Fungal Melanin Modulation of Calcium-Mediated Responses in Lung Epithelium

PROJECT SUMMARY Invasive pulmonary fungal infections carry unacceptably high mortality rates and contribute to over $2.4 billion in direct healthcare costs in the US alone. The most prominent fungal pathogens causing lung disease include Aspergillus fumigatus (Af). Although it is well demonstrated that the immune system is critical to a proper response to invading fungi, the airway epithelium remains a vastly understudied component of antifungal immunity. We previously demonstrated that the recognition of Aspergillus lacking the outer layer of 1,8-dihydroxy naphthalene (DHN) melanin by airway epithelial cells leads to higher transmigration of neutrophils. Our preliminary data demonstrate that purified Af melanin (termed melanin ghosts; MG) blocked the secretion of IL-8 and GROα by the epithelium, muted calcium fluxing, reduced phosphorylation of key signaling molecules, induced distinct changes in the transcriptional signatures differentially in airway epithelial cell subtypes, altered actin filamentation, and dampened the production of reactive oxygen species (ROS). These data argue that fungal melanin actively triggers distinct immunomodulatory responses in primary human epithelial cells. Thus, our overall testable hypothesis is that Af melanin blocks calcium fluxing, which silences inflammatory circuits (i.e., exocytosis pathways, TLR and CLR signaling pathways, and ROS production) in airway epithelial cells to subvert the innate immune response. To address this hypothesis, we propose the following three specific aims: [1] dissect the mechanism of how Af fungal melanin inhibits chemokine secretion, [2] determine the TLR and CLR signaling pathways affected by Af melanin; and [3] reveal the mechanism by which melanin blocks ROS production in lung epithelial cells. This work will identify the mechanisms by which fungal melanin halts chemokine secretion by airway epithelial cells and define the pathway(s) altered by this suppression through the lens of calcium signaling. Completion of this proposal will provide novel therapeutic targets for invasive fungal infections caused by melanized fungi. Furthermore, lung diseases with the hallmark of a hyper- inflammatory state, such as asthma, could be treated with synthesized melanin to dampen exacerbated inflammation.