FACULTY OF HEALTH AND MEDICAL SCIENCES UNIVERSITY OF COPENHAGEN PhD Thesis Mucosal adhesion and immune activation by gut bacteria in inflammatory bowel diseases Stina Rikke Jensen 2012 1 PhD Thesis: Mucosal adhesion and immune activation by gut bacteria in inflammatory bowel diseases By: Stina Rikke Jensen Supervisors: Susanne Brix Anders Elm Pedersen Department of Systems Biology, Technical University of Denmark Faculty of Health and Medical Sciences, University of Copenhagen 2 PREFACE! This!PhD!thesis!is!based!on!work!performed!at!Technical!University!of!Denmark!in!collaboration!with! University!of!Copenhagen!and!Herlev!Hospital!in!2010@2012.!The!work!was!financially!supported!by!Faculty! of!Health!and!Medical!Sciences,!University!of!Copenhagen.!! First!of!all,!I!wish!to!thank!my!two!supervisors,!Susanne!Brix!and!Anders!Elm!Pedersen,!for!profitable! scientific!discussions,!inspiration!and!guidance.!In!particular,!I!wish!to!thank!my!daily!supervisor,!Susanne! Brix,!who!believed!in!me!from!the!very!beginning!and!gave!me!all!the!opportunities!to!get!excited!about! cutting@edge!research,!thanks!a!lot!!A!special!thanks!goes!out!to!Lisbeth!Nielsen!Fink,!who!has!been!an! incredible!support!and!mentor!for!the!scientific!work,!but!also!for!me!personally.!I!also!wish!to!thank! Monica!Hansen!Hovmøller!for!helping!in!the!lab,!for!careful!reading!of!this!thesis!and!not!the!least!for!being! the!amazing!friend!you!are.!My!good!friend!and!colleague!Anna!Hammerich!Thysen!has!been!indispensable! in!the!data!generation;!I!owe!you!a!lot.!You!have!been!of!incredible!assistance,!especially!when!the!time!in! lab!was!running!out!for!me.!I!am!also!grateful!to!Casper!Steenholdt!from!Herlev!Hospital!for!great!support,! especially!when!the!time!was!difficult,!and!chief!physician,!Jørn!Brynskov,!for!giving!me!the!opportunity!to! work!closely!with!the!patients.!I!also!wish!to!thank!Professor!Karen!A!Krogfelt!and!her!group!for!their! important!contribution!to!the!scientific!work.!Many!thanks!to!Rikke!Brandt!for!training!me!in!the!lab,! reading!this!thesis,!and!for!your!always@so@positive!attitude.!I!also!owe!Lisbeth!Buus!Rosholm!a!massive! thanks!for!her!very!skilful!expertise!and!for!helping!me!whenever!needed.!!In!general,!thanks!to!all!my! colleagues!in!the!group,!Systems!Biology!of!Immune!Regulation!at!DTU,!for!the!fantastic!teamwork.!Also,! thanks!to!DTU!FOOD!and!the!Sommer!lab!at!DTU!for!allowing!me!to!make!use!of!your!microbial!lab! facilities.! My!friends!and!family!have!been!an!indispensable!support;!without!you!I!could!not!have!gotten!so!far.!I!am! forever!grateful!for!your!support,!Rasmus.!Thanks!for!all!the!fun!times!we!have!together,!Pernille,!Anja!and! Tina,!it!provides!me!with!a!lot!of!energy.!! Finally,!my!deepest!feeling!goes!to!Erwin;!for!your!endless!support,!love,!and!for!having!faith!in!me.!! ! Stina!Rikke!Jensen! November!2012! ! ! ! 3 CONTENTS! LIST!OF!PUBLICATIONS!...............................................................................................................................................!6! SUMMARY!IN!ENGLISH!..............................................................................................................................................!7! SUMMARY!IN!DANISH!...............................................................................................................................................!9! ABBREVIATIONS!......................................................................................................................................................!11! INTRODUCTION!.......................................................................................................................................................!13! ! CHAPTER!1!...............................................................................................................................................................!16! Gut!tolerance!contra!inflammation!.............................................................................................................................!17! Local!gut!inflammation!............................