N0d2 Gene Expression in Paneth Cells and Monocytes

Introduction: Mutations in the NOD2 gene are associated with the development of Crohn’s disease, an inflammatory disorder of the gastrointestinal tract. The NOD2 protein induces cellular activation in response to the bacterial antigen muramyl dipeptide (MDP). The NOD2 gene is mainly expressed by circulating blood monocytes although NOD2-associated Crohn’s disease involves mainly the terminal ileum. Paneth cells, which are most numerous in the terminal ileum, are specialised intestinal epithelial cells that secrete antimicrobial peptides in response to bacterial products and are critically important in enteric antibacterial defence. I hypothesised that Paneth cells express the NOD2 gene: this thesis describes the expression and quantification of the NOD2 gene in Paneth cells in inflammatory diseases such as Crohn’s disease and necrotizing enterocolitis. I also identified factors that regulate NOD2 gene expression in intestinal epithelial cells and peripheral blood mononuclear cells (PBMC). Methods: In situ hybridisation and immunohistochemistry were used to localize NOD2 mRNA and protein expression in intestinal tissue. Laser capture microdissection (LCM), and calcium chelation followed by mechanical disruption were used to isolate intestinal crypt and villus epithelial cells. Real-time RT-PCR was used to determine NOD2 gene expression in intestinal epithelial cells and PBMC. Results: NOD2 mRNA and protein expression was readily detected in Paneth cells in normal and Crohn’s disease affected terminal ileum; NOD2 was also expressed by monocytes, but not by mature macrophages in the lamina propria or within granulomas. NOD2 mRNA levels were enriched in isolated crypt compared to villous epithelial cells, and NOD2 expression was mainly detected in LCM-acquired Paneth cells but not villous epithelial cells. In vitro, tumour necrosis factor alpha (TNFa) upregulates NOD2 gene expression in intestinal epithelial cells. There is a possibility that Paneth cell antimicrobials are reduced in patients with NOD2-related Crohn’s disease. Conclusions: Paneth cells express NOD2 and could therefore play an important and hitherto unrecognized role in the development of NOD2-associated Crohn’s disease.