The Toll Like Receptor (TLR) family of pathogen recognition molecules plays a critical role in recognizing and responding to potential pathogens, initiating anti-microbial inflammatory responses that often result in tissue damage. TLRs also respond to microbial products in the absence of live bacteria, and can induce an inflammatory response. The central hypothesis of the previous and current proposals is that TLR activation in the cornea results in development of neutrophil infiltrates, corneal opacities, and subsequent loss of corneal clarity. In addition to the importance of understanding these responses in relation to active bacterial infection, it is highly likely that this process is involved in sterile forms of keratitis associated with contact lens wear, including peripheral ulcer, red eye and corneal infiltrates. Studies described in the current proposal will focus on identifying key regulatory mediators of LPS / TLR4 activation in the cornea using in vitro analysis of human corneal epithelial cells (HCEC) in addition to murine models of corneal inflammation.
Aim 1 will examine the MD-2 co-receptor for TLR4, and will focus on the role of intra-epithelial dendritic cells, and regulation of MD- 2 expression by HCEC.
Aim 2 will examine the sub-cellular responses to LPS activation, focusing on the role of TIR containing adaptor molecules and MAP kinases.
Aim 3 will examine the role of these adaptor molecules in Pseudomonas aeruginosa keratitis, and will examine and will correlate P. aeruginosa and S. marcescens Lipid A composition with TLR4/MD-2 reactivity In addition to increasing our understanding of how the corneal epithelium responds to LPS and other bacterial products, results from the proposed studies will identify potential cell surface and intracellular candidates for targeted anti-inflammatory intervention.
Bacterial keratitis is a serious cause of visual impairment in the USA and worldwide, and contact lens wear is a major risk factor. Given the very large number of contact lens wearers in the USA (34 million) and worldwide (~140 million), even a low percentage of side effects translates into a large number of affected individuals. The Toll Like Receptor (TLR) family of pathogen recognition molecules plays a critical role in recognizing and responding to potential pathogens, initiating anti-microbial inflammatory responses that often result in tissue damage. TLRs also respond to microbial products in the absence of live bacteria, and can induce an inflammatory response in the cornea. Proposed studies will focus on signaling induced lipopolysaccharide (LPS, endotoxin), which activates TLR4 and is the major stimulatory component of Gram negative bacteria, and will examine intracellular responses in human corneal epithelial cells and in a murine model of corneal inflammation. Proposed experiments will also examine TLR responses to Pseudomonas aeruginosa and Serratia marcescens, which cause bacterial keratitis. Results of the proposed studies will also identify potential cell surface and intracellular candidates for targeted anti-inflammatory intervention.
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