Modulation of intestinal barrier by intestinal microbiota

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PharmacologicalResearch

journalhomepage:www.elsevier.com/locate/yphrs

Invitedreview

Modulationofintestinalbarrierbyintestinalmicrobiota:Pathologicalandtherapeuticimplications

JaneM.M.Natividad,ElenaF.Verdu∗

FarncombeFamilyDigestiveHealthResearchInstitute,McMasterUniversity,Hamilton,Canada

article

info

abstract

Articlehistory:

Received12July2012

Receivedinrevisedform11October2012Accepted13October2012

Keywords:

IntestinalbarrierfunctionIntestinalmicrobiotaCellrenewal

ApicaljunctionalproteinsAntimicrobialsMucin

Mammalsandtheirintestinalmicrobiotapeacefullycoexistinamutualisticrelationship.Commensalbacteriaplayanactiveroleinshapingandmodulatingphysiologicalprocessesinthehost,whichinclude,butarenotrestrictedto,theimmunesystemandtheintestinalbarrier.Bothplayacrucialroleincon-tainingintestinalbacteriaandotherpotentiallynoxiousluminalantigenswithinthelumenandmucosalcompartment.Althoughmutualismdefinestherelationshipbetweenthehostandtheintestinalmicro-biota,disruptionsinthisequilibriummaypromotedisease.Thus,alterationsingutmicrobiota(dysbiosis)havebeenlinkedtotherecentincreasedexpressionofobesity,allergy,autoimmunity,functionalandinflammatorydisorderssuchasirritablebowelsyndrome(IBS)andinflammatoryboweldisease(IBD).Inthisarticle,wereviewtheevidencesupportingaroleofgutmicrobiotainregulatingintestinalbarrierfunction.Wediscussthehypothesisthatmicrobialfactorscanmodulatethebarrierinwaysthatcanpre-ventorpromotegastrointestinaldisease.Abetterunderstandingoftheroleoftheintestinalmicrobiotainmaintainingafunctionalintestinalbarriermayhelpdeveloptargetedstrategiestopreventandtreatdisease.

Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.

Contents1.

2.

3.

Intestinalmicrobiotaandintestinalbarrier:importantdeterminantsofhost’shomeostasis......................................................1.1.Intestinalepithelialcelltypes,guthomeostasisandinflammation.........................................................................1.2.Intestinalbarrierarchitecture,guthomeostasisandinflammation.........................................................................1.3.Antimicrobialpeptides,guthomeostasisandinflammation................................................................................1.4.Mucuslayer,guthomeostasisandinflammation............................................................................................1.5.Microbialrecognition,guthomeostasisandinflammation..................................................................................Regulationoftheintestinalbarrierbygutmicrobiota..............................................................................................2.1.Microbialeffectsonintestinalepithelialcellrenewal.......................................................................................2.2.Microbialeffectsonintestinalpermeability.................................................................................................2.3.Microbialeffectsonantimicrobialproteinexpression......................................................................................2.4.Microbialeffectsonmucuslayer.............................................................................................................Conclusion............................................................................................................................................References...........................................................................................................................................

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1.Intestinalmicrobiotaandintestinalbarrier:importantdeterminantsofhost’shomeostasis

residentbacteriainthegastrointesti-Therearemorethan

naltractthatestablishalife-long,bi-directionalandsymbiotic

1014

∗Correspondingauthorat:FarncombeFamilyDigestiveHealthResearchInstitute,McMasterUniversity,1280MainStreetWest,Room3N8,Hamilton,Ontario,CanadaL8N3Z5.Tel.:+19055259140x20051;fax:+19055223454.

E-mailaddress:verdue@mcmaster.ca(E.F.Verdu).

relationshipwithourcells[1].Thisecosystem,termedcollectivelytheintestinalmicrobiota,playsakeyroleinthematurationoftheimmunesystemandinotherphysiologicalprocessesinclud-ingneuro-motorandintestinalbarrierfunction[2,3].Conversely,alterationsingutcolonizationatthetimeofbirthordisturbancesofthiscomplexecosysteminadulthoodmayresultinallergyandinflammation[4].

Thehosthasevolvedwithimmuneandphysiologicaladapta-tionstomaintainconstantvigilanceagainstpotentiallyharmfulluminalantigenswhilepreventingthedevelopmentofuncon-trolledinflammation.Assuch,theintestinalbarrierprovidesthe

1043-6618/$–seefrontmatter.Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.phrs.2012.10.007

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Fig.1.Intestinalbarrierasmodulatorofintestinalhomeostasis.Theintestinalbarrierisequippedwithseverallevelsofdefensemechanismstolimitluminalantigentranslocation.Thisincludesasinglelayerofsemi-permeableepithelialcells,apicaljunctions(adherensandtightjunctionsproteins)thatbindepithelialcellstogetherandregulateparacellularantigenandmoleculeentryacrosstheepithelium.Intestinalepithelialcellstransportantigensandmoleculesfromthelumenintothemucosathroughthetranscellularpathway.SpecializedepithelialcellscalledM-cellsmediateconstantsamplingofluminalantigens.Gobletcells,Panethcellsandenterocytessecretemucinsandantimicrobialpeptidesthatassembleintoamucuslayer.Mucinsandantimicrobialsarelocalizedwithintheunstirredmucuslayer.IntestinalepithelialcellssecreteIgA,whichareproducedbyplasmacellsinthelaminapropria.Intestinalepithelialcellsarealsoequippedwithanumberofmicrobialrecognitionreceptors(MRR)suchasTLRandNOD-likereceptorsthatcanrecognizespecificmicrobialassociatedmolecularpatterns.(MAMP).Basalrecognitionofintestinalmicroorganismsbyintestinalepithelialcellsinducessecretionofcytokinesandotherimmunemediators,whichmayhelpintheinductionaregulatoryresponseagainstintestinalmicrobiotaandmaintenanceofintestinalhomeostasis.

firstlineofdefense,byphysicallyseparatingtheinternalmilieufromthegutlumenandthroughactivemechanismssuchasmicro-bialrecognition,productionofantimicrobialpeptides,mucus,andsecretionofantibodies(Fig.1).

Regulationofbarrierintegrityandfunctionismediatedbyendogenousandexogenousfactors,suchascytokines,chemicalsanddrugs[5–9].Pathogensandtheirtoxinsalsohavetheabilitytodirectlyorindirectlymodulatetheintestinalbarrier[10–12].Recently,commensalbacteriahavebeenproposedaskeymodula-torsofintestinalbarrierfunction[13,2].

