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

Arielle M Bryan,Jeehyun Karen You,Guangtao Li,Jihyun Kim,Ashutosh Singh,Johannes Morstein,Dirk Trauner,Nívea Pereira De Sá,Tyler G Normile,Amir M Farnoud,Erwin London,Maurizio Del Poeta

doi:10.1016/j.jbc.2021.101411

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 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 phagocytosis, 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 raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C.neoformans.

Highlights

We found that cholesterol and SMenriched ordered domains may be important for the function of Fcγ receptors (FcγRs), the class of immune receptors activated by immunoglobulin G (IgG) immune complexes (IgGICs)
Since depletion treatments perturbed SM–cholesterol complexes and significantly affected the FcγR-mediated cell signaling without altering the amount of FcγR IIB and III localized to the cell surface, it appears that SM and cholesterol-dependent lipid rafts are critical for facilitating FcγR-mediated cell signaling and function
Lipid rafts do not appear to be critical for phagocytosis mediated by complement serum opsonization, which was not affected by cholesterol depletion or bacterial sphingomyelinase (bSMase) treatment

Summary

Introduction

These findings provide more directed insights into the role of cholesterol- and SM-rich lipid rafts in mediating FcγR activation and IgG-dependent phagocytosis of C. neoformans. When treated macrophages were coincubated with C. neoformans cells opsonized with anti-GXM IgG, we found that we were able to restore phagocytosis by cholesterol or 7dehydrocholesterol repletion, whereas coprostanol repletion did not restore phagocytosis (Fig. 3B). When macrophages were coincubated with C. neoformans cells opsonized with antiGXM IgG immediately following AzoSM exchange (no light exposure), we observed phagocytosis comparable to untreated control cells.

Results

Conclusion