Activated immune and inflammatory cells that are in intimate contact with the epithelium lead to changes in the absorptive, secretory and barrier functions of the epithelium. Barrier function is quite important as the epithelium normally separates potentially noxious stimuli in the lumen from the underlying tissue. Therefore, loss of this function is important in the pathogenesis of peptic ulcers or colitis caused by luminal acid or flora respectively. The general hypothesis is that immune-mediated damage to gastrointestinal epithelial cells is an important component of a multi-factorial process that culminates in gastrointestinal disease associated with chronic inflammation. Evidence exists showing that, during infection with H. pylori, lymphoepithelial interactions enhance local oxidative stress that induces a redox-sensitive signaling pathway leading to epithelial cell death by apoptosis. Therefore, the specific hypothesis for these proposed studies is that gastric mucosal immune responses modify the expression of pro-apoptotic genes that contribute to epithelial cell damage through the induction of apoptosis. While substantial evidence in support of this hypothesis has been obtained, one of the challenges is to validate the in vitro evidence with in vivo studies. The advent of new technologies to isolate discrete subsets or even individual cells combined with advances in gene array techniques, make it possible to develop innovative approaches to study molecular immunopathogenesis in vivo. In the current proposal, we outline a plan to develop unique methods to isolate human epithelial cells and to characterize the regulation of gene expression by local immune mechanisms that lead to epithelial cell death. We propose a multidisciplinary approach combining expertise in rare-event analysis and gene expression with mucosal immunity. The hypothesis will be examined in the following specific aims: (1) Aim 1 will define the expression of redox-sensitive, pro-apoptotic genes in isolated epithelial cells; and (2) Aim 2 will compare acute versus chronic changes in epithelial gene expression at the single-cell level. This project aims to develop techniques in rare- event analysis that will become an integral part of research on the mucosal immunopathogenesis of gastrointestinal disease in humans. The molecular mechanisms controlling the susceptibility of epithelial cells will be defined on the single cell level and set the stage for future studies on the role for T cells in regulating the expression of genes that define the susceptibility of the host to disease.
