PROJECT SUMMARY/ABSTRACT: Fungal keratitis is an infection of the corneas by pathogenic fungal species that is notoriously resistant to treatment and could lead to permanent blindness. Conidia (fungal spores) are ubiquitous in the environment and can infect healthy individuals following ocular trauma or unsanitary contact lens use. Once in the corneal stroma, conidia germinate resulting in rapid hyphae formation and tissue infiltration. Neutrophils are the first cells recruited to the corneas, and play a critical role in the host response against infection. While they are necessary to eliminate fungi, neutrophils also contribute to severe tissue damage. Current treatment includes antifungal agents followed by corticosteroids to suppress the inflammatory response. However, if the pathogen persists, this could lead to rapid hyphae invasion in the absence of an immune response. Therefore, there is a need for a more targeted treatment for inflammation to better control the balance between fungal clearance and tissue damage. To address this issue, we used Nanostring gene array analysis to analyze inflammatory genes that might be up- or down-regulated in neutrophils responding to fungal keratitis. We found that sorted neutrophils from the corneas of Aspergillus-infected mice highly upregulated Interleukin-1α (IL-1α) by >10,000 fold compared to bone marrow neutrophils. IL-1α is a pro-inflammatory cytokine that is critical for various infections. It is released by corneal epithelial cells as an alarmin to initiate and amplify inflammation, and can indirectly recruit neutrophils. IL-1α secretion is independent of the ER/golgi transport system. Many aspects of its biogenesis, regulation, and secretion are not well understood. Based on preliminary data, I hypothesize that IL-1α plays a critical role in the immune response during fungal keratitis. In this proposal, I will address three aims to support my hypothesis: 1) determine the importance of neutrophil-derived IL-1α during the inflammatory stages of Aspergillus keratitis, 2) define the role of corneal epithelial cells-derived IL-1α during disease, and 3) dissect the mechanism for IL-1α secretion from live neutrophils. The proposed research will address many unknown aspects of IL-1α, corneal epithelial cells, and neutrophil biology in response to fungal keratitis to provide a better understanding of the inflammatory processes involved.