Identification of proteases involved in pathogenesis of inflammatory bowel disease, deciphering molecular pathways altered by their hyperactivity – IBD-ase

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders of the gut, which are characterized by an uncontrolled inflammatory response to lumenal content. The main forms of IBD are Crohn’s disease and ulcerative colitis. Only palliative care is available to the several million patients afflicted with IBD worldwide, and the short-term goal of medical treatments is to bring symptoms under control to prevent complications. The mechanisms of IBD pathogenesis are not fully understood, and the development of more effective, ideally curative treatments for IBD and other inflammatory diseases depends upon a better understanding of the regulation of the inflammatory response.
Several studies have demonstrated a crucial role for proteases, notably trypsin-like and elastolytic activties, in the maintenance of a chronic inflammatory response of the gastrointestinal tract and associated pain. Therefore, the control of the protease/anti-protease balance appears to be crucial to the development of IBD. The potential therapeutic benefits of protease inhibitors are highlighted by the association of over-expression of the elastase inhibitor, ELAFIN, with a strong protective effect against colitis.
Since several years, I have carried out research programs on the pathophysiology of a genodermatosis, Netherton syndrome (NS), where defective control of protease activity leads to severe dysregulation of inflammation and immunity. We have dissected the respective contribution of three hyperactive serine proteases to the NS phenotype, and we identified elastase 2 (ELA2) as a new epidermal protease whose expression has not been reported in any epithelium previously.
The present project proposes to investigate the regulation of protease activity in chronically inflamed bowel diseases, based on my expertise on the functional characterization of proteases, and on the strong rationale recently presented for a role of proteases in IBD.
Our preliminary data indicate that, in both human and mice, ELA2 is expressed in the monolayer intestinal epithelium under physiological and ulcerative conditions. Moreover, the elastolytic activity is increased in monolayer intestinal epithelial cells during colitis. Consequently, further characterization of ELA2 hyperactivity will focus on its capability to induce pro-inflammatory mediators. To provide an in vivo demonstration of the involvement of ELA in colitis, an ELA2 conditional knockout mouse model will be developed. In addition, the protein targets of ELA2 activity will be identified by using an innovative proteomic approach (ICAT, Isotope-Coded Affinity Tag). This study should bring forth new insights into bowel homeostasis, and reveal new therapeutic targets for IBD treatment.
The project will be extended to the identification of hyperactive proteases released within the intestinal tissue of IBD patients. Proteolytic activities will be detected by zymographic analysis of gel-fractionated protein extracts, and then assigned to specific proteases using mass spectrometry. The characterization of the hyperactive proteases hyperactive in the inflamed gut, their substrates, and the consequences of their increased activity will advance our knowledge of the biological cascades initiated by protease dysregulation in IBD. We expect our analysis of protease hyperactivity to identify control points in the mechanisms of disease that could be addressed by the development of specific inhibitors, as the basis of novel therapeutic approaches for IBD.