Evidence for the involvement of NOD2 in regulating colonic epithelial cell growth and survival

AIM: To investigate the function of NOD2 in colonic epithelial cells (CEC). METHODS: A combination of in vivo and in vitro analyses of epithelial cell turnover in the presence and absence of a functional NOD2 protein and, in response to enteric Salmonella typhimurium infection, were used. shRNA interference was also used to investigate the consequences of knocking down NOD2 gene expression on the growth and survival of colorectal carcinoma cell lines. RESULTS:In the colonic mucosa the highest levels of NOD2 expression were in proliferating crypt epithelial cells. Muramyl dipeptide (MDP), that is recognized by NOD2, promoted CEC growth in vitro . By contrast,the growth of NOD2-deficient CECs was impaired. In vivo CEC proliferation was also reduced and apoptosis increased in Nod2-/- mice, which were also evident following enteric Salmonella infection. Furthermore, neutralization of NOD2 mRNA expression in human colonic carcinoma cells by shRNA interference resulted in decreased survival due to increased levels of apoptosis. CONCLUSION: These findings are consistent with the involvement of NOD2 protein in promoting CEC growth and survival. Defects in proliferation by CECs in cases of CD may contribute to the underlying pathology of disrupted intestinal homeostasis and excessive inflammation.

Characterization of colonic dendritic cells in normal and colitic mice

AIM: Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC.METHODS: Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2-/-) mice that develop colitis.RESULTS: In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c+, CD11b+,B220-, CD8α-) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40,and had high endocytic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN).The majority (＞85%) of DC in the colon and MLN of IL2-/-mice were type 1 myeloid, and expressed high levels of MHC class Ⅱ, CD80, CD86, CD 40, DEC 205, and CCR5molecules and were of low endocytic activity consistent with mature DC.CONCLUSION: These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.