Adefectinintestinalbarriercanleadtopersistentimmuneacti-vation.Indeed,intestinalbarrierdysfunctionhasbeensuggestedtoplayapathogenicroleinanumberofintestinaldiseasesinclud-ingceliacdisease(CD),colorectalcancer,irritablebowelsyndrome(IBS)andinflammatoryboweldisease(IBD)[14].Althoughtheexactroleofintestinalbarrierdysfunction,particularlyinthecon-textofIBDpathogenesis,isstillamatterofdebate,animalmodelsindicatethatitcouldplayaprimaryoradjuvantrole.Thisdoesnotnecessarilyimplythatitisthesolefactorcausingdisease,butthatbarrierdysfunctionmayexacerbateinflammationinthepresenceofothertriggersandcompromisemucosalhealingafterinflamma-tion[15].

1.1.Intestinalepithelialcelltypes,guthomeostasisandinflammation

Theintestinalbarrierconsistsofapolarizedandpermeablemonolayerofepithelialcells.Therearefourmajorintestinalepithe-lialcelltypes,whicharisefromthemulti-potentstemcellswithinthecrypt:(1)absorptiveenterocyteswhichmakeupmorethan80%ofallepithelialcells;(2)mucousandtrefoilfactorproducing

gobletcells;(3)hormoneproducingenteroendocrinecells;and(4)antimicrobialandgrowthfactorproducingPanethcells.

WiththeexceptionofPanethcells,allintestinalepithelialcelltypesarelocatedinthesmallandlargeintestineandundergocon-tinualrenewalevery3–5days[16].Panethcells,ontheotherhand,areprimarilylocatedatthebaseofsmallintestinalcryptandarerenewedapproximatelyevery18–23days[17].TheM(microfold)cells,characterizedbythelackofmicrovilli,overlaythePeyer’spatchesfoundinsmallintestineaswellasisolatedlymphoidfol-licles(ILF)inthesmallandlargeintestine.Underhomeostaticconditions,awiderangeofmicroorganismsandmacromoleculescangainentrythroughtheM-cellsensuringconstantsamplingbyimmunecells[18].

Thedevelopmentofknockoutmiceaswellastheabilitytoinvivoablatethedevelopmentofspecificintestinalcelllineageshavehighlightedtheroleofintestinalcelltypesinmainte-nanceofhealth.Murineatonalhomologue1(Atoh1orMath1)isanimportanttranscriptionfactorinvolvedindifferentiationofsecretoryintestinalepithelialcells.Math1nullmicelackgoblet,enteroendocrineandPanethcellsintheintestine[19].Inter-estingly,homozygousMath1knockoutmice(Math1−/−),butnotheterozygousmice,dieshortlyafterbirth[19].Math1isalsoessen-tialforthedevelopmentofcertainneuronalcelltypes[20].Thus,decreasedsurvivalofMath1−/−micecannotbesolelyattributedtothelossofsecretoryepithelialcellsintheintestine.Instead,ithasbeensuggestedthatrespiratoryfailureduetolossofbrain-stemneuronsunderlietheprematuredeathofMath1−/−mice[20].Theimportanceofsecretoryintestinalepithelialcells,however,hasbeenunderscoredinmicethatlacktheMath1geneinintestinalepithelialcells[20].TheseintestinalepithelialspecificMath1−/−micefailedtosurvivepast14daysofage.Itisunknownwhether

44J.M.M.Natividad,E.F.Verdu/PharmacologicalResearch69 (2013) 42–51

theintestinalmicrobiotainfluencesthesurvivalofmicelackingMath1.However,itispossiblethatthelackofsecretoryepithelialcells,suchasPanethcells,impairsantimicrobialdefenseleadingtoincreasedmorbidity.ItisunknownwhetherMath1−/−micecanthrivenormallywhenmaintainedundergerm-freeconditions.

AnessentialroleofPanethcellsinlimitingmucosalpene-trationofintestinalbacteriahavebeenunderlinedbystudiesshowingthatuponoralchallengewithpathogenicbacteria,Panethcell-deficienttransgenicmice(CR2-tox176)displayedincreasednumbersoflivebacteriainmesentericlymphnodes(MLN)com-paredtowild-typemice,suggesting[21,22].ThemechanismbywhichPanethcellslimitbacterialpenetrationmayinvolvecellactivationthroughpatternrecognitionreceptorsandsecretionofantimicrobials[22].

Ontheotherhand,theimportanceofgobletcellsinmaintenanceofhealthhavebeenemphasizedbyexperimentalobservationthatmicethatlackKruppel-likefactor4(Klf4)gene,atranscriptionfactornecessaryforterminaldifferentiationofgobletcells,dieshortlyafterbirth[23].Klf4−/−neonatalmicedisplayedgobletcellhypoplasiaandabnormalexpressionofMuc2.Thesemicealsodis-playedotherabnormalities,whichlikelycontributedtoincreasedmorbidityandmortality[23].Inagreementwiththis,micewithspecificintestine-epithelialdeletionofKlf4genewereviablebutstilldisplayedsignificantlowernumbersofgobletcellsandalteredintestinalepithelialhomeostasischaracterizedbydysregulatedcellproliferation,migration,differentiationandpositioningofintesti-nalepithelialcells[24].ItremainstobedeterminedwhetherthereducednumbersofgobletcellsinKlf4−/−micerendersthemsus-ceptibletointestinalinflammation.Incontrast,transgenicmiceinwhich60%ofgobletcellswereablatedbytheexpressionofanattenuateddiphtheriatoxingenedrivenbytheITFpromoterwereprotectedagainstexperimentalcolitis,andthisparadoxicalresulthasbeenattributedtotheincreasedproductionoftrefoilfactorpeptidesbyremaininggobletcells[25].

Thus,animalstudiesdemonstratekeyrolesofintestinalepithe-lialcelltypesinmaintenanceofbarrierpropertiesandhealth.However,redundancyexistsandtherearecompensatorymech-anismsthatdeveloptomaintainhomeostasisintheabsenceofaspecificintestinalepithelialcelllineage.

1.2.Intestinalbarrierarchitecture,guthomeostasisandinflammation

Theintegrityandstructureoftheepithelialcellislargelymod-ulatedbymicrotubulesandactin.Microtubulesplayacentralroleinmaintainingcellularintegrity,directingintracellulartransportandsecretion,andcoordinatingorganellemovements.Ontheotherhand,actinregulatesepithelialpermeabilitythroughinteractionswithapicaljunctionalproteins,whicharecomposedoftightjunc-tions,intermediateoradherensjunctions,desmosomesormaculaadherens,andgapjunctions(Fig.2)[26,27].Apicaljunctionalproteinsplayanimportantroleinthemaintenanceofepithelialpolarity.Bothadherensjunctionsanddesmosomesareresponsibleinbindingepithelialcellstogether.Adherensjunctionsalsohaveacrucialroleincellrenewalandarenecessaryforproperassemblyofthetightjunctions[28,27].

