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J. J. Mun, C. Tam, D. J. Evans, S. M. J. Fleiszig; Genomic Analysis of Corneal Epithelial Cell Responses to Human Tear Fluid and Pseudomonas aeruginosa Antigens. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3893.
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Healthy corneas are resistant to infection by Pseudomonas aeruginosa. We have shown that human tear fluid can protect corneal epithelial cells against P. aeruginosa invasion, cytotoxicity and traversal. Since tear pre-exposure is sufficient for the protective effect, the data suggest tear-modulation of epithelial cell defenses. As a first step toward deciphering the mechanism of tear protective activity, we explored how gene expression in human corneal epithelial cells is impacted by human tear fluid.
Human corneal epithelial cells were incubated with human tear fluid or cell culture media for 6 h, or to tear fluid or media for 16 h followed by 3 h incubation with P. aeruginosa culture supernatant (antigens). Total RNA was isolated from the epithelial cells, first strand cDNA synthesized and used to generate cRNA. The cRNA was fragmented and applied to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays (University of California, Berkeley Functional Genomics Laboratory). Genes upregulated by at least 10-fold compared to control were categorized by function and pathway using Onto-Express and Pathway-Express programs, on the Intelligent Systems and Bioinformatics Laboratory database (or the NCBI database).
Tear fluid alone (6 h) upregulated 76 genes in corneal epithelial cells by 10-fold or more. Pre-exposure to tear fluid alone (16 h) followed by exposure to P. aeruginosa antigens upregulated 47 genes. Both groups of upregulated genes contained significant numbers encoding transcriptional regulators, cytokine receptors, proteases and protease inhibitors, transmembrane-, structural-, and tumor-related proteins, and an antimicrobial protein. Some upregulated genes were involved in Jak-STAT signaling, cytokine-cytokine receptor interactions, cancer biology, tight junctions, and Toll-Like Receptor and MAPK signalling.
Tear fluid is likely to play multiple roles in defending the ocular surface against infection including direct effects on microbial virulence, and indirect effects such as modulating the antimicrobial defenses of the corneal epithelium. We have found that the expression of many genes is impacted by tear fluid exposure. Determining which gene products play a role in tear induced defense against infection will be the next stage of this project.
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