Microbial induction of CARD15 expression in intestinal epithelial cells via toll-like receptor 5 triggers an antibacterial response loop.
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.
Begue
B
B
INSERM, U793, Paris, France.
Dumant
C
C
Bambou
J C
JC
Beaulieu
J F
JF
Chamaillard
M
M
Hugot
J P
JP
Goulet
O
O
Schmitz
J
J
Philpott
D J
DJ
Cerf-Bensussan
N
N
Ruemmele
F M
FM
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*
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