This is a competitive renewal application to further investigate the mechanisms by which IgE-independent activation of mast cells contributes to ocular inflammation. Mast cells have garnered much attention over the past decade for their diverse IgE-independent effector function in the setting of non-allergic inflammatory diseases. Nevertheless, fundamental questions remain unanswered regarding the function and immune mechanisms of mast cells in mediating ‘non-allergic’ ocular pathologies. Our laboratory has made substantial progress in understanding how ocular mast cells contribute to the non-allergic inflammatory response. Reports from our lab provide evidence that (i) epithelial cell-derived interleukin-33 activates mast cells in an IgE- independent manner and that mast cells (ii) as resident immune cells initiate neutrophil infiltration by releasing CXCL2, and (iii) promote pathological growth of blood and lymph vessels in part by secreting high levels of VEGFs. Moreover, our robust preliminary findings indicate that mast cells augment the tissue-damaging function of neutrophils and that deficiency of mast cells results in faster regression of mature pathological corneal vessels. However, the exact mechanisms by which mast cells interact with neutrophils and vascular cells to promote their pathological functions are not well defined. In accordance with our laboratory’s expertise in immunological studies and our well-established murine models of corneal injury and neovascularization, we propose a series of novel experiments to decipher the function of mast cells as an orchestrator of inflammation and tissue damage. Our pilot investigations show that mast cells express high levels of neutrophil (granulocyte)-stimulating factors, GM-CSF and G-CSF. In Aim 1, we will test the hypothesis that Mast cell-derived granulocyte stimulating factors (GM-CSF and G-CSF) promote the release of tissue-damaging cytokines and enzymes by neutrophils following corneal injury. Specifically, we will assess the effect of GM-CSF versus G-CSF deficient mast cells (using CRISPR-Cas9) and neutralizing antibody treatment on (i) neutrophil effector function and survival using our standardized in vitro co-culture assays and (ii) neutrophil-mediated tissue damage in the corneal injury model. In Aim 2, based on our preliminary data of mast cells expressing high levels of vessel stabilizing Angiopoietin 1 (Ang1), we will test the hypothesis that Mast cells juxtaposing pathological vessels prolong vascular endothelial cell (VEC) survival and maintain vessel integrity by secreting Ang1. Specifically, we will determine the effect of (i) mast cells and VECs interaction on Ang1-Tie2 ligand-receptor axis, (ii) Ang1-deficient mast cells on the stability of pathological blood vessels. We will also evaluate the relative therapeutic efficacy of topical application of Ang1 blocked versus general mast cell inhibitor in accelerating vessel regression. It is anticipated that the completion ...