Paracellulartransportacrossthebarrieriscontrolledbyapi-caljunctionproteins,whichdynamicallyrespondtodifferentstimuliincludingpathogens,commensalbacteriaandbacterialproducts.Epithelialcellsallowasmallamountofluminalanti-genstopasstranscellularlyacrosstheepitheliumeitherthroughreceptor-mediatedendocytosisornon-selectivelyviafluid-phaseendocytosis.Thisprocessiscalledtranslocation.Asmalldegreeofbacterialmucosaltranslocationisnormalandcontributestothephysiologicalsamplingofluminalcontentbythehost’simmunesystem[29].However,whenthehostcannoteffectivelycontaingutbacteriatothemucosalcompartment,increasedimmuneacti-vationandinflammationmayoccur[5].

Animalmodelslackingspecificcomponentsofapicaljunc-tionalproteinshighlighttheimportanceofthesemoleculesinthemaintenanceofintestinalhomeostasis.Expressionofdomi-nantnegativeN-cadherininthemouseintestinalepitheliumledtospontaneousdevelopmentoftransmuralenteritis,similartoCrohn’sdisease[30].Also,micelackingintracellularmicrobialsensors,Nod1andNod2,displayeddecreasedcolonicE-cadherinexpression,paralleledbyincreasedparacellularpermeabilityanddecreasedantimicrobialproteinproduction[15].ThebarrierdefectinNod1−/−;Nod2−/−micewasinsufficienttocausespontaneousinflammation,howeveritincreasedsusceptibilitytosubsequent

Fig.2.Intestinalepithelialbarrierstructure.(A)Theintegrityandstructureofepithelialcellislargelymodulatedbythecytoskeleton.Withinthecell,twotypesofcytoskeletalproteinsarepresent:(1)␣-integrin,whichpolymerizestoformmicrotubules;(2)actinthatpolymerizestoformfilamentousactin.Actinfilamentsformadensecross-linkedactincortexattheapicalregionoftheepithelialcells,andplayaroleinregulatingepithelialpermeabilitythroughinteractionswithapicaljunctionalproteins.(B)Apicaljunctionalcomplexcomposedoftightjunctions,intermediateoradherensjunctions,desmosomesormaculaadherens,andgapjunctionsboundepithelialcellstogetherandmaintainepithelialpolarity.Adherensjunctioniscomposedofcadherins,suchasE-cadherin(alsocalledcadherin-1),andisboundtoalphaandbetacatenins.Moreover,adherensjunctionsarenecessaryforproperassemblyofthetightjunctions,whicharejunctionalproteinsthataremostapicallylocated.Thetightjunctionsarecomposedoftransmembraneproteinsoccludins,claudins,andjunctionaladhesionmolecule(JAM)thatarelinkedtotheactincytoskeletonthroughzonulaoccludens(ZO)proteins.

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acuteexperimentalcolitis[15].Similarly,micethatlackedJAM,oneofthecomponentsoftightjunction,didnotspontaneouslydevelopintestinalinflammation,butexpressedincreasedsensi-tivitytocolitis[31,32].Ontheotherhand,SAMP1/YitFcmice,whichexhibitedincreasedpermeabilityinileumandaberrantexpressionofthetightjunctionproteinsClaudin-2andoccludin,spontaneouslydevelopedileitissimilartoCrohn’sdisease[33–35].Whenraisedundergerm-freeconditions,SAMP1/YitFcmicestilldisplayedalteredilealpermeabilitysuggestingthatthebarrierphe-notypeisindependentofthepresenceofgutbacteria[36,37].Takentogether,thesestudiessuggestthatadenovodefectinbarrierintegritymaybeinsufficienttodisruptintestinalhomeostasisinawaythatleadstodiseasebutitmayincreasetheriskorseverityofinflammation,particularlyinthepresenceofothertriggers.Compromisedbarrierintegrityhasalsobeenobservedduringactiveinflammation[8,10,38].Theexactcontributionofbarrierdysfunctionatthisstageofdiseaseremainsuncertain.Inflamma-torymarkerssuchascytokinescandisruptpersebarrierintegrityandfunction[6,27].Adefectinbarrierfunctionduringactiveinflammationmaybeaconsequenceofdisease,andalthoughaninflammation-amplifyingrolecannotberuledout,aprimaryroleinthedevelopmentofinflammationisdifficulttoascertain.

1.3.Antimicrobialpeptides,guthomeostasisandinflammation

Epithelialcellssecreteawidevarietyofmolecules,whicharenaturalantibioticswithbroadantimicrobialactivity.Panethcells,primarilylocatedatthebaseofsmallintestinalcrypts,arethemainsourceofantimicrobialsintheintestine.About70%ofPanethcells’granulescontain␣-defensinsandtherestisamixtureoflysozymes,secretoryphospholipaseA2,C-typelectinssuchasRegIII-␥andRegIII-␤,calprotectinsandribonucleasessuchasangiogenin-4(Ang-4)[39].EnterocytesalsohavetheabilitytosecreteantimicrobialsincludingRegIII-␥,cathelicidins,smallamountsof␣-defensins,and␤-defensins[15,40].Antimicrobialsarepresentinboththesmallandlargeintestine,andcaneitherbeconstitutivelyexpressedorinducible(Table1).

