The intestinal epithelium represents a primary physical barrier, and is the first line of defense against ingested toxins and bacterial products. In addition, a growing body of evidence suggests that intestinal epithelial cells (IEC) actively participate in gut inflammation. Dysregulation of epithelial function can disrupt the homeostasis of normal mucosal immunity and drive adaptive immune responses to a pathogenic disease state. In the last funding period, we demonstrated that epithelial phenotypic alterations and increased small intestinal permeability occur early, before the onset of inflammation in SAMP1/YitFc (SAMP) mice. In addition, bone marrow chimeras (BMCs) of irradiated SAMP mice reconstituted with control AKR bone marrow exhibit significant ileitis, suggesting that an innate defect in epithelial function may represent the primary source of disease susceptibility in SAMP mice. The central hypothesis of Project 5 is that dysregulation of intestinal epithelial barrier function leads to aberrant innate immune responses that drive the activation of pathogenic effector cells, resulting in chronic intestinal inflammation similar to that observed in Crohn's disease (CD). To test this hypothesis, we will perform three specific aims: 1) Determine if the increase in small intestinal permeability of SAMP mice is a consequence of environmental factors or genetic predisposition. In vivo and in vitro analyses of epithelial permeability will compare SAMP mice raised under germ-free conditions to SPF-raised mice. We will also determine whether a genetic component contributes to the susceptibility of increased small intestinal permeability, utilizing consomic mice generated by Project 2 that carry BL76 chromosomal intervals associated with disease severity on the SAMP background. Finally, to facilitate fine gene mapping of disease susceptibility genes, Affymetrix chip technology will be used to compare the relative expression of epithelial-derived genes from isolated IEC of consomic and native SAMP mice. 2) Characterize the relative contribution of epithelial tight junction (TJ) and adherens junction (AJ) proteins comprising the apical junctional complex (AJC) in SAMP mice, and if alteration in their expression affects ileitis. Based on our preliminary findings, we will characterize the TJ proteins ZO-1, claudins 1-4 and occludin, and the AJ proteins E-cadherin and beta-catenin, using real-time RT-PCR, Western blotting and immunohistochemical techniques. In addition, consomic mice that possess BL/6 chromosomal intervals containing genes of the AJC will be evaluated for expression of TJ and AJ proteins, as well as the state of intestinal inflammation. 3) Evaluate the specific role of hematopoietic cells in the development of ileitis. in the presence of epithelial barrier dysfunction. Phenotypic and functional characterization will be performed on immune cells from BMCs, and the ability of specific T/B cell populations to adoptively transfer disease will be determined. Finally, we will investigate whether ileitis and activation of pathogenic adaptive immune responses are a consequence of uncontrolled translocation of microbial products across a leaky epithelial barrier. The overall goal of Project 5 is to elucidate the precise role of the epithelium and barrier dysfunction in the pathogenesis of chronic ileitis, and to facilitate the design of future therapeutic strategies based on manipulation of the intestinal epithelial barrier.
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