Toll-like receptors and intestinal inflammation

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The human gut harbors a large collection of commensal microbes. Several clinical observations and animal experiments suggested that intestinal bacteria play a major role in the pathogenesis of chronic bowel inflammation. Enteroinvasive pathogens such as Salmonella, Shigella, Yersinia, and Listeria can invade the epithelium and provoke inflammatory responses. Flagellin, a major component of bacterial flagellar, is released from various bacteria, including commensal or enteroinvasive microbes, and stimulates Toll-like receptor (TLR) 5 that is highly expressed in intestinal epithelium. Our recent results showed in non- transformed human colonocytes, as well as normal human colon that flagellin stimulation specifically induces proinflammatory gene expression, a prominent characteristic of the pathophysiology of IBD. In spite of these findings, the mechanisms by which bacterial flagellin signals proinflammatory responses in colonocytes and native colonic mucosa remain poorly understood. The central hypothesis of this proposal is that compromised epithelial barrier function, either by injury or by erosive pathogens, enables bacterial flagellin to penetrate the leaky epithelium and activate basolateral TLR5 leading to colonic inflammation. Our goals are to elucidate the signaling pathways by which flagellin exert its proinflammatory action(s) in human colonocytes and examine the participation of bacterial flagellin in the development and progress of colonic inflammation in mouse colitis models. Aim 1 will study the signal transduction pathway(s) leading to IL-8 and MIP-3a gene expression in response to flagellin in non-transformed human colonic NCM460 cells. In this aim we will identify the specific kinase(s) involved in MEK phosphorylation in response to flagellin exposure and examine their involvement in TLR5-dependent IL-8 and MIP-3a gene expression. Experiments to determine the signaling mechanism(s) by which the phosphatase MKP-1 amplifies flagellin-induced proinflammatory responses are also proposed. Aim 2 will examine the role of PI-3K signaling in TLR5-associated IL-8 and MIP-3a gene expression in human colonocytes. Aim 3 will examine the role of flagellin in the progress of colitis using mouse models of colitis and determine whether the basolateral or luminal aspect of the human and mouse colonic mucosa is responsive to flagellin. Our proposed studies will provide significant information relevant to the pathogenesis of colonic inflammation, including IBD.