Studiesinanimalshaverevealedthatepithelial-derivedantimi-crobialpeptidesareimportantinthepreventionandclearanceofintestinalpathogens.Thisisevidentinmicelackingcryptidins,whichisamurinecounterpartofhuman␣-defensins,andRegIII-␥proteins[41–44].Similarly,thecontributionof␣-defensinstohostdefenseagainstentericpathogenshasbeendemonstratedinastudyshowingthattransgenicmiceexpressinghuman␣-defensinHD5arefullyresistanttoorallethalinfectionwithSalmonellatyphimurium.Impaired␣-defensinexpressionmayleadtoareductionofantibacterialactivity,makingsusceptiblehostsmorevulnerabletointestinalbacterialinvasionandattachment.Indeed,antimicrobialpeptidesarestrategicallylocatedattheepithelialsurfacealongthemucuslayer[45].Theirlocationhints

totheirroleinkeepingintestinalbacteriaatbay,andinpreven-tingepithelialcellstressandimmuneactivation.IthasbeenshownthatalackofRegIII-␥wasassociatedwithanincreasednumberofbacteriainclosecontactwiththeepithelium[45].Increasedmicrobial–epithelialcontactinRegIII-␥−/−micewasparalleledwithheightenedadaptiveimmuneresponse,implyingthatalossofbacterialsegregationinducesunnecessaryimmuneactivation[45].ItispossiblethattheincreasedimmuneactivationinRegIII-␥−/−micereflectsacompensatorymechanismmountedbythehosttocompensateforthelackofRegIII-␥expression.Itremainstobedeterminedwhetherthisadaptationcouldcontributetodiseaseifoverwhelmed,particularlyinthepresenceofintestinalinjuryorinflammatorytriggers.

UsingtransgenicmicethatexpressHD5,ithasbeensuggestedthat␣-defensinmaybeinvolvedinpreventingmucosa-associatedbacteriasuchassegmentedfilamentousbacteria(SFB)tocolonizeincloseproximitywiththeepithelium,leadingtoTh17activation.HD5transgenicmice,however,stillexpressmousecryptidins.Asaresult,theobservationsfoundintheHD5transgenicmicemaynotnecessarilyreproducewhatishappeningunderphysiologicalconditions[46,47].Overall,antimicrobialpeptideslikelycontributetointestinalhomeostasisbyactingasnaturalhostantibioticsthatprotectthevulnerablemucosalsurfacefrommicrobialinvasionandpenetrationandinappropriateimmuneactivation.

Inadditiontoitsroleasnaturalhostantibiotics[48],antimi-crobialpeptideshaveimmuno-modulatoryproperties[49,50].Forinstance,cathelicidinhasbeenshowntomodulatemacrophagesinvitrointoananti-inflammatoryphenotype[51].Antimicrobialpeptidesmayalsobeessentialinregulatingintestinalmicrobiotacomposition[52].Inparticular,secretionofAng-4,whichpreferen-tiallytargetsgram-positivebacteriawhilesparinggram-negativebacteria,hasbeensuggestedtoshapethecompositionoftheintestinalmicrobiotaatthetimeofweaningwhentheproportionofgram-positivebacteriadecreaseandgram-negativeanaerobicorganismsbecomeestablished[48].Likewise,HD5mayalsoinflu-encethecompositionofgutmicrobiota[46,47].Altogether,thesestudieshighlighttheroleofepithelialderivedantimicrobialsasactiveinnateimmunemediatorsandregulatorsofintestinalmicro-biotacomposition.

Inbothanimalmodelsandclinicalstudies,antimicrobialpep-tidesseemtobeup-regulatedduringactiveinflammationoratthesiteofinflammation[53–56].Thismayreflectadefensemechanismagainstinvadingbacteriaduringmucosalbreaksorpermeabilityincreases.Ontheotherhand,increasedantimi-crobialpeptideproductionduringactiveinflammationcouldbepotentiallydetrimentalandaffectbeneficialbacteriawithanti-inflammatoryeffects[57].Thusfar,thebruntofevidencesupportstheconceptthatabalancedexpressionofantimicrobialpep-tidesiscrucialtomaintainintestinalhomeostasisandpreventinflammation.

Table1

Expressionpatternofepithelial-derivedantimicrobials.

Family

Members

Localization

Transcriptionallyinduced

␣-Defensins␣-Defensins␣-Defensins␣-Defensins␤-Defensin␤-Defensin␤-Defensin

␤-1,4-Glycosidase

Phospholipidsn-2enteraseC-typelectinC-typelectinRibonucleaseCathelicidins

HD-5(human)HD-6(human)

Cryptidins(mouse)

Cryptidinsrelatedpeptides(mouse)HBD-1(human)HBD-2(human)mBD-3(mouse)HBD-3(human)LysozymeCsPAL2

RegIII-␣orHIP/PAP(human)RegIII-␥(mouse)RegIII-␤(mouse)ANG-4

LL37(human)

Panethcells,smallintestinalandcolonicenterocytesPanethcells,smallintestinalandcolonicenterocytesPanethcellsPanethcells

ColonicenterocytesColonicenterocytesColonicenterocytesPanethcellsPanethcells

Panethcells,smallintestinalandcolonicenterocytesPanethcells,smallintestinalandcolonicenterocytesPanethcells

Panethcells,smallintestinalandcolonicenterocytes

NoNoNoNoNoYesYesNoNoYesYesYesNo

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Table2

Regionalexpressionofintestinalmucins.

MUCtype

Membranebound/secreted

LocalizationMUC1(human,mouse)MembraneboundLargeintestineMUC2(human,mouse)

Secreted

Smallandlargeintestine

MUC3A(human,mouse)

Membranebound/secreted

Smallandlargeintestine

MUC3BSmalland(human,mouse)

Membranebound/secreted

largeintestine

MUC4Small(human,mouse)MembraneboundintestineMUC11Smalland(human,mouse)

Membranebound

largeintestine

MUC12(human,mouse)Membranebound

LargeintestineMUC13(human,mouse)

Membranebound/secreted

Smallandlargeintestine

MUC15(human,mouse)

Membranebound

Smallandlargeintestine

MUC17(human,mouse)MembraneboundLargeintestineMU19(human,mouse)Secreted

LargeintestineMUC21(human,mouse)

Membranebound

Largeintestine

1.4.Mucuslayer,guthomeostasisandinflammation

Twomucuslayerscanbefoundwithintheintestine(Fig.1)[58].Theunstirred(inner)mucuslayer,isapproximately100␮mthick,isfirmlyadherent,richinepithelial-derivedantimicrobialsandmucin,andhaslowbacterialdensity.Thestirred(outer)mucuslayeriscomposedofmucinanddilutedantimicrobials,andsomebacteriaareabletopenetratethislayer.Mucinconstitutesavis-coelasticgelthatlinestheintestinalepithelialbarrier.Thethicknessandcompositionofthemucuslayervariesdependingontheloca-tionwithintheintestinaltract[59].Thehighestmucusviscosityisfoundinthedistalcolon[59].Interestingly,microbialdensityincreasesalongtheproximaltodistalpartsoftheintestinaltract,reachingupto1012bacteriapermlofluminalcontentinthecolon[60,61].Itisunclearwhethertheincreasedviscosityatthedistalcolonreflectsadefensemechanismofepithelialcellstoprotectagainstpotentiallyinvadingbacteria,orwhetheritisthedirectresultofhighermicrobialload.