................................................................................................................!18! Inflammatory!bowel!diseases!..................................................................................................................................!18! Organization!of!the!gut!epithelium!and!mucosal!immune!system!..............................................................................!19! Epithelial!barrier!functions!......................................................................................................................................!20! Gut!mucosal!immune!system!..................................................................................................................................!22! Maturation!of!dendritic!cells!and!T!cell!activation!..................................................................................................!22! Host!recognition!by!cell@surface!receptors!..................................................................................................................!23! TLRs!..........................................................................................................................................................................!24! CLRs!..........................................................................................................................................................................!25! NLRs!.........................................................................................................................................................................!26! Host!effector!responses!...............................................................................................................................................!27! Gut!epithelial!effector!responses!............................................................................................................................!27! Host!tolerance!to!gut!commensals:!Treg!cells!.........................................................................................................!28! Immune!effector!response!to!microbial!infection!induces!cell!death!.....................................................................!29! Crohn’s!disease:!Th1/Th17!inflammation!................................................................................................................!30! Ulcerative!colitis:!Th2!(/Th17)!inflammation!...........................................................................................................!32! The!gut!microbiota!.......................................................................................................................................................!33! Bifidobacteria!...........................................................................................................................................................!33! Faecalibacterium!prausnitzii!....................................................................................................................................!34! Secreted!products!by!gut!commensals!....................................................................................................................!35! E.!coli!Nissle!1917!.....................................................................................................................................................!35! The!significance!of!E.!coli!in!inflammatory!bowel!diseases!.....................................................................................!36! References!....................................................................................................................................................................!38! ! ! ! 4 CHAPTER!2………………………………………………………………………………………………………………………………………………………………….47! CHAPTER!3…..……………………………………………………………………………………………………………………………………………………………..58! CHAPTER!4…..……………………………………………………………………………………………………………………………………………………………..63! CHAPTER!5…..……………………………………………………………………………………………………………………………………………………………..70! CHAPTER!6…..……………………………………………………………………………………………………………………………………………………………..85! CHAPTER!7…..