Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic organism associated with bacterial keratitis, especially in extended wear contact lens users. Our goal is to determine the molecular mechanisms for development of bacterial keratitis, specifically, the role of Toll-like receptor (TLR) 4, which at present remains underexplored in the eye. We hypothesize that TLR4 plays a critical role in the innate immune response to P. aeruginosa in the cornea and that the activation and signaling through this TLR will regulate disease outcome.
Three specific aims are proposed: 1) To test the hypothesis that TLR4 regulates inflammatory angiogenesis in the infected cornea. 2) To test the hypothesis that TLR4 regulates apoptosis in the infected cornea. 3) To test the hypothesis that TLR4 regulates antimicrobial peptides in the infected cornea. Experiments will include use of techniques such as real time RT-PCR, short interfering RNA (siRNA), as well as plate counts, enyme linked immunosorbant assay (ELISA), myeloperoxidase assay (MPO) for neutrophil (PMN) quantitation, dual antibody immunostaining, histopathology, Tunel assay, DNA laddering, NF?B functional assays and Western blotting. Our long-term objective is to understand the interactions between bacteria and the immune response in the mouse cornea so that non-conventional therapies against keratitis can be developed for human patients. Since in the United States alone the incidence of microbial keratitis is 25,000-30,000 cases annually, with cost of treatment estimated at $15-30 million, and with emerging bacterial resistance, the studies are of relevance to human health and have considerable medical and economic impact.

Public Health Relevance

P. aeruginosa is an opportunistic, gram-negative pathogen associated with bacterial keratitis, especially in extended wear contact lens users. Elucidating the precise role of TLR4 in microbial keratitis, including regulation of innate and adaptive immune responses, modulation of inflammatory angiogenesis, apoptosis and antimicrobial peptide production, will provide substantive insight into the pathogenesis of bacterial keratitis. Ultimately, the findings from this proposal will be useful clinically in development of better treatments to reduce bacterial keratitis and prevent blindness.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY016058-07
Application #
8206825
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
2005-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
7
Fiscal Year
2012
Total Cost
$361,152
Indirect Cost
$123,552
Name
Wayne State University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Li, Cui; McClellan, Sharon A; Barrett, Ronald et al. (2014) Interleukin 17 regulates Mer tyrosine kinase-positive cells in Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci 55:6886-900
Hazlett, Linda D; Jiang, Xiaoyu; McClellan, Sharon A (2014) IL-10 function, regulation, and in bacterial keratitis. J Ocul Pharmacol Ther 30:373-80
Jiang, Xiaoyu; McClellan, Sharon A; Barrett, Ronald et al. (2014) HGF signaling impacts severity of Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci 55:2180-90
Berger, Elizabeth A; McClellan, Sharon A; Vistisen, Kerry S et al. (2013) HIF-1* is essential for effective PMN bacterial killing, antimicrobial peptide production and apoptosis in Pseudomonas aeruginosa keratitis. PLoS Pathog 9:e1003457
Foldenauer, Megan E B; McClellan, Sharon A; Berger, Elizabeth A et al. (2013) Mammalian target of rapamycin regulates IL-10 and resistance to Pseudomonas aeruginosa corneal infection. J Immunol 190:5649-58
Jiang, Xiaoyu; McClellan, Sharon A; Barrett, Ronald P et al. (2011) VIP and growth factors in the infected cornea. Invest Ophthalmol Vis Sci 52:6154-61
Hazlett, Linda D; Hendricks, Robert L (2010) Reviews for immune privilege in the year 2010: immune privilege and infection. Ocul Immunol Inflamm 18:237-43
Hazlett, Linda D; McClellan, Sharon A; Barrett, Ronald P et al. (2010) IL-33 shifts macrophage polarization, promoting resistance against Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci 51:1524-32
Zhou, Zimei; Wu, Minhao; Barrett, Ronald P et al. (2010) Role of the Fas pathway in Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci 51:2537-47
Wu, Minhao; McClellan, Sharon A; Barrett, Ronald P et al. (2009) Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis. J Immunol 183:8054-60

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