Chalmers-Watson, T.A.; (2009) The role of the NOD2 gene in the pathogenesis of Crohn’s disease. Doctoral thesis , UCL (University College London).

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Abstract

Introduction: Crohn’s disease is characterised by an abnormal inflammatory response possibly induced by components of enteric bacteria, in genetically susceptible individuals. Mutations in the NOD2 gene are strongly associated with Crohn's disease, although the mechanisms by which these mutations cause Crohn’s disease remain unknown. Peripheral blood monocytes (PBMC), a key component of the innate immune system, highly express NOD2, as do intestinal epithelial Paneth cells. The ligand for NOD2 is Muramyl dipeptide (MDP), a component of bacterial peptidoglycan. MDP has been shown to be a powerful priming agent for subsequent stimulation by lipopolysaccharide (LPS) in cell lines and this effect has been linked to NOD2. Studies of primary mononuclear cells in Crohn’s disease comparing the functional effects of the NOD2 mutations had not previously been reported. Aims: To determine the effect of inherited mutations in the NOD2 gene on the cellular responses of freshly extracted PBMC to MDP and other bacterial ligands including mycobacteria. To examine the effect of MDP pre treatment ‘priming’ on PBMC responses to subsequent LPS stimulation in both normal and Crohn’s disease affected patients expressing wild type and mutant NOD2 proteins. Methods: PBMC from healthy controls (n=12), and Crohn’s disease affected patients who were genotypically either wild type (n=12), heterozygous (n=11) or homozygous (n=5) for the common disease-causing NOD2 mutations. PBMC were stimulated with bacterial products in vitro, with or without prior stimulation or ‘priming’ with MDP. The transcription of selected cytokine genes was determined by real time quantitative RT-PCR Results: MDP is a weak stimulant of inflammatory responses in PBMC whereas LPS evoked much stronger responses. Responses to MDP were particularly reduced in PBMC homozygous for the NOD2 mutations. Priming with MDP reduced the inflammatory response of normal PBMC to subsequent LPS stimulation. In PBMC carrying two mutant NOD2 alleles this modulatory effect was reversed and MDP priming caused the inflammatory response to be enhanced. Conclusion: MDP priming significantly modulates responses of monocytes to LPS. This effect is altered in patients with Crohn’s disease – possibly related to mutations in the NOD2 gene. This modulatory effect may explain in part the pro-inflammatory consequence of mutations in the NOD2 gene and could provide mechanistic understanding of how mutations in the NOD2 gene may cause Crohn’s disease.

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