CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells.
OBJECTIVE
Mutations in the CARD15/NOD2 gene, a putative intracellular pattern recognition receptor, have been linked to the risk for Crohn's disease. Because intestinal epithelial cells play a role as the barrier to luminal microorganisms, we investigated the expression and function of CARD15/NOD2 in intestinal epithelial cells.


METHODS
Expression of CARD15/NOD2 messenger RNA (mRNA) in intestinal epithelial cell lines and primary intestinal epithelial cells was assessed by reverse-transcription polymerase chain reaction (RT-PCR). Regulation of expression of CARD15/NOD2 by cytokines was determined by Northern blot using the SW480 cell line. Active CARD15/NOD2 protein in SW480 cells was assessed by the combination of immunoprecipitation and immunoblotting using anti-CARD15/NOD2 antisera. To identify the functional role of CARD15/NOD2 in intestinal epithelial cells, gentamicin protection assays of Salmonella typhimurium were performed using Caco2 cells stably transfected with either wild-type CARD15/NOD2 or the 3020insC mutant associated with Crohn's disease.


RESULTS
CARD15/NOD2 mRNA was expressed in both intestinal epithelial cell lines and primary intestinal epithelial cells. CARD15/NOD2 mRNA and protein were up-regulated by tumor necrosis factor alpha (TNFalpha) in SW480 cells. The number of viable internalized S. typhimurium in Caco2 cells stably transfected with CARD15/NOD2 expression plasmid was lower than untransfected Caco2 cells or MOCK transfectant. In contrast, expression of a variant associated with Crohn's disease was unable to constrain bacterial survival.


CONCLUSIONS
CARD15/NOD2 is expressed in intestinal epithelial cells and may serve as a key component of innate mucosal responses to luminal bacteria as an antibacterial factor. Failure in this activity may contribute to the development of Crohn's disease.
Hisamatsu
Tadakazu
T
Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Suzuki
Manabu
M
Reinecker
Hans-Christian
HC
Nadeau
William J
WJ
McCormick
Beth A
BA
Podolsky
Daniel K
DK
D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004755:Enterobacteriaceae; D004847:Epithelial Cells / Q000382:microbiology / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000382:microbiology / Q000503:physiopathology; D007413:Intestinal Mucosa / Q000166:cytology / Q000382:microbiology* / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger / Q000032:analysis; D012480:Salmonella Infections / Q000382:microbiology / Q000503:physiopathology; D012486:Salmonella typhimurium
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger