T1769 Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A Novel Role in Intestinal Inflammation

Abstract

Background and aims: Mucosal immunity and epithelial integrity are interconnected factors involved in intestinal homeostasis. The hedgehog (Hh) family of morphogens is essential in embryogenesis, but it has been also associated with inflammation and tissue repair. We hypothesized that the Hh-pathway could affect the inflammatory response and cell survival, crucial factors in IBD pathogenesis. Methods: Endoscopic biopsies were obtained from the inflamed colon of 14 patients with Crohn's disease (CD), 12 ulcerative colitis (UC), and 15 controls. Frozen sections of mucosal samples were analyzed by immunohistochemistry using antibodies against Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Gli-1. Double immunofluorescence was used for co-localization studies. RT-PCR was used for analyzing the expression of Gli-1 mRNA. Tissue cultures of colon samples were performed during 24h in the presence of Shh peptide, anti-Shh antibody, cyclopamine, a specific Hh inhibitor, or diluent. Supernatants were analyzed for cytokine production, by ELISA, and total protein was extracted from tissue for caspase-3 activity assay. Results: CD colonic mucosa showed a lower expression of Gli-1 mRNA compared to UC and controls (p=0.03). Hh-proteins were characteristically expressed at the superficial epithelium, and a marked reduction was observed in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria, low densities of Hh-family members basically do not co-localize with infiltrating T-cells or macrophages. Baseline levels of cytokines in culture supernatants were higher in IBD compared to controls. In CD colon explants, treatment with Shh peptide resulted in a significant decrease in the levels of TNF-alpha (p=0.04), IL-17 (p=0.01), and TGF-beta (p=0.03). Caspase-3 activity was higher in CD and significantly decreased after blockade of Hh, either with anti-Shh antibody(p=0.04) or with cyclopamine (p=0.03), in contrast to UC and controls. Levels of Gli-1 negatively correlated to caspase-3 activity (CC -0.75, p=0.01). Conclusion: Normal colonic mucosa constitutively expresses Hh-proteins, which appear to constitute a key pathway for intestinal homeostasis. Hh-signaling affects cytokine production and the control of apoptosis within the colonic mucosa. Disruption of the intestinal Hhpathway might be implicated in the pathogenesis of CD.