The Role of Toll-Like Receptors in the Pathogenesis of Dry Eye

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DESCRIPTION (provided by applicant): The primary objective of the proposed research plan is to provide the Principal Investigator (PI) with the comprehensive didactic and laboratory training necessary to develop into an independent researcher in ocular surface inflammation. The multidisciplinary training plan will be under the guidance of mentors Alison McDermott, PhD and Stephen Pflugfelder, MD. The PI is an Optometrist with a background in corneal immunology and this proposed research program will expand upon the her previous scientific experience through studies to investigate the role of Toll-like receptors (TLRs) in ocular surface inflammation in dry eye syndrome. The activation of TLRs has been shown to stimulate the production of cytokines and chemokines, initiating an inflammatory reaction. The scientific focus of this proposal will investigate the general hypothesis that TLRs stimulate inflammation in dry eye syndrome. The specific aims are: (1) To establish TLR 2, 4, 5 and 9 expression pattern in human conjunctival epithelial cells from subjects with mild, moderate, and severe dry eye, Sjogren's syndrome, and age matched normal subjects. Conjunctival impression cytology samples will be collected from human subjects and TLR mRNA and protein expression will be determined by real time RT-PCR and flow cytometry respectively; (2) To determine the expression and localization pattern of TLR 2, 4, 5 and 9 in primary human conjunctival and corneal epithelial cells and in normal human conjunctival and corneal epithelial cell lines in response to hyperosmolar stress and cytokine treatment by real time RT-PCR and confocal microscopy; (3) To compare the TLR 2, 4, 5 and 9 expression pattern among control C57BL/6 mice with experimental dry eye (EDE) induced by desiccating stimulus C57BL/6 mice. Additionally this specific aim will determine if EDE TLR 2, 4, and 9 deficient mice have reduced ocular surface inflammation as measured by a.) a reduction in MMP-9 and cytokine (IL-1beta and TNF-alpha) mRNA and protein expression and b.) epithelial cell apoptosis and goblet cell loss compared to wild type EDE C57BL/6 mice. These studies will help understand the role of TLRs in the pathogenesis of dry eye while also providing the PI with a training program to develop the skills necessary to become a successful independent investigator.