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

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 by aiding in clearance but can also potentially serve as a niche for their 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 [SM]) have been implicated in facilitating phagocy tosis, we evaluated whether these ordered domains govern macrophages' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raftpromoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cisAzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fc gamma receptor (Fc gamma R) function, whereby IgG immune complexes crosslink 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 SM 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.

Automated summary

The study showed that lipids like sphingomyelin and cholesterol play a crucial role in macrophages' ability to phagocytose Cryptococcus neoformans. Phosphorylation was found to modulate the phagocytic activity of macrophages.