The active phase of Salmonella-associated gastroenteritis along with more chronic states of inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, is characterized histologically by polymorphonuclear leukocyte (PMN) migration into and across the epithelial lining of the intestine. These events result in acute inflammation of the epithelium and subsequent epithelial dysfunction. The degree of PMN transmigration into intestinal crypts and the formation of crypt abscesses is indicative of disease severity and is used clinically to evaluate the activity of IBD. It is unclear what signals induce such directional movement of PMN across the intestinal epithelium. Previously it was speculated that endogenous chemoattractants in the lumen, such as bacterial-derived n-formyl-peptides, triggered such PMN responses perhaps as a consequence of altered permeability characteristics in IBD. However, we have recently shown that epithelial cells themselves can send such signals to underlying PMN and that these signals are regulated by enteric flora, such as S. typhimurium (St). The broad long term objectives of this proposal are to investigate the molecular events by which epithelial cells can signal to PMN and orchestrate their directed migration. Since St and ulcerative colitis share similar idiopathologies, once we understand the basis of such transcellular signaling important in promoting disease flares of St pathogenesis, it may be possible to develop novel therapeutic strategies aimed at treatments for and ameliorating IBD.
The specific aims of this proposal are ultimately directed at achieving this goal, and are three-fold:
Specific Aim 1 is designed to examine events occurring at the interface of St and the apical membrane of intestinal epithelial cells required to generate novel transcellular signals to PMN. Fluorescent/confocal microscopic approaches will be used to identify specific surface events required to initiate the signaling cascade. Ligand binding techniques will be used to probe for epithelial cell surface proteins which interact with St. The third part of this aim will be to functionally screen a panel of specific monoclonal antibodies for their ability to block St-epithelial interactions and/or resulting PMN recruitment. Such data should provide information as to the target molecules on apical membranes of enterocytes whose activation initiates transcellular signaling to PMN.
Specific Aim 2 is designed to identify St strain-related genetic characteristics which permit initiation of mucosal inflammation. Two different genetic approaches will be utilized to allow identification of genetic elements which are required for St to produce clinical gastroenteritis. Current evidence indicates that a tandem of different signals is required to orchestrate PMN transmigration across the lamina propria matrix and subsequently across the epithelium. The first part of Specific Aim 3 is designed to examine whether chemokine gradients are formed across the epithelial matrix, while the second part of this aim is designed to purify and identify a novel PMN chemotactic factor important for directing PMNs across the intestinal epithelium.