……………………………………………………………………………………………………………………………………………………………..97! Discussion!.....................................................................................................................................................................!98! References!..................................................................................................................................................................!103! ! ! ! ! 5 LIST%OF%PUBLICATIONS!! The!data!of!this!thesis!comprise!five!papers:!three!published,!and!two!manuscripts.! Jensen!SR,!Nielsen!OH,!Brix!S.!Are!NOD2!polymorphisms!linked!to!a!specific!disease!endophenotype!of! Crohn's!disease?!Inflamm!Bowel!Dis!2011!Nov;!17(11):2392@401.!! Jensen!SR,!Fink!LN,!Struve!C,!Sternberg!C,!Andersen!JB,!Brynskov!J,!Nielsen!OH,!Brix!S.!Quantification!of! specific!E.'coli!in!gut!mucosa!from!Crohn's!disease!patients.!J!Microbiol!Methods!2011!Jul;!86(1):111@114.!! Jensen!SR,!Fink!LN,!Nielsen!OH,!Brynskov!J,!Nielsen!OH,!Brix!S.!Ex'vivo!intestinal!adhesion!of!E'coli!LF82!in! Crohn's!disease.!Microbial!Pathogenesis!2011!Dec;!51(6):426@31.! Jensen!SR,!Mirsepasi!H,!Thysen!AH,!Brynskov!J,!Krogfelt!KA,!Petersen!AM,!Pedersen!AE,!Brix!S.!Functional! divergence!in!innate!immune!activating!properties!of!E.'coli!strains!from!patients!with!ulcerative!colitis! and!Crohn’s!disease.!(Manuscript!in!submission!to!Inflamm!Bowel!Dis)! Jensen!SR,!Hovmøller!MH,!Thysen!AH,!Brandt!R,!Pedersen!AE,!Brix!S.!Rewiring!inflammatory!signatures!in! dendritic!cells!induced!by!viable!pathogenic!E.'coli!via!fermentation!products!from!gut!commensal! ! bacteria.!(Manuscript!in!preparation)! ! ! 6 SUMMARY&IN&ENGLISH! A!defective!regulation!at!the!gut!barrier!line!together!with!an!altered!gut!microbial!colonization!might! propagate!to!excessive!immune!activation.!Inflammatory!bowel!diseases!include!two!disease!phenotypes! known!as!Crohn’s!disease!(CD)!and!ulcerative!colitis!(UC).!CD!and!UC!are!two!distinct!diseases,!but!share!in! part!similar!pathogenesis,!wherein!chronic!gut!inflammation,!reduced!mucosal!barrier!integrity!and! disturbance!in!the!microbiota!have!been!implicated.!An!increase!in!E.!coli!strains,!in!parallel!with!a!decrease! in!anaerobic!gut!commensals,!have!become!a!hallmark!of!an!inflamed!gut!environment!and!might! contribute!to!the!induction!of!persistent!pro@inflammatory!immune!responses.!While!CD!is!described!by!a! typical!Th1/Th17!mediating!immune!response,!UC!is!manifested!by!a!Th2!effector!response,!which!indicates! the!involvement!of!different!disease!triggers!in!CD!and!UC,!respectively.! The!aims!of!this!thesis!were:!1)!to!investigate!if!differences!in!bacterial!adhesion!patterns!to!gut!mucosa!are! disease!associated!and!if!pathogenic!E.!coli!adhesion!can!be!averted!by!non@pathogenic!E.!coli,!2)!to! examine!the!inflammation@inducing!properties!of!clinical!E.!coli!isolates!from!different!intestinal!locations! and!disease!origins,!and!3)!to!study!the!regulatory!effect!of!anaerobic!gut!commensals!and!their!secreted! products!on!the!host!response!to!disease@linked!E.!coli.!! The!main!findings!were:!1)!the!CD@associated!E.!coli!LF82!showed!similar!adhesion!properties!to!gut!mucosa! from!CD!and!healthy!controls.!The!non@pathogenic!E.!coli!Nissle!(ECN)!was!not!able!to!exclude!E.!coli!LF82! adhesion!to!ileal!mucosa!from!CD!or!healthy!controls,!2)!intestinal!E.!coli!isolates,!irrespective!of!their!site! of!location!(ileum/!colon/!faeces),!disease!origin!(diseased/!non@diseased)!or!pathogenicity,!had!the!same! inflammation@inducing!properties!in!dendritic!cells!