Gobletcellsaretheprimarysourceofmucinsintheintes-tine.Thereare20differentmucinsthathavebeenidentified,andnineofthoseareexpressedbybothmurineandhumanintestineandareeithersecretedormembranebound(Table2).Mucinsaredividedintoneutralandacidicsubtypes.Acidicsubtypesarefurtherclassifiedassulfated(sulfomucins)ornonsulfated(sialomucins)groups.MUC(in)2isthepredominantmucinfoundinthelargeintestine.Interestingly,miceknockoutfortheMUC2genesponta-neouslydevelopcolitis[62].Ontheotherhand,micenullforMUC13genesdonotspontaneouslydevelopintestinalinflammationbuthaveshowntobemoresusceptibletoexperimentalcolitis[63].Incontrast,IL-10−/−micecrossedtohumanMUC1-transgenicmicedevelopmoreseverespontaneouscolitiswithahigherincidenceofcoloncancerdevelopmentascomparedtoIL-10−/−mice[].Thesestudieshighlightthatsubsetsofmucinshavedistinctfunc-tionsinthemaintenanceofintestinalhomeostasisandintheirroleindiseaseprogression.

Themucuslayerisalsocomposedofdiverseproteinssuchasglycoproteinsandpeptidesofthetrefoilfactorfamily(TFF).Thesepeptidesaresecretedbygobletcellsandenterocytesandhavebeenshowntoprotecttheepitheliumfromavarietyofinsults[65].MicelackingTFF3,onetypeofTFFpeptide,showedincreasedsuscepti-bilitytoexperimentalcolitis.TheunderlyingmechanismcouldberelatedtotheimpairedabilityofTFF3−/−micetohealthemucosaandpromoteregeneration.Therefore,TFFpeptidesareimportantinitiatorsofmucosalhealingcontributingtointestinalhomeostasis.

Theroleofthemucuslayerininfluencingintestinalmicrobiotacompositionhasnotyetbeenfullydefined,butitispossiblethat

differentialexpressionofmucinbothincompositionandlocationacrossthegastrointestinaltracthasaroleinintestinalcolonizationaswellasinshapingthecompositionofintestinalmicrobiota.

1.5.Microbialrecognition,guthomeostasisandinflammation

Intestinalepithelialcellsexpressawidevarietyofpatternrecog-nitionreceptorssuchasToll-likereceptors(TLR)andNOD-likereceptors.Expressionofthesereceptors,however,istightlycon-trolledtopreventinappropriateimmuneactivationwhilestillallowingconstantvigilance.Forexample,TLR5,whichrecognizeflagellin,hasbeenfoundtobeexclusivelyexpressedatthebasolat-eralsideofepithelialcellswhilesomeTLRssuchasTLR9andTLR7aswellasNOD-likereceptorsareintracellular,ensuringthatthesereceptorscanonlybeactivatedbyinvadingantigensormicroor-ganisms[66].Theimportanceofinnateimmuneactivationatthelevelofintestinalepitheliumishighlightedbythedevelopmentofspontaneousinflammationinspecificpathogenfree(SPF)micewithintestinalepithelialcelldeletionofthecomponentsoftheIKKcomplex,whichisadownstreamsignalingpathwayofbothPRRandupstreamkinaserequiredforNF-␬␤activation[67].Basalrecogni-tionofcommensalbacteriabyintestinalepithelialcellsinfluencesthesecretionoftheimmuno-regulatorycytokinessuchasTSLPandTGF-␤,whichhavetheabilitytodampenpro-inflammatoryresponses[66,68,69].Moreover,theanaerobiccommensalbac-teriumBacteroidesthetaiotaomicronhasbeenshowntoattenuateintestinalinflammation.Theanti-inflammatorycapabilityofB.thetaiotaomicronmaybeattributedtoitsabilitytoactivatePPAR-␥dependentpathwayinintestinalepithelialcells[70].Thisdatasug-gestthatactivationofPPAR-␥pathwayinintestinalepithelialcells,perhapsusingPPAR-␥agonist,maybebeneficialinamelioratingintestinalinflammation.

2.Regulationoftheintestinalbarrierbygutmicrobiota

Compositionalchangesinintestinalmicrobiota(dysbiosis)havebeenfoundinpatientswithceliacdisease,IBSandIBD[14].Ithasbeenproposedthatdysbiosismayplayaroleinthepathogenesisofthesediseases.Sofartheevidenceofaroleofdysbiosisingastroin-testinaldiseaseislimitedtoassociation.Itispossiblethatdysbiosiscontributestointestinalinflammationbydisturbingthesignalsthatmaintainbarrierfunction.Abetterunderstandingofhowtheintestinalmicrobiotamodulatetheseprocesseswillhelpdeveloptherapeutictargetstopreventortreatgastrointestinaldisease.

2.1.Microbialeffectsonintestinalepithelialcellrenewal

Abalancebetweencellproliferationandcelldeathisrequiredfornormalintestinalbarrierfunction.Anumberofstudiescompar-inggerm-freeandconventionallyraisedanimalshaveshownthatlackofintestinalmicrobiotagreatlyimpairsintestinalmorphology,architectureandcellrenewalproperties[3,2].Astrikingdifferencebetweenmicerearedundergerm-freeandconventionalconditionsisthatthetotalintestinalsurfaceareaissignificantlydecreasedintheabsenceofgutbacteria[71,72].Thisisparalleledbyaberrantintestinalmorphologywithshorterilealvilliandsmallerintesti-nalcryptsingerm-freemice[73].Moreover,therateofturnoverofilealandPeyer’spatchesingerm-freemicewasfoundtobelowerthaninmiceraisedinconventionalizedconditions[73].Eightdaysaftercolonization,cellrenewalrevertedtothesamedegreeasthatofconventionalmice[74].Decreasedcellrenewalmayhavedetri-mentalconsequencesonepithelialproliferationandregenerativeability.Indeed,germ-freemicehavebeenshowntobemoresus-ceptibletodextransulfatesodium(DSS)-colitis[75],underscoringthatintestinalbacteriaconferprotectionagainstintestinalinjurybyregulatingepithelialcellturnoverandrestitution.

