Corneal infections caused by the Gram negative bacteria Pseudomonas aeruginosa, the most common cause of contact lens wearing-associated infectious keratitis, and the fungus Candida albicans occur in the USA and worldwide, causing painful, sight-threatening disease. Furthermore, among all human P. aeruginosa infections, more than 13% are multidrug-resistant. This highlights the importance of better understanding the innate immune responses to opportunistic pathogens of the cornea. The objective of this proposal is to defining the protective role of the novel interleukin-36 subfamily of cytokines, close relatives of the IL-1 subfamily, in microbial keratitis and their targets, the dendritic cell. Our preliminary data show that IL-36?, ?, and their receptor IL-36R are required for a proper response to infection, and that down-regulation of their natural inhibitor, IL-36R antagonist, protects the cornea from P. aeruginosa infection. While the IL-1 subfamily is known as one of the most important mediators of innate immunity and inflammation and has been studied extensively with several drugs being developed from this body of research, little is known regarding the role of IL-36R in protective mucosal innate immunity. Based on the preliminary data and published studies, the hypothesis that IL-36R signaling promotes corneal innate defenses against microbial keratitis and antagonizes IL-1? in limiting infection-induced inflammation is postulated and will be tested with three specific aims: 1. To determine the expression and cellular sources of IL-36?, -36? and -36Ra and cell type(s) bearing IL- 36R in the cornea in response to microbial infection. This can be tested by assessing the cellular sources of IL- 36?, ?, and IL-36Ra, and cell types expressing IL-36R in the cornea during murine Pa- and Ca-infection using ELISA of epithelial and corneal extracts, immunohistochemistry. Primary human corneal epithelial cells will be used to assess the means by which IL-36 cytokines are secreted with particular focus on exosomes. The presence of IL-1 cytokines in corneal scrapings from keratitis patients will also be assessed. 2. To decipher the role(s) of IL-36 family members in corneal innate immune responses to infection in vivo and in vitro. This can be tested by assessing the role(s) of IL-36?, ? or IL-36R blockade (using neutralizing antibodies, siRNA, and/or genetic deletions) in mouse models of P. aeruginosa and C. albicans keratitis in vivo and bone marrow derived dendritic cell isolated from wild-type and knockout mice. 3. To assess the therapeutic potential of IL-36? on innate immunity, inflammation resolution, and/or angiogenesis in infected corneas. This can be tested by assessing the effects of topical application of IL-36? in combination with IL-1Ra on the outcome of microbial keratitis in B6 mice. The therapeutic potential of IL-36 stimulated, dendritic cell produced IL-23, and/or ?? T cell produced IL-22 will also be tested. The results from this proposal should improve our understanding of IL-36/IL-36R axis and dendritic cells in the regulation of corneal innate immunity at the molecular and cellular levels and may lead to the development of therapies that limit the progress/pathogenesis of microbial keratitis.

Public Health Relevance

Microbial keratitis is a leading cause of sight-threatening ocular infections commonly associated with contact lens wear. This proposal is to assess the role of a novel subfamily of the IL-1 family of cytokines, IL-36, and their secretion carrier exosomes in evoking protective innate immunity against keratitis-causing pathogens. The knowledge gained will be critical for understanding the biology of this novel, little known, and potential vital subfamily of cytokines and for developing new adjunctive therapeutics using IL-36 or its downstream effectors for treating microbial keratitis.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Mckie, George Ann
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Wayne State University
Schools of Medicine
United States
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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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