Project 1 Mutations of CARD15, a gene encoding NOD2, are susceptibility factors in Crohn?s disease. We observed previously that the peptidoglycan-induced IL-12 response of antigen-presenting cells (APCs) from NOD2-deficient mice is increased. Nevertheless, NOD2-deficient mice do not develop spontaneous colitis, suggesting that enhanced innate immunity alone is not sufficient for induction of colitis. In this study, we investigated the effect of NOD2 deficiency on adaptive immune responses specific to an antigen. We show that ovalbumin (OVA)-presenting NOD2-deficient APCs stimulated with peptidoglycan enhances IFN-_amma production by co-cultured OVA-specific CD4+ T cells and reduces apoptotic cell death of the latter cells. Similarly, APCs cultured with Esherichia coli expressing OVA (ECOVA) induce enhanced IFN-_ production by OVA-specific CD4+ T cells. Then, in in vivo studies, we demonstrate that NOD2-deficient mice adoptively transferred OVA-specific CD4+ T cells and administered intra-rectal ECOVA, but not control Esherichia coli, develop severe colitis. The latter was marked by the clonal expansion of OVA-specific CD4+ T cells in mesenteric lymph nodes and colonic lamina propria producing large amounts of IFN-_amma. These findings indicate that interacting innate and adaptive immune responses to commensal organisms are required for the development of colitis in NOD2 deficiency. Project 2: In previous studies we showed that ulcerative colitis (UC) is characterized by an atypical Th2 immune response characterized by NKT cell production of IL-13. It remained unclear, however, how such secretion leads to epithelial dysfunction. In intial studies we showed that lamina propria mononuclear cells from patients with UC produce far greater amounts of IL-13 (985 +/- 73 pg/mL) than comparable cells from controls or patients with Crohn's disease. In studies of the effect of such IL-13 on epithelial cell function we performed extensive studies of IL-13 on HT-29/B6 epithelial cell monolayers. First we showed that IL-13 had a dose-dependent effect on transepithelial resistance of the monolayers (reduction to 60% +/- 4%), whereas IL-4 had no such effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally to the change in resistance. Second, we showed that IL-13 caused a decrease in epithelial restitution velocity from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h. Parallel changes were observed in human samples in that the latter manifested a 9-10-fold increase in claudin-2 expression. These clearly show that IL-13 impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity; thus, its over-secretion in UC helps explain the epithelial cell dysfunction in this disease.
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