title: Microbial induction of CARD15 expression in intestinal epithelial cells via toll-like receptor 5 triggers an antibacterial response loop.
abstract: With the discovery of CARD15 as susceptibility gene for Crohn's disease (CD) a first link to a potential defect in the innate immune system was made. In this work we aimed to analyze enterocyte NOD2/CARD15 expression and regulation in response to bacterial motifs and the consequences of the most common CD-specific CARD15 mutation on antibacterial responses of normal intestinal epithelial cells (IEC). Under normal conditions, IEC lines and ileal enterocytes did not express NOD2/CARD15 mRNA or protein, contrary to IEC derived from inflammatory CD sections. In vitro analyses revealed that the simple contact with non-pathogenic commensal E. Coli K12 was sufficient to induced NOD2/CARD15 mRNA and protein in human IEC (HIEC). We identified bacterial flagellin interacting with TLR5 as major motif in this regulation of NOD2/CARD15. E. Coli mutants not expressing flagellin (DeltaFliC) failed to induce CARD15. Similarly, in HIEC transfected with a plasmid encoding dominant negative TLR5, no CARD15 induction was observed after K12 contact. Isolated TLR2 or TLR4 stimulation had no or only a marginal effect on NOD2/CARD15 expression. NOD2/CARD15 negative HIEC were unresponsive to muramyl dipeptide (MDP), but once NOD2/CARD15 was induced, HIEC and Caco2 cells responded to intra or extracellular MDP presentation with the activation of the NFkB pathway. IEC transfected with the Crohn-specific CARD15 mutant (F3020insC, FS) failed to activate NFkB after MDP-challenge, in contrast to CARD15WT IEC. In response to MDP, IEC induced a massive antibacterial peptide (ABP) response, seen in the apical release of CCL20. This was completely abolished in IEC carrying CARD15FS. These data suggest a critical role of NOD2/CARD15 in the bacterial clearance of the intestinal epithelium while CD-specific mutated NOD2/CARD15 causes an impaired epithelial barrier.
authors.affiliation: INSERM, U793, Paris, France.
authors.forename: B
authors.initials: B
authors.lastname: Begue
authors.forename: C
authors.initials: C
authors.lastname: Dumant
authors.forename: J C
authors.initials: JC
authors.lastname: Bambou
authors.forename: J F
authors.initials: JF
authors.lastname: Beaulieu
authors.forename: M
authors.initials: M
authors.lastname: Chamaillard
authors.forename: J P
authors.initials: JP
authors.lastname: Hugot
authors.forename: O
authors.initials: O
authors.lastname: Goulet
authors.forename: J
authors.initials: J
authors.lastname: Schmitz
authors.forename: D J
authors.initials: DJ
authors.lastname: Philpott
authors.forename: N
authors.initials: N
authors.lastname: Cerf-Bensussan
authors.forename: F M
authors.initials: FM
authors.lastname: Ruemmele
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000378:metabolism; D020816:Amino Acid Motifs; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism; D018938:Caco-2 Cells; D002478:Cells, Cultured; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology*; D004926:Escherichia coli / Q000378:metabolism*; D005786:Gene Expression Regulation; D019073:HT29 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000378:metabolism*
chemical_list: D023181:Antimicrobial Cationic Peptides; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger; D051198:Toll-Like Receptor 5; D000119:Acetylmuramyl-Alanyl-Isoglutamine