CARD15/NOD2 Functions as an Antibacterial Factor in Human Intestinal Epithelial Cells

Gastroenterology. 2003 Apr;124(4):993-1000. doi: 10.1053/gast.2003.50153.

Abstract

Background & aims: 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Caco-2 Cells
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Enterobacteriaceae*
  • Epithelial Cells / microbiology
  • Epithelial Cells / physiology
  • Gene Expression / physiology
  • HT29 Cells
  • Humans
  • Inflammatory Bowel Diseases / microbiology
  • Inflammatory Bowel Diseases / physiopathology
  • Intestinal Mucosa / cytology
  • Intestinal Mucosa / microbiology*
  • Intestinal Mucosa / physiology*
  • Intracellular Signaling Peptides and Proteins*
  • Nod2 Signaling Adaptor Protein
  • RNA, Messenger / analysis
  • Salmonella Infections / microbiology
  • Salmonella Infections / physiopathology
  • Salmonella typhimurium

Substances

  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • NOD2 protein, human
  • Nod2 Signaling Adaptor Protein
  • RNA, Messenger