Pathogenesis of Fungal Keratitis

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Abstract Pathogenic Fusarium and Aspergillus molds are an important cause of vision loss in the USA and worldwide. Preliminary data from an unbiased gene array analyses (Nanostring and RNA sequencing) showed an anticipated increase in expression of multiple genes in neutrophils isolated from Fusarium and Aspergillus infected mouse corneas compared with bone marrow neutrophils from the same mice. However, we found an unexpected significant increase in expression of the pro-inflammatory cytokine IL-1 alpha, and of Spp1/osteopontin (OPN), which has known pro-inflammatory activity, and which exists as a secreted (sOPN) and intracellular (iOPN). Elevated IL-1 alpha and OPN was confirmed by Q-PCR, ELISA and intracellular flow cytometry. Aim 1 will characterize the membrane, secreted and intracellular activities the pro- and cleaved forms of IL-1 alpha in murine and human neutrophils, and in Fusarium and Aspergillus keratitis. Aim 2 will identify regulators of OPN expression, and use OPN-/- and iOPN knock-in mice to examine the role of the intracellular versus secreted forms of this protein in human neutrophils and in fungal keratitis. Aim 3 will characterize the role of neutrophil extracellular traps (NETs), which can limit hyphal growth, but also have the potential to contribute to tissue damage. Proposed experiments will identify the molecular pathways leading to NET formation in infected corneas, and using mice that lack the enzyme required for histone citrullinatation and decondensation (PAD4), we will examine the role of NETs in Fusarium and Aspergillus keratitis. Collectively, the results of these proposed studies will increase our understanding of the pathogenesis of this blinding disease, and will potentially identify novel targets for immune intervention to prevent neutrophil mediated tissue damage and loss of corneal clarity.