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Theexactmolecularandcellularsignalingpathwaybywhichintestinalmicrobiotaregulatecellrenewalisstillunclear.However,indirectevidencesuggeststhatmicrobialassociatedmolecularpat-terns(MAMP)inducerelevantsignalsforbarrierfortification.MicelackingbacterialsensingsuchasToll-likereceptorsandNod-likereceptorsaremoresusceptibletocolitiscomparedtowild-typecontrols[15,76,77].Increasedsusceptibilitytocolitishasbeenasso-ciatedwithalteredcellrenewalandrestitution[76,77].Depletionofgutmicrobiotausingbroad-spectrumantibioticsinbothwild-typeandmicedeficientineitherNod2orMyD88,anadaptorproteinforTLRreceptors,didnotameliorateDSSinducedintestinalinjurybutratherpromotedmoreseveremorbidity[77,76].OralingestionofbacterialproductssuchasLPSormuramyldipeptide(MDP),aligandforNod2receptors,priororduringDSSchallenge,protectedwild-typemicethatreceivedoralantibioticsand/ormicelackinginnatebacterialsensors[77,78].Onelimitationinmostofthesestudiesisthefactthatmicrobialrecognitionisalsodefectiveinothercells,particularlyhematopoieticcells.Thus,thespecificcon-tributionofmicrobialrecognitionincolitisatthelevelofintestinalepitheliumisstillunclear.Nevertheless,thesestudiessuggestthatmicrobialsignalsareimportantforprotectionagainstintestinalinjury,likelythroughcellrenewalandpromotionofwoundheal-ing.IL-10−/−micespontaneouslydevelopintestinalinflammationinSPFconditions[79–82].Colitisonsethowever,isdelayedifIL-10−/−micearere-derivedgerm-free,highlightingthatintestinalbacteriaarenecessaryfortheinductionofintestinalinflammationinthismodel[79,82].

ArecentstudyhasindicatedthattheprobioticbacteriumLacto-bacillirhamnosusGGpromotescellrenewalandaugmentsmucosalrepairfollowingDSSinducedcolitisviareactiveoxygenspeciesgenerationinepithelialcells[83].Itishoweverunknownwhethercommensalbacteriadirectlyorindirectlysignaltotheepithelialcellstopromotecellrenewalandwoundhealing.Certaincom-ponentsofthemicrobiotagenerateavarietyofshortchainfattyacids(SCFA)suchasbutyrate,succinateandpropionate,whichhaveacrucialroleinregulatingcolonicepithelialcellgrowthanddifferentiationandareimportantenergysourcesforcolono-cytes[84].Overall,maintenanceofintestinalbarrierhomeostasisrequirescomplexinteractionsbetweenthemicrobiota,hostgenes,andotherenvironmentalfactorssuchasdiet.

2.2.Microbialeffectsonintestinalpermeability

Theabilityoftheintestinalmicrobiotatoaffectintestinalper-meabilityhasbeenmostlysupportedbytheuseofprobiotics,exogenouslyadministeredbacteriathatarethoughttohavebenefi-cialeffectsonthehost.Acuteorpsychologicalstressinducesbarrierdysfunctioncharacterizedbyincreasedmacromolecularfluxandchangesinionsecretion[85].OraladministrationofprobioticscontainingLactobacillusspp.preventedstress-inducedintestinalbarrierchanges[86].Somestudieshavereportedincreasedper-meabilityinDSScolitisaccompaniedbychangesinapicaljunctionexpressionandre-organization,beforetheappearanceofnotableinflammation,suggestingthatintestinalbarrierdysfunctionmayprecedetheonsetofcolitis[87,88].AdministrationofeitherEscherichiacoliNissle1917orL.rhamnosusduringDSScolitispreventedcolitis-associatedintestinalbarrierdysfunctioninmice[,90].TreatmentwithVSL#3,whichiscomposedofeightpro-bioticbacteria,protectedtheepithelialbarrierinmiceafterDSScolitisinduction[91].Theseeffectshavenotbeendescribedwithallprobioticbacteria,andwehaveshownthatadministrationofadefinedprobiotic,BifidobacteriumbreveNCC2950,didnotmodulatethealteredpermeabilityobservedinnaiveNod1−/−;Nod2−/−mice[15].Interestingly,B.brevewasabletopreventincreasedseverityofDSScolitis,butthemechanismofprotectionwasunrelatedtoacorrectionofthebaselinepermeabilitydefectobservedinthese

mice[15].Mostofthestudiesshowthattheeffectofprobioticsonintestinalpermeabilitycannotbeobservedincontrolanimals[15,91],suggestingthatprobioticsmaybemoreeffectiveforthepreventionofbarrierchangesandinflammation.

IthasbeensuggestedthatintestinalbarrierdysfunctionmayprecedetheonsetofspontaneousintestinalinflammationinmicedeficientforIL-10gene[81,82].TheprimarydefectinIL-10−/−micewasdependentonthepresenceofintestinalmicrobiotaasgerm-freeIL-10−/−micedidnotdisplayincreasedintestinalperme-ability[82].LactobacillushavebeenshowntobereducedinIL-10−/−mice[92].Specifically,administrationofLactobacillusreuteriduringneonatalperiodattenuatedthedevelopmentofcolonichistologi-calinjuryinIL-10−/−mice[92].Itis,however,unknownwhetherreducedinflammationwasaccompaniedbyimprovementinintestinalpermeability.AdministrationofLactobacillusplantarumwasshowntobeeffectiveinamelioratingtheincreasedcolonicpermeabilityandchangesintightjunctionalexpressionduringactivediseaseinIL-10−/−mice[81].Moreover,administrationofL.plantarumtogerm-freemicebeforecolonizationwithSPFandcon-tinuedprobiotictherapythereafterdecreasedhistologicalinjuryinIL-10−/−mice.Similarly,Mdr1a−/−mice,anotheranimalmodelofspontaneouscolitis,displayedintestinalbarrierdefectbeforetheonsetofhistologicalinjury[93].AdministrationofStreptococcusthermophilusandLactobacillusacidophiluspreventedincreasedper-meability,whichwasaccompaniedbygeneralimprovementinthehealthofmice[94].Overall,thesestudiesshowthatspecificbacte-riacanregulateofintestinalbarrierfunction,andthiscanpositivelyinfluencethedevelopmentofintestinalinflammation.Moreworkisneededonspecificmechanismsofactionbywhichthesespecificprobioticstrainsmodulatetheintestinalbarrier.

