The goal of this project is to study the role and mechanisms by which sphingomyelin synthases (Sms1 and Sms2) are involved in controlling the infection caused by the pathogenic fungus Cryptococcus neoformans (Cn). Our laboratory has pioneered studies dealing with the role of fungal sphingolipids in the regulation of infectious diseases. In addition to studying fungal sphingolipids, we have recently discovered that certain host sphingolipids play a key role in controlling the immune response against the human fungal pathogen Cn. One of the host sphingolipid shown to regulate immune responses is sphingomyelin (SM) produced by sphingomyelin synthase (SMS), encoded by the SMS1 and SMS2 genes.1,2 SMS transfers a choline phosphate moiety from phosphatidylcholine (PC) to ceramide, producing SM and diacylglycerol (DAG) (Fig. 1).3,4 These lipids have been implicated in many cellular functions including the activation of pro-inflammatory responses,5 suggesting that the regulation of SMS activity in immune cells may assume a critical role in controlling infections. In fact, we have shown previously that the DAG produced by SMS mediates the in vitro extracellular killing of Cn by phagocytic cells possibly through a protein kinase D (PKD) dependent mechanism, and the SMS-DAG-PKD signaling pathway mediates the secretion of antimicrobial peptides by phagocytic cells, particularly defensins ( Progress Report, new Fig. 9, and6,7). Very intriguingly, our current studies also revealed a key role for SM in the regulation of cholesterol-rich membrane rafts in macrophages. We found that depletion of SM in the outer membrane of macrophages dramatically decreases phagocytosis (Figs. 3 and 5) and displaces the Fcg receptor (FcgR) from a punctuated, clustered to a diffused and homogeneous distribution (Fig. 7). This phenomenon was validated when cholesterol was depleted (Fig. 7), corroborating the association of the FcgR with lipid rafts. The resulting effect of these depletions is a significant decrease of antibody-mediated phagocytosis of Cn (Figs. 3, 4 and 5). Importantly, the displacement of the FcgR was also observed in alveolar macrophages isolated from mice lacking Sms1 (sms1-/-) or Sms2 (sms2-/-), which also showed a decrease of antibody-mediated phagocytosis (new Fig. 12). Taken together, these results suggest that SMS regulates the internalization of Cn cells by macrophages through the production of SM, which, at the plasma membrane, stabilizes cholesterol-rich lipid rafts (new Fig. 8) for anchoring the FcgR. Deletion of Sms1 (sms1-/-) or Sms2 (sms2-/-) renders the animals significantly hypersusceptible to Cn infection (Fig. 10). Upon inhalation, Cn cells are rapidly replicating in the lung and quickly disseminating to the brain of sms1-/- or sms2-/- mice (new Fig. 11). Preliminary flow cytometry shows a different immuno cellular composition in sms2-/- compared to WT lungs (new Fig. 15), and the sms2-/- mice cannot form an efficient lung granuloma (new Fig. 13). These results suggest that the regulation of SMS at the Cn-macrophage interface in the lung environment may have a key role in the regulation of the overall host immunity and the outcome of the disease. Based on these studies, we hypothesize that, through the production of SM and DAG, SMS regulates the phagocytosis/killing of Cn, stimulating an effective host immune response against Cn. Thus, we propose the following aims: 1) To determine the mechanism by which SMS regulates phagocytosis and killing of Cn by phagocytic cells; and 2) To establish the role and mechanism(s) by which SMS regulates the overall host immunity against Cn. By studying the role of SMS during Cn-phagocytes interaction and during the infection we will be able to identify new mechanisms of host protection with important insights into the development of new immunotherapies to better control this life-threatening disease.
Fungal infections have dramatically increased during the last decade afflicting both immunodeficient and immunocompetent individuals including military personnel. This proposal focuses on a better understanding of host resistance and susceptibility with important implications for a better understanding of the pathophysiology of fungal infections and the developing of new therapeutic strategies.
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