(DCs)!characterized!by!a!Th1/Th17!driving!effector! response.!However,!two!E.!coli!strains!from!UC!patients!showed!distinct!cytopathic!properties!in!host!cells,! which!were!not!identified!with!the!other!clinical!E.!coli!strains,!and!3)!secreted!products!from!anaerobic!gut! commensals,!but!not!the!bacteria!themselves,!had!a!significant!suppressive!effect!on!the!pro@inflammatory! DC!response!induced!by!pathogenic!E.!coli!LF82.! In!conclusion,!this!PhD!thesis!provides!data!showing!that!intestinal!tissue!from!CD!and!healthy!controls!is! subject!to!similar!E.!coli!adhesion!patterns,!suggesting!that!enhanced!bacterial!adhesion!by!E.!coli!LF82!is! not!linked!to!the!pathogenesis!of!CD.!Moreover,!exclusion!of!E.!coli!LF82!adhesion!to!ileal!mucosa!by!non@ pathogenic!ECN!was!not!proven!to!be!effective;!on!the!contrary,!ECN!exerted!inflammation@inducing! properties,!similar!to!E.!coli!LF82!in!DCs.!Generally,!an!increased!presence!of!E.!coli!in!the!gut!mucosal! immune!compartment!might!enhance!the!risk!of!intestinal!inflammation;!however,!certain!disease@ ! ! 7 associated!E.!coli!strains!may!also!be!involved!in!the!initial!inflammatory!phase.!Finally,!a!homeostatic!gut! environment!comprising!certain!anaerobic!commensals!and!their!fermentation!products!have!a!crucial! function!to!control!E.!coli@induced!inflammation,!and!thereby!possibly!play!a!role!in!sustaining!gut! homeostasis.! ! ! ! ! ! 8 SUMMARY'IN'DANISH! Defektiv!regulering!af!tarmbarrieren!sammen!med!en!ændret!mikrobiel!kolonisering!kan!føre!til!en! overdreven!immun!aktivering.!Inflammatoriske!tarmsygdomme!(IBD)!inkluderer!to!sygdomsfænotyper,! kendt!som!Crohn’s!sygdom!(CD)!og!colitis!ulcerosa!(UC).!CD!og!UC!er!to!forskellige!sygdomme,!men!deler!til! dels!den!samme!patogenese!beskrevet!ved!kronisk!tarm!inflammation,!reduceret!tarm!barriere!integritet! og!ændring!i!mikrobiotaen.!Et!øget!antal!af!E.!coli!stammer,!parallelt!med!en!reduktion!i!anaerobe!tarm! kommensale,!er!blevet!et!kendetegn!for!et!inflammatorisk!tarmmiljø,!og!kan!medvirke!til!et!vedvarende! pro@inflammatorisk!immunrespons.!Mens!CD!er!beskrevet!ved!et!typisk!Th1/Th17!medierende! immunrespons,!er!UC!manifesterede!ved!et!Th2!effektor!respons,!hvilket!indikerer!involvering!af!forskellige! sygdoms@initiatorer!i!hhv.!CD!og!UC.!!! Formålene!med!denne!ph.d.@afhandling!var:!1)!at!undersøge!om!forskelle!i!bakterielle!adhæsionsmønstre!til! tarmvæv!er!sygdoms@associerede,!og!hvorvidt!patogene!E.!coli!adhæsion!kan!hindres!af!non@patogene!E.! coli,!2)!at!undersøge!inflammations@inducerende!egenskaber!af!kliniske!E.!coli!isolater!fra!forskellige! tarmområder!og!sygdoms!oprindelser,!og!3)!at!undersøge!den!regulatoriske!effekt!af!anaerobe!tarm! kommensale!og!deres!secernerede!produkter!på!vært!responset!til!sygdoms@relateret!E.!coli.! Hovedfundne!var:!1)!den!sygdoms@associerede!E.!coli!LF82!udviste!samme!adhæsions!egenskaber!til! tarmvæv!fra!CD!og!raske!kontroller.!Den!ikke@patogene!E.!coli!Nissle!(ECN),!var!ikke!i!stand!til!at!ekskludere! E.!coli!LF82!adhæsion!til!ileal!væv!fra!CD!eller!raske!kontroller,!2)!E.!coli!isolater,!uanset!tarmområde!(ileum,! kolon/!fæces),!sygdoms!oprindelse!(sygdomsramte/raske)!eller!patogenitet,!udviste!de!samme! inflammations@inducerende!egenskaber!i!dendritiske!celler!(DCs)!karakteriserede!ved!et!Th1/Th17!drevet! effektor!respons.!To!E.!coli!stammer!fra!UC!patienter!udviste!cytopatiske!egenskaber!i!værtsceller,!hvilket! ikke!kunne!genfindes!ved!de!øvrige!kliniske!E.!coli!stammer,!3)!secernerede!produkter!fra!anaerobe!tarm! kommensale,!men!ikke!bakterierne!selv,!havde!en!signifikant!suppressiv!effekt!på!det!pro@inflammatoriske! DC!respons,!induceret!af!patogene!E.!coli!LF82.!! Afslutningsvis,!data!fra!denne!ph.d.@afhandling!viser,!at!tarmvæv!fra!CD!og!raske!kontroller!er!udsat!for!de! samme!E.!coli!adhæsionsmønstre,!hvilket!tyder!på!at!øget!bakteriel!adhæsion!af!E.!coli!LF82!ikke!er!linket!til! patogenesen!af!CD.!Desuden,!eksklusion!af!E.!coli!LF82!adhæsion!af!ikke@patogene!ECN!kunne!ikke!påvises,! tværtimod!udviste!ECN!inflammations@inducerende!egenskaber!på!tilsvarende!vis!som!E.!coli!LF82!i!DCs.! Generealt,!øget!tilstedeværelse!af!E.!coli!i!tarmslimhindens!immunsystem!kan!være!med!til!at!øge!risikoen! for!inflammation,!hvor!visse!sygdoms@associerede!E.!coli!stammer!har!potentialet!til!at!være!medvirkende!i! ! ! 9 den!begyndende!inflammatoriske!fase.!Endeligt,!et!homeostatisk!tarmmiljø!bestående!af!visse!anaerobe! kommensale!og!deres!fermenterings!produkter!har!en!afgørende!funktion!i!kontrollering!af!E.!coli@ inducerede!inflammation,!og!dermed!spiller!en!væsentlig!rolle!i!opretholdelse!af!tarm!homeostasen.!!!!!! ! ! ! ! ! 10