Componentsoftheresidentintestinalmicrobiotamayalsoinflu-enceintestinalbarrierfunction.ThisisevidentinarecentstudyshowingthatMyD88−/−;Ticam−/−mice,whichlackToll-likerecep-torssignaling,raisedingnotobioticenvironmentandcolonizedwithdefinedmicrobiotadonotdisplayalteredbarrierfunction[29].ThisobservationcontrastswithotherstudiesusingMyD88−/−micerearedandmaintainedunderSPFcondition[29,77,95].Itispossi-blethatmicecolonizedwithamicrobiotaoflimiteddiversity,lackorganismscapableofadverselymodulatingintestinalpermeability.ThemaintenanceofepithelialbarrierhasalsobeensuggestedtobeTLR2mediatedandassociatedwithrestorationandincreasedexpressionofZO-1[96,97].Mono-colonizationofgerm-freemicewithB.thetaiotaomicronmodulatedexpressionofgenesneces-saryforintestinalbarrierfortification[98].Thesechangesincludeup-regulationofsmallproline-richprotein-2,whichactsascross-bridgingproteinslinkedtodesmoplakin,acomponentoftheapicaljunctionalproteindesmosones(Fig.2)[98].Itisunclearwhetherandhowtheexpressionofmicrobialinducedbarrier-relatedgenestranslatesintoproteinexpressiontomodulateintestinalhomeo-stasis.Similarly,mono-colonizationofgerm-freewithE.coliNissle1917resultedinup-regulationofZO-1butnotZO-2inintesti-nalepithelialcells[90].TheincreasedexpressionofZO-1wasnotobservedingerm-freemicemono-colonizedwithotherstrainsofE.coli.SpecificprobioticsandothercommensalbacteriaareabletosecreteuniquemoleculessuchasSCFA,whichcanhavebeneficialeffectsontheintestinalbarrier[99–101].Lysatesoftheprobio-ticLactobacilluscaseiDN-114,butnotL.plantarum,amelioratedDSScolitisbypreventingtheincreaseinpermeabilityandpre-servingZO-1expressioninmice.Thesechanges,however,wereaccompaniedbyanincreaseinbutyrateproducingbacteria[102].Thus,itisunclearwhethertheimprovementinintestinalperme-abilityandepithelialintegritywasdirectlymediatedbythelysateorweresecondarytothechangeingutmicroenvironment.Never-theless,thedatacollectivelysuggestthatcertainbacterialspeciesproducemetabolitesthatcaninfluenceintestinalpermeabilityandintegrity.

48J.M.M.Natividad,E.F.Verdu/PharmacologicalResearch69 (2013) 42–51

Althoughthebruntofdatasuggestaroleofprobioticsinmod-ulatingbarrierfunctionandintegrity,moststudieshavefocusedonpreventativeeffectsbeforeadministrationofaninfectiousorinflammatorychallenge.Therearestillconsiderablegapsinknowledgeonthemechanismsbywhichprobioticsandothercom-mensalbacteriaaffectintestinalpermeability.Moreimportantly,informationislackingondosage,timingandformulationofexoge-nouslyadministeredmicroorganismstopreventandtreatdisease[103,104].

2.3.Microbialeffectsonantimicrobialproteinexpression

Antimicrobialproteinshaveadualroleinlimitingtheinva-sionofpathogensaswellasmodulatingtheintestinalmicrobiotacomposition.Certainsubsetsofintestinalepithelialcells-derivedantimicrobialproteinsareindependentandothersareinfluencedbythegutmicrobiota,emphasizingthebi-directionalinteractionbetweenintestinalmicrobiotaandintestinalbarrier.

GnotobioticstudieshaverevealedthatPanethcell-derivedantimicrobialssuchaslysozymeandsPLA2areexpressedundergerm-freeconditions.Similarly,cryptidinsareexpressedintheabsenceofgutmicrobiota,suggestingthatbaselineexpressionofthesemoleculesdonotrequiresignalsfromgutmicrobiota[105,106].However,cryptidin-relatedsequences(CRS)havebeenshowntobesignificantlyhigherinconventionallyraisedmicecomparedtomiceraisedingerm-freeconditionssuggestingthatgutmicrobiotamayinfluencethelevelofexpressionofcryptidins[106].Thus,itseemsthequantity,andquality,ofcryptidinscanbeaffectedbythemicrobiota.Expressionof␣-defensinsandcryp-tidinsissignificantlylowerinhumanneonatesandun-weanedmiceandrats,butincreasedramaticallyinadulthood[107–109].Duringthefirstfewyearsoflife,eachindividualundergoesatransitionalintestinalcolonization,characterizedbydynamicandrandomcolonizationwithadistinctivemicrobialcombination[110,111].Thecolonizationpatternsstabilizeintime,witheachindividualeventuallypossessingauniquefingerprintofdiverseintestinalmicrobiota[110,111].Theincreaseinexpressionof␣-defensinsoccursconcomitantlywithcolonizationanddevel-opmentofastablegutmicrobiota,implyingthatcomponentsofmicrobiotamayhavearoleintheregulationofintestinal␣-defensins[110–112].Itwillbeinterestingtoknowwhetherthe␣-defensinsfromneonateshavethesamebiologicalactivityasadult␣-defensins.Indeed,␣-defensinsarepost-translationallymodifiedintomaturedefensinsbymatrilysin(MAT)inmiceandtrypsininhumansuponsecretion[43,113].Interestingly,MATexpressionisundetectableingerm-freemice,implyingthatmicro-bialstimuliarerequiredforexpressionoffunctionalcryptidins[114].

Panethcell-derivedantimicrobials,including␣-defensins,arereleaseduponexposuretolivebacteriaandtheirantigens,suchaslipopolysaccharide(LPS)andmuramyldipeptide(MDP)[115].Incontrast,antimicrobialsecretionbyPanethcellsisnotinfluencedbyfungalorprotozoalsignals[115,116].Theexactsignalingpath-waythatleadstomicrobial␣-defensinexpressionisunclearbutclinicalandexperimentalevidencesuggeststhatinnatemicrobialsensors,particularlyNod2receptors,maybeinvolved[56,117,118].Likewise,microbialsignalingthroughNod2receptorsisessentialfortheexpressionofotherkindsofPanethcells’antimicrobials,namelyDefcr4andDefcr10[119,120].

