Unique Function of Protein C in Controlling Epithelial Tight Junction Expression and in Regulating Mucosal Healing in Experimental Colitis

Abstract

of NHE3 to the development of colitis, immune activation and mucosal homeostasis in IL10-/mice. Methods: We back-crossed NHE3-/mice to 129/SvEv genetic background for more than ten generations with no apparent changes to their survival or phenotype as compared to the original mixed background. We then crossed them with IL-10-deficient mice on the same genetic background. This double knockout (DK) model allows for the development of an unrestrained activation of the Th-1/Th-17-mediated immune response to further study the consequence of NHE3 loss on the inflammatory process and epithelial integrity. It also represents a conjunction of several key factors implicated in the pathogenesis of IBD: genetic predisposition and loss of immune tolerance to colonic commensal bacteria as well as defective barrier function. Mice at 10 weeks of age were used in the study. Results: Histological examination of H&E stained proximal and distal colon and immunohistochemistry with a Ly-6B.2-specific antibody established important architectural alterations including a massive neutrophil infiltration in IL-10-/xNHE3-/as compared to IL-10-/xNHE3+/+ or IL-10+/+xNHE3-/-. Consistently, we observed increased colonic expression of neutrophil collagenase MMP-8 as well as of the potent mouse neutrophil chemoattractant MIP-2 in DK mice. Colonic IFNγ and IL-17 mRNA expression as well as protein secretion were increased in the distal colon of IL-10-/-xNHE3-/mice compared to NHE3 or IL-10 single KO. Interestingly, IL-10-/-xNHE3-/colonic epithelium had increased hallmarks of apoptosis, including significantly increased number of cleaved caspase-3 positive surface epithelial cells in the proximal and distal colon (>2.5 fold and >3.5 fold increase as compared to NHE-3+/+xIL10-/or NHE3-/-xIL-10+/+, respectively). Conclusions: These results highlight the importance of NHE3 in the maintenance of intestinal integrity and in modulating the inflammatory process in IL-10-deficient mice. These novel observations may be of great importance to the pathogenesis of IBD, where NHE3 inhibition, associated with genetic predisposition such as IL-10R deficiency, may influence the extent of the epithelial barrier defect and contribute to the ultimate degree of inflammation.