Pseudomonas aeruginosa keratitis is a sight-threatening corneal disease associated with trauma and contact-lens wearing. Although an antibiotic regimen can eliminate the infectious organisms, the host inflammation that occurs, if not suppressed, can cause corneal scarring that can potentially lead to permanent vision loss in some patients. Previous studies from the laboratory revealed that human corneal epithelial cells (HCECs) sense Pseudomonas via recognition of flagellin, the structural protein of bacterial flagella, by Toll-like receptor-5 (TLR5) and respond to Pseudomonas-challenge by producing pro-inflammatory cytokines. To date, how the corneal inflammatory response is modulated after TLR stimulation is largely unresolved. We recently discovered that a prior exposure to flagellin resulted in cell reprogramming, as manifested by decreased production of pro-inflammatory cytokines and augmented expression of antimicrobial molecules in HCECs and in development of protective mechanisms, including enhanced bacterial clearance and downregulated expression of cytokines/chemokines, and accelerated inflammation resolution in the cornea of Pseudomonas-infected C57BL/6 (B6) mice. We hypothesize that TLR-mediated epithelial reprogramming is a key determinant of corneal innate defense and the underlying mechanisms can be exploited as novel approaches for anti-inflammatory and/or anti-infection therapies. To understand flagellin-induced cell reprogramming and ocular protective mechanisms, we propose the following Specific Aims: 1) To assess how the TLR5-mediated signaling pathways are modulated by the IRAK family of proteins and PI3K/AKT and what roles they play in acquiring tolerance phenotype in HCECs. This will be achieved by using siRNA and specific inhibitors to define the role of signal transducing molecules of the TLR-pathway in HCEC reprogramming and its consequences in vitro. 2) To determine how flagellin-induced epithelial reprogramming differentially regulates the expression of antimicrobial genes and modulates neutrophil infiltration and stromal cell activation in the cornea in response to Pseudomonas infection. This will be done using in vitro bacterial killing assay and co-culture of epithelial-neutrophil and epithelial-stromal cells. 3) To exploit the underlying mechanisms of tolerance/reprogramming as novel approaches for anti-inflammatory and anti-infection therapies in murine model of Pseudomonas keratitis. C57BL/6 mouse model of Pseudomonas keratitis, along with gene knockout mice, will be used to test this hypothesis and to determine therapeutic potential of flagellin in preventing infection- and inflammation-caused corneal tissue damage. The results of this study will provide a logical basis for the design of novel anti-inflammatory and anti-infection therapies for prophylaxis and/or treatment of bacterial keratitis.

Public Health Relevance

. This study explores the possibility of using flagellin as a prophylactic measure to prevent microbial keratitis and as an adjuvant therapy to traditional antibiotic regimen to suppress the ongoing inflammation after bacterial infection of the cornea. In the light of emerging antimicrobial resistance, the controversial role of corticosteroids, and an increased outbreak of contact solution-associated microbial keratitis, this study is of paramount importance and may lead to clinical trials for the use of flagellin in preventing and/or treating infectious keratitis.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Anterior Eye Disease Study Section (AED)
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Shen, Grace L
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Wayne State University
Schools of Medicine
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Ross, Bing X; Gao, Nan; Cui, Xinhan et al. (2017) IL-24 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas. J Immunol 198:3536-3547
Dong, Chen; Gao, Nan; Ross, Bing X et al. (2017) ISG15 in Host Defense Against Candida albicans Infection in a Mouse Model of Fungal Keratitis. Invest Ophthalmol Vis Sci 58:2948-2958
Gao, Nan; Liu, Xiaowei; Wu, Jiayin et al. (2017) CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13. Angiogenesis 20:505-518
Gao, Nan; Yan, Chenxi; Lee, Patrick et al. (2016) Dendritic cell dysfunction and diabetic sensory neuropathy in the cornea. J Clin Invest 126:1998-2011
Gao, Nan; Lee, Patrick; Yu, Fu-Shin (2016) Intraepithelial dendritic cells and sensory nerves are structurally associated and functional interdependent in the cornea. Sci Rep 6:36414
Dandekar, Aditya; Qiu, Yining; Kim, Hyunbae et al. (2016) Toll-like Receptor (TLR) Signaling Interacts with CREBH to Modulate High-density Lipoprotein (HDL) in Response to Bacterial Endotoxin. J Biol Chem 291:23149-23158
Yan, Chenxi; Gao, Nan; Sun, Haijing et al. (2016) Targeting Imbalance between IL-1? and IL-1 Receptor Antagonist Ameliorates Delayed Epithelium Wound Healing in Diabetic Mouse Corneas. Am J Pathol 186:1466-80
Gao, Nan; Kumar, Ashok; Yu, Fu-Shin X (2015) Matrix Metalloproteinase-13 as a Target for Suppressing Corneal Ulceration Caused by Pseudomonas aeruginosa Infection. J Infect Dis 212:116-27
Tolle, Leslie; Yu, Fu-shin; Kovach, Melissa A et al. (2015) Redundant and cooperative interactions between TLR5 and NLRC4 in protective lung mucosal immunity against Pseudomonas aeruginosa. J Innate Immun 7:177-86
Sun, Haijing; Mi, Xiaofan; Gao, Nan et al. (2015) Hyperglycemia-suppressed expression of Serpine1 contributes to delayed epithelial wound healing in diabetic mouse corneas. Invest Ophthalmol Vis Sci 56:3383-92

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