Human␤-defensinisconstitutivelyexpressedincolonicepithe-lialcells.Mouse␤-defensin(mBD)1,mBD3,andmBD4havebeendetectedinthetongueandstomachofgerm-freemicebutfullcomparisonbetweenexpressionof␤-defensininotherpartsofintestinaltractbetweengerm-freeandconventionalmicehavenotyetbeenfullystudied[121].However,uponmono-colonizationofgerm-freemicewithCandidaalbicans,up-regulationofmBD1,

mBD3,andmBD4expressionhasbeenobserved.Expressionofhuman␤-defensin(hBD)2,butnothBD1,requiresbacterialorinflammatorystimuli[122–124].TheprobioticVSL#3,severalLac-tobacillusspecies,E.coliNissle1917andE.colistrainDSM17252(Symbioflor2)havebeenshowntoinduceexpressionandsecretionofhBD2inhumancolonicepithelialcelllines[125–128].Further-more,increasedfecallevelsofhBD2havebeenobservedinhealthyhumanswhoreceivedSymbioflor2twicedailyfor3weekscom-paredtoplacebotreatedindividuals[125].NF-␬␤pathwayandTLR2dependentsignalinghavebeenshowntobeinvolvedintheinductionofhBD2inepithelialcells[127,129].Similarly,bacterialflagellinderivedfromE.coliNissle1917wasshowntoinducehBD2.FlagellinisrecognizedbyTLR5,henceitispossiblethatthisreceptorplaysaroleintheinductionofhumanhBD2inintestinalepithelialcells.

ExpressionoftheantimicrobialribonucleasesAng-4andtheC-typelectinRegIII-␥andRegIII-␤issignificantlylowerorunde-tectableinthesmallintestineofgerm-freemicecomparedtoconventionallyraisedmice,inferringthattheirexpressionisunderthecontrolofthegutmicrobiota[22,130,131].Uponcolonizationofgerm-freemice,RegIII-␥expressionincreased[22,130,131].TheimportanceofmicrobialregulationofRegIIIproteinexpressionisalsosupportedbystudiesshowingthatmicelackingtheMyD88gene,butnotmicelackingNod2signaling,havesignificantlylowerRegIII-␥inthesmallintestinecomparedtocontrols[22].Spe-cificpathogen-freeNod1−/−;Nod2−/−micehavesignificantlylowerRegIII-␥expressioninthecoloncomparedtoheterozygotelitter-mates,suggestingthatNodreceptors,perhapsNod1,alsoinfluencecolonicRegIII-␥expression[15].TheprobioticB.breveNCC2950wasabletoup-regulateRegIII␥inNod1−/−;Nod2−/−mice.ItremainstobedeterminedwhetherB.breveisabletoup-regulateHIP/PAP,whichisthehumancounterpartofRegIII-␥.Mono-colonizationofgerm-freemicewithB.thetaiotaomicroninducedafive-foldincreaseinRegIII-␥expressionwhilemicecolonizedwithBifidibac-teriumlongumrepressedRegIII-␥expression[132].SuppressionofRegIII-␥byB.longum,however,wasreverteduponsimultaneouscolonizationofgerm-freemicewithbothB.longumandB.thetaio-taomicron[132].RegIII-␥hasbroadantimicrobialactivityagainstgram-positivebacteria.AsB.longumisagram-positivebacterium,itispossiblethatitsabilitytodown-regulateRegIII-␥mayrepre-sentitscopingmechanismtosurviveinthegut[132].Overall,thesestudiesindicatethatspecificbacteriahavetheabilitytodifferen-tiallyregulateantimicrobialexpression.

2.4.Microbialeffectsonmucuslayer

Germ-freemicehavelowernumbersofmucin-secretinggob-letcellsinthececumcomparedtoconventionallyraisedanimals[133].Moreover,germ-freemicedisplayedthinner,lessstableandcompactmucuslayercomparedtocolonizedanimals[71,134–139].ExposuretobacterialproductsLPSandpeptidoglycanincreasedthemucuslayerthicknessofgerm-freemicetothelevelsimilartoconventionallyraisedmice[136].Takentogether,thesefindingssuggestthatthecompositionofthegutendogenousmicrobiotamayregulatethemucuslayer.

AdministrationofVSL#3toratsinducedincreasedMUC2expressionandsecretion[140].AmongthebacteriainVSL#3,theexpressionseemedtobestronglymediatedbytheLactobacillibacteria[140].ItremainstobedeterminedtheexactmechanismbywhichLactobacilliinducedexpressionofmucin.Moreover,VSL#3up-regulatedtheexpressionandsecretionofMUC1,2and3inratbutnotinmice[140,141].Similarly,mucinexpressionorsecretionwasnotchangeduponinoculationofB.brevetomice,emphasiz-ingthedifferentialeffectsofprobioticsinregulatingmucuslayercomposition[15].

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Mucinsareessentialinpreventingluminalbacteriafromadher-ingorpenetratingtheintestinalepithelium.Thispropertyreliesonmucins’carbohydratecomponents.Comparisonbetweengerm-freeandconventionallyraisedanimalrevealedthatgerm-freemicehaveadifferentglycosylationprofile[142].Itisstilluncleartowhatextenttheresidentcommensalsengageinthecontrolofmucinglycosylation.ExposuretoLPSfromE.coliO55:B5stimu-latedthereleaseofmucinsingerm-freemice[143].Furthermore,mono-colonizationofgerm-freemicewithB.thetaiotaomicronstimulatedtheexpressionofcomponentsofmucuslayerincluding␣,2-fucosyltransferasemRNAandsynthesisoffucosylatedglyco-conjugatesonsmallintestinalepithelialcells[144].Theincreaseinmucinfucosylation,allowedB.thetaiotaomicrontobetterinter-actwithintestinalepithelialcells.Thus,thereseemstobeabi-directionalintestinalbarrier–microbiotainteraction,whichiscrucialforthemaintenanceofhealth.

3.Conclusion

Thecomplexcommunityofmicroorganismsresidingwithinthelumenoftheintestinaltracthasamutualisticrelationshipwiththehost.Disruptionofitscompositionandhost–microbialinterac-tionscanalterhostfunctionsandhasbeenproposedtocontributetoanumberofgastrointestinalandinflammatorydiseases.Itisnowrecognizedthatthemicrobiotainfluencesintestinalphysiol-ogy,includingtheintestinalbarrier,andthatthisrelationshipisbi-directional.However,thespecificmolecularandcellularmech-anismsremaintobedetermined.Understandingtheinteractionbetweenintestinalepithelialbarrierandgutmicrobiotawillbepivotalforthedevelopmentofnewprophylacticandtherapeuticagentsforchronicinflammation.Thesestrategiesmaybebasedontheuseofspecificbacterialspecieswithbarriermodulatingcapacitytopreventintestinalinflammation.Importantquestionsregardingmodeandtimeofdeliveryofpotentiallybeneficialbacte-ria,aswellastheinfluenceofhostgenotypeinthetherapeuticresponseachieved,needtobeaddressedinthefuture.

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