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bioRxiv · 10.1101/2021.11.02.466923

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Bryan, A. M.·You, J. K.·Li, G.·Kim, J.·Singh, A.·Morstein, J.·Trauner, D.·Pereira de Sa, N.·Normile, T. G.·Farnoud, A. M.·London, E.·Del Poeta, M.
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Abstract

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores, whereby macrophages either aid in clearance or serve as a niche for its dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high order plasma membrane domains enriched in cholesterol and sphingomyelin) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages ability to phagocytose C. neoformans. We found that cholesterol or sphingomyelin depletion resulted in significantly deficient IgG-mediated phagocytosis of the fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency while a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable sphingomyelin (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis whereas raft-inhibiting conformation (cis-AzoSM) significantly blunted phagocytosis in a reversible manner. We observed that the effect on phagocytosis may be mediated by facilitating Fc{gamma} receptor (Fc{gamma}R) function, whereby IgG immune complexes cross-link to Fc{gamma}RIII, resulting in tyrosine phosphorylation of FcR {gamma}-subunit (FcR{gamma}), an important accessory protein in the Fc{gamma}R signaling cascade. Correspondingly, cholesterol or sphingomyelin depletion resulted in decreased FcR{gamma} phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcR{gamma} phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating Fc{gamma}RIII-mediated phagocytosis of C. neoformans.

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Bryan, A. M., You, J. K., Li, G., Kim, J., Singh, A., Morstein, J., Trauner, D., Pereira de Sa, N., Normile, T. G., Farnoud, A. M., London, E., Del Poeta, M.. 2021-11-02. Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages. https://doi.org/10.1101/2021.11.02.466923

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