The long-term goal of this application is to understand the mechanisms underlying the induction of the inflammatory reaction and breakdown of the epithelial barrier in the cornea upon infection. Recent data has shown that challenging human corneal epithelial (HCE) cells with lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa (PA), a major cause of bacterial keratitis, increases monolayer permeability and alteration of tight junction (TJ) status. LPS and PA challenges also lead to activation of NF-kappaB, a transcription factor activated by Toll-like receptor 4 (TLR4, the predominant LPS receptor in mammals). Furthermore, PA infection resulted in the loss of epithelial barrier function in cultured pig corneas. The current application will test the hypothesis that in the cornea TLRs confer responsiveness of HCE cells to pathogens, and PA challenge-induced TLR signaling, through activation of NF-kappaB and/or mitogen-activated protein kinase (MAPK), contributes to infection-induced epithelial barrier breakdown. The following studies will be carried out. (1) Alterations of barrier properties in corneal epithelial cells upon PA infection will be assessed using HCE cells cultured on Transwell filters as a model epitheliuin. Parameters to be measured include transepithelial electrical resistance, and paracellular flux, alterations in TJ proteins ZO-1 and ZO-2. (2) Signal transduction pathway(s) that couples PA challenge to alteration of epithelial barrier function in HCE cells will be characterized using antibodies to detect cell surface expression of TLRs and co-receptor CD 14, and pharmacological reagents to inhibit activation of NF-kappaB and MAPK upon HCE cell infection. (3) Whether infection-induced epithelial responses, including barrier breakdown, can be inhibited by modification of TLR-mediated signaling will be assessed. A combination of transfection of TLRs and their mutants and the neutralizing antibodies will be used to assess epithelial response to PA challenge in Transwell model epithelium. TLR neutralizing antibodies, along with other biological and pharmacological reagents, will also be applied to corneal organ culture to determine if infection-induced epithelial responses and barrier breakdown can be inhibited. An understanding of how TLRs transmit signals that lead to epithelial responses, including modulation of barrier function, may allow the development of therapeutic agents that prevent breakdown or enhance recovery of barrier function during infection and, as an adjuvant therapy, eliminate the corneal scarring and vision loss associated with bacterial keratitis.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
7R01EY014080-04
Application #
6792197
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Shen, Grace L
Project Start
2001-09-30
Project End
2007-08-31
Budget Start
2004-09-01
Budget End
2007-08-31
Support Year
4
Fiscal Year
2004
Total Cost
$302,000
Indirect Cost
Name
Wayne State University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
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Xu, Ke-Ping; Yu, Fu-Shin X (2007) Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing. Invest Ophthalmol Vis Sci 48:2242-8
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Kumar, Ashok; Zhang, Jing; Yu, Fu-Shin X (2006) Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology 117:11-21
Yu, Fu-Shin X; Hazlett, Linda D (2006) Toll-like receptors and the eye. Invest Ophthalmol Vis Sci 47:1255-63
Xu, Ke-Ping; Yin, Jia; Yu, Fu-Shin X (2006) SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells. Invest Ophthalmol Vis Sci 47:2832-9

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