Microbial keratitis is a common cause of vision loss. The cornea is exquisitely sensitive to inflammation- mediated damage and therefore has strong innate defenses. However, when the epithelial barrier function is breached, opportunistic bacterial and fungal pathogens can gain access to the epithelial cell layers and colonize in the cornea, leading to infection. During the initial grant period, flagellin, the ligand of Toll-like receptor 5 (TLR5), was used to fortify corneal innate immunity and render resistance to a broad spectrum of keratitis causing pathogens, including Pseudomonas aeruginosa, Candida albicans and Aspergillus fumigates. This highly inducible and robust innate mucosal protection can be attributed to flagellin-induced genomic reprogramming in the epithelium which, in turn, interacts with professional immune cells to control inflammation and to eradicate invading pathogens. The hypothesis in this application is that flagellin, through TLR5, stimulates protective corneal innate mucosal immunity that involves epithelial cells, infiltrated PMNs, and activated dendritic cells, collectively conferring robust resistance to microbial keratitis.
Three specific aims are proposed to test this hypothesis: 1) To determine how flagellin-induced reprogramming is regulated in corneal epithelial cells and in the cornea. This can be assessed by using the chromatin immunoprecipitation assay and histone deacetylase inhibitors for gene-specific chromatin modification and by using siRNA and knockout mice for delineating the role of activating transcription factor-3, a transcription factor induced by fg, in regulating the innate immune response in the cornea. 2) To determine how the epithelium participates in and coordinates flagellin-induced mucosal innate immune defense against microbial keratitis. The role of the epithelium and its interaction with dendritic cells in innate defense will be determined by using various transgenic and knockout mice, bone marrow reconstitution, and cell depletion. 3) To determine how flagellin induces protection against non-flagellated pathogens in the cornea. The therapeutic potential of flagellin and its mechanisms of action will be characterized by using mouse models of fungal keratitis (Candida and Aspergillus as pathogens and post-infection topical flagellin application) and double TLR knockout mice. The results of the proposed study should shed light on corneal innate immunity and its induction, and may lead to the development of new prophylactic/therapeutic modalities for preventing and treating microbial keratitis.

Public Health Relevance

This proposal is to determine the mechanisms underlying flagellin-induced protective corneal mucosal immunity and to explore its therapeutic potential to treat microbial keratitis, a common cause of vision loss. The knowledge gained will be critical for the long-term goal of developing mechanism-based, efficacious prophylactic/therapeutic modalities for preventing and treating infectious keratitis.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01EY017960-06
Application #
8655872
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mckie, George Ann
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Wayne State University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Liu, Xiaowei; Gao, Nan; Dong, Chen et al. (2014) Flagellin-induced expression of CXCL10 mediates direct fungal killing and recruitment of NK cells to the cornea in response to Candida albicans infection. Eur J Immunol 44:2667-79
Bettahi, Ilham; Sun, Haijing; Gao, Nan et al. (2014) Genome-wide transcriptional analysis of differentially expressed genes in diabetic, healing corneal epithelial cells: hyperglycemia-suppressed TGF?3 expression contributes to the delay of epithelial wound healing in diabetic corneas. Diabetes 63:715-27
Gao, N; Sang Yoon, G; Liu, X et al. (2013) Genome-wide transcriptional analysis of differentially expressed genes in flagellin-pretreated mouse corneal epithelial cells in response to Pseudomonas aeruginosa: involvement of S100A8/A9. Mucosal Immunol 6:993-1005
Yoon, Gi Sang; Dong, Chen; Gao, Nan et al. (2013) Interferon regulatory factor-1 in flagellin-induced reprogramming: potential protective role of CXCL10 in cornea innate defense against Pseudomonas aeruginosa infection. Invest Ophthalmol Vis Sci 54:7510-21
Gao, Nan; Yin, Jia; Yoon, Gi Sang et al. (2011) Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair. Am J Pathol 179:2243-53
Xu, Keping; Yu, Fu-Shin X (2011) Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats. Invest Ophthalmol Vis Sci 52:3301-8
Gao, Nan; Kumar, Ashok; Guo, Hui et al. (2011) Topical flagellin-mediated innate defense against Candida albicans keratitis. Invest Ophthalmol Vis Sci 52:3074-82
Yin, Jia; Huang, Jenny; Chen, Cynthia et al. (2011) Corneal complications in streptozocin-induced type I diabetic rats. Invest Ophthalmol Vis Sci 52:6589-96
Yu, Fu-shin; Cornicelli, Matthew D; Kovach, Melissa A et al. (2010) Flagellin stimulates protective lung mucosal immunity: role of cathelicidin-related antimicrobial peptide. J Immunol 185:1142-9
Yin, Jia; Yu, Fu-Shin X (2010) LL-37 via EGFR transactivation to promote high glucose-attenuated epithelial wound healing in organ-cultured corneas. Invest Ophthalmol Vis Sci 51:1891-7

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