Research Project
10164794
Ocular injury is a leading cause of corneal blindness, resulting in millions of cases of visual impairment globally each year. Currently, the uncontrolled immune activation and tissue damage that occur following ocular injury are treated with non-specific anti-inflammatory drugs (e.g. corticosteroids), which are rife with deleterious side effects such as delayed wound healing and infection. Our research aims to identify the specific cellular and molecular factors that mediate early immune cell activation and infiltration following ocular injury, so that novel therapeutics may be developed. Our group?s recently published reports and preliminary data have identified tissue-resident mast cells as the primary reservoir of neutrophil-chemoattractants at the cornea, and have established that instant release of preformed CXCL2 by mast cells is critical for early neutrophil migration (<1 hour) following injury. Early-recruited ?scout? neutrophils subsequently release chemoattractant factors that drive a robust second phase of neutrophil infiltration, amplifying the innate immune response. Excessive recruitment and activation of neutrophils is known to cause deleterious inflammation and damage corneal architecture. These observations pose a critical question: How do mast cells sense tissue injury and promote early neutrophil recruitment? Our data show that damaged corneal epithelial cells (but not resident stromal fibroblasts or macrophages) release inflammatory mediators that activate mast cells. In accordance with our laboratory?s expertise in immunological assays and utilizing a well-characterized murine model of sterile corneal stromal injury, we propose a series of novel experiments to decipher the role of mast cells as sensors of tissue injury. In Aim 1, we will test the hypothesis that danger-associated inflammatory molecules IL33, IL36? and HMGB1 released from corneal epithelial cells activate mast cells in an IgE- independent manner. Specifically, we will (i) establish that necrotic epithelial cells stimulate mast cells relative to healthy and IL1?-treated epithelial cells using mast cell-specific tryptase and ?-hexosaminidase release assays; and (ii) identify the key danger-associated molecules expressed by corneal epithelial cells that activate mast cells. In Aim 2, we will test the hypothesis that in vivo blockade of IL36? function will be more effective, relative to IL33 and HMGB1, in suppressing mast cell activation, resulting in reduced neutrophil infiltration and tissue damage. Specifically, we will determine the effect of (i) silencing select mast cell- activating mediators, and (ii) silencing receptors for select mediators on mast cells, on mast cell activation and early neutrophil recruitment during corneal injury. We will also evaluate the therapeutic potential of local blockade of select mediators. It is anticipated that this research will have significant translational impact due to the high prevalence of ocular injury and inflammatory disease, as well as the relevance of mechanisms governing neutrophil infiltration to non-ocular tissues.
