisposition,miscommunicationbetweeninnateandadap-tiveimmunity,exposuretoenvironmentaltriggers,andlossoftheintestinalbarrierfunctionsecondarytodysfunctionofintercellularTJseemtobeallkeyingredientsinvolvedinthepathogenesisofautoimmunediseases.BothinCDandT1Dgliadinmayplayaroleincausinglossofintestinalbarrierfunctionand/orinducingtheautoimmuneresponseingenet-icallypredisposedindividuals.Thisnewtheoryimpliesthatoncetheautoimmuneprocessisactivated,itisnotautoperpe-tuating,rathercanbemodulatedorevenreversedbypreventingthecontinuousinterplaybetweengenesandenvironment.SinceTJdysfunctionallowsthisinteraction,newtherapeuticstrategiesaimedatreestablishingtheintesti-nalbarrierfunctionofferinnovative,unexploredapproachesforthetreatmentofthesedevastatingdiseases.WorkpresentedinthisreviewwassupportedinpartsbygrantsfromtheNationalInstitutesofHealthGrantsDK-48373andDK-078699toAF.1.PerlA(2004)Pathogenesisandspectrumofautoimmunity.MethodsMolMed102:12.ChristenU,vonHerrathMG(2004)Induction,accelerationorpreventionofautoimmunitybymolecularmimicry.MolImmunol40:111311203.FasanoA(2001)Pathologicalandtherapeuticimplicationsofmacromoleculepassagethroughthetightjunction.InTightJunctions.CRCPress,Inc,BocaRaton,pp6974.YuQH,YangQ(2009)Diversityoftightjunctions(TJs)betweengastrointestinalepithelialcellsandtheirfunctioninmaintainingthemucosalbarrier.CellBiolInt33:785.FasanoA(2001)Intestinalzonulin:opensesame!Gut49:1591626.FasanoA,Shea-DonohueT(2005)Mechanismsofdisease:theroleofintestinalbarrierfunctioninthepathogenesisofgastroin-testinalautoimmunediseases.NatClinPractGastroneterolHepatol2:4167.PlengeRM(2010)Unlockingthepathogenesisofceliacdisease.NatGenet42:2818.DragoS,ElAsmarR,DePierroMetal(2006)Gliadin,zonulinandgutpermeability:effectsonceliacandnon-celiacintestinalmucosaandintestinalcelllines.ScandJGastroenterol41:4084199.MadaraJL,TrierJS(1980)Structuralabnormalitiesofjejunalepithelialcellmembranesinceliacsprue.LabInves43:25410.SzakálDN,GyorffyH,AratóAetal(2010)Mucosalexpressionofclaudins2,3and4inproximalanddistalpartofduodenuminchildrenwithcoeliacdisease.VirchowsArch456:24511.TripathiA,LammersKM,GoldblumSetal(2009)Identificationofhumanzonulin,aphysiologicalmodulatoroftightjunctions,asprehaptoglobin-2.ProcNatlAcadSciUSA106:1679912.WoltersVM,AlizadehBZ,WeijermanMEetal(2010)Intestinalbarriergenevariantsmaynotexplaintheincreasedlevelsofantigliadinantibodies,suggestingothermechanismsthanalteredpermeability.HumImmunol71:39213.MäkeläM,VaaralaO,HermannRetal(2006)Enteralvirusinfectionsinearlychildhoodandanenhancedtype1diabetes-associatedantibodyresponsetodietaryinsulin.JAutoimmun14.MojibianM,ChakirH,LefebvreDE,CrookshankJA,SonierB,KeelyE,ScottFW(2009)Diabetes-specificHLA-DR-restrictedproinflammatoryT-cellresponsetowheatpolypeptidesintissuetransglutaminaseantibody-negativepatientswithtype1diabetes.Diabetes58:178915.WestallFC(2007)AbnormalhormonalcontrolofguthydrolyticenzymescausesautoimmuneattackontheCNSbyproductionofimmune-mimicandadjuvantmolecules:acomprehensiveexplanationfortheinductionofmultiplesclerosis.MedHypotheses68:36416.YokoteH,MiyakeS,CroxfordJL,OkiS,MizusawaH,YamamuraT(2008)NKTcell-dependentameliorationofamousemodelofmultiplesclerosisbyalteringgutflora.AmJPathol173:1714172317.EdwardsCJ(2008)Commensalgutbacteriaandtheetiopatho-genesisofrheumatoidarthritis.JRheumatol35:147718.AbreuMT(2010)Toll-likereceptorsignalingintheintestinalepithelium:howbacterialrecognitionshapesintestinalfunction.NatRevImmunol10:13119.FasanoA(2008)Physiological,pathological,andtherapeuticimplicationsofzonulin-mediatedintestinalba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