Role of host sphingolipids against fungal infections

Northport Va Medical Center, Northport NY

Investigators

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Abstract

ABSTRACT The goal of this project is to study the role of the host sphingomyelin synthases (SMSs) pathway in controlling the infection by Cryptococcus neoformans (Cn). A mouse model of lung cryptococcal granuloma that recapitulates the human granuloma was developed. When these mice become immunocompromised, Cn Dgcs1 disseminates to the brain, causing meningo-encephalitis.1 Thus, Cn Dgcs1 mimics the physiopathology of the infection in immunocompromised subjects with which cryptococcosis is mostly associated.2 SMSs transfer a choline phosphate moiety from phosphatidylcholine (PC) to ceramide, producing SM and diacylglycerol (DAG),3, 4 encoded by SMS1 and SMS2 genes.5, 6 SM and DAG have been implicated in many cellular functions including the activation of pro-inflammatory responses,7 suggesting that the regulation of SMS activity in immune cells may assume a critical role in controlling infections. Very intriguingly, we found that SM stabilizes cholesterol-rich membrane rafts in macrophages. Depletion (or substitution) of SM in the outer membrane of macrophages dramatically decreases phagocytosis and displaces the Fcg receptor (FcgR) from a clustered to a diffused and homogeneous distribution. This phenomenon was validated when cholesterol was depleted, 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. The displacement of the FcgR was observed in alveolar macrophages isolated from mice lacking Sms1 (sms1-/-) but this phenotype was more profound in mice lacking Sms2 (sms2-/-). In fact, macrophages from sms2-/- show a profound decrease of antibody-mediated phagocytosis compared to macrophages from sms1-/-. Taken together, these results suggest that SMS2 regulates the internalization of Cn cells by macrophages through the production of SM, which, at the plasma membrane, stabilizes cholesterol-rich lipid rafts for anchoring the FcgR. MALDI imaging shows that SM is highly enriched in the macrophage ring surrounding the granuloma, where phagocytosis by these immune cells is essential for controlling Cn and single cell RNA sequencing shows that SMS2 is particularly upregulated in macrophages at the granuloma. Intriguingly, the sms2-/- mice cannot form an efficient lung granuloma and Cn cells are escaping and disseminating to the central nervous system. Preliminary flow cytometry shows a different immuno cellular composition in sms2-/- compared to WT mice. These results are supported by recent genomic data obtained by sequencing healthy patients affected by cryptococcosis showing the presence of genetic mutations in the sphingolipid metabolizing genes. These results suggest that the regulation of SMS at the Cn-macrophage interface in the lung environment has a key role in the regulation of the host immunity controlling the granuloma organization and the overall 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 and the granuloma site, stimulating an effective host immune response controlling Cn. Thus, we propose the following aims: 1) To determine the immune mechanism by which SMS regulates the organization of Cn granuloma, and 2) To determine the mechanism by which SMS regulates phagocytosis and killing of 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.