INTESTINAL INFLAMMATION ORCHESTRATED BY PATHOGENS

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The active phase of both Salmonella-associated gastroenteritis and chronic states of inflammatory bowel disease IBD), 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 triggers directional movement of PMN across the intestinal epithelium. Towards this end, we have recently shown that epithelial cells themselves can send such signals to underlying PMN and these signals are regulated by enteric flora, such as S. typhimurium. The broad long term objectives of this proposal are to investigate the molecular mechanism by which epithelial cells in response to microbial pathogens can signal to PMN and orchestrate their directed migration. Once we begin to understand the basis of such transcellular signaling important in promoting disease flares of S. typhimurium pathogenesis, it may be possible to develop novel therapeutic strategies aimed at treatments for and ameliorating IBD. The specific aims are ultimately directed at achieving this goal, and are three-fold. Specific Aim 1 is designed to determine the nature of S. typhimurium virulence factors and define their contribution to the epithelial orchestration of mucosal inflammation. Specifically, we will delineate how S. typhimurium SipA, SopB, and SopA secreted proteins interfere with the signaling pathways which lead to epithelial orchestration of mucosal inflammation by expression of these proteins in epithelial cells using adenoviral expression vectors. Functional effect of expression of these proteins on orchestration of proinflammatory events which govern PMN transepithelial migration will be correlated with morphological consequences by both confocal and electron microscopy. Specific Aim 2 is designed to identify the signal transduction cascades which lead to the release of the proinflammatory chemoattractant PEEC and will employ several different approaches which include determining the relationship between S. typhimurium invasion and the apical epithelial release of PEEC, examination of the role of the JNK-pathway, determining the effects of small GTPase (cdc42, rac-1, and Arf6) expression on the ability of S. Typhimurium to induce PMN transepithelial migration by expression of dominant inhibitory mutants using adenoviral expression vectors, examining the role of phosphinositide signaling, and determining whether the ability of S. typhimurium to elicit PEEC secretion correlates with their ability to induce an increase in intracellular calcium in model intestinal epithelia. Specific Aim 3 is designed to characterize a recently identified pro-inflammatory PMN chemoattractant. The first part of this aim will elucidate the structure of PEEC utilizing HPLC purification, NMR analysis, mass spectrometry and sequence analysis, while the second part of this aim will define PEEC's relationship to other PMN chemoattractants including its ranking in the PMN chemoattractant hierarchy, will determine whether PEEC is able to activate other immune-type cells as well as assess the role of PEEC in inflammation.