Cholesterol and sphingomyelin are critical for Fcγ receptor–mediated phagocytosis of Cryptococcus neoformans by macrophages
2021; Elsevier BV; Volume: 297; Issue: 6 Linguagem: Inglês
10.1016/j.jbc.2021.101411
ISSN1083-351X
AutoresArielle 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,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoCryptococcus 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. 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. Cryptococcus spp. are major opportunistic fungal pathogens that cause life-threatening meningoencephalitis in immunocompromised individuals (1Perfect J.R. Casadevall A. Cryptococcosis.Infect. Dis. Clin. North Am. 2002; 16 (v-vi): 837-874Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 2Eisenman H.C. Casadevall A. McClelland E.E. New insights on the pathogenesis of invasive Cryptococcus neoformans infection.Curr. Infect. Dis. Rep. 2007; 9: 457-464Crossref PubMed Scopus (38) Google Scholar). Cryptococcus neoformans has been reported to cause approximately 223,100 cases of meningitis globally, resulting in 181,000 deaths every year (3Rajasingham R. Smith R.M. Park B.J. Jarvis J.N. Govender N.P. Chiller T.M. Denning D.W. Loyse A. Boulware D.R. Global burden of disease of HIV-associated cryptococcal meningitis: An updated analysis.Lancet Infect. Dis. 2017; 17: 873-881Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). C. neoformans mediated host damage at the molecular and cellular level results in deficient immune effector cell function, disruption of endothelial barriers, and dissemination (4Casadevall A. Coelho C. Alanio A. Mechanisms of Cryptococcus neoformans-mediated host damage.Front. Immunol. 2018; 9: 855Crossref PubMed Scopus (29) Google Scholar). Despite the substantial morbidity and mortality associated with cryptococcal meningoencephalitis, there is still much that is unknown about the delicate interactions between host immune cells and fungal pathogens like C. neoformans. Pulmonary macrophages comprise the first line of host defense in response to C. neoformans, as infection begins with inhalation of fungal spores commonly found in urban environments. A healthy host can typically kill and clear the pathogen. However, the fungus can be a facultative intracellular pathogen that survives within macrophages in immunocompromised individuals (e.g., HIV and AIDS patients) (2Eisenman H.C. Casadevall A. McClelland E.E. New insights on the pathogenesis of invasive Cryptococcus neoformans infection.Curr. Infect. Dis. Rep. 2007; 9: 457-464Crossref PubMed Scopus (38) Google Scholar, 5Nelson B.N. Hawkins A.N. Wozniak K.L. Pulmonary macrophage and dendritic cell responses to Cryptococcus neoformans.Front. Cell Infect. Microbiol. 2020; 10: 37Crossref PubMed Scopus (11) Google Scholar). In these cases, the macrophages can serve as a niche for the replication of the pathogen and may facilitate its dissemination to the central nervous system where the disease becomes fatal (6Kechichian T.B. Shea J. Del Poeta M. Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice.Infect. Immun. 2007; 75: 4792-4798Crossref PubMed Scopus (94) Google Scholar, 7Casadevall A. Cryptococci at the brain gate: Break and enter or use a Trojan horse?.J. Clin. Invest. 2010; 120: 1389-1392Crossref PubMed Scopus (57) Google Scholar, 8Chretien F. Lortholary O. Kansau I. Neuville S. Gray F. Dromer F. Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.J. Infect. Dis. 2002; 186: 522-530Crossref PubMed Scopus (161) Google Scholar, 9Luberto C. Martinez-Marino B. Taraskiewicz D. Bolanos B. Chitano P. Toffaletti D.L. Cox G.M. Perfect J.R. Hannun Y.A. Balish E. Del Poeta M. Identification of App1 as a regulator of phagocytosis and virulence of Cryptococcus neoformans.J. Clin. Invest. 2003; 112: 1080-1094Crossref PubMed Scopus (108) Google Scholar). C. neoformans can impair mitochondrial function, alter protein synthesis, or undergo nonlytic exocytosis from macrophages (10Ben-Abdallah M. Sturny-Leclere A. Ave P. Louise A. Moyrand F. Weih F. Janbon G. Memet S. Fungal-induced cell cycle impairment, chromosome instability and apoptosis via differential activation of NF-kappaB.PLoS Pathog. 2012; 8e1002555Crossref PubMed Scopus (33) Google Scholar, 11Coelho C. Souza A.C. Derengowski Lda S. de Leon-Rodriguez C. Wang B. Leon-Rivera R. Bocca A.L. Goncalves T. Casadevall A. Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans.J. Immunol. 2015; 194: 2345-2357Crossref PubMed Scopus (32) Google Scholar, 12Ma H. Croudace J.E. Lammas D.A. May R.C. Expulsion of live pathogenic yeast by macrophages.Curr. Biol. 2006; 16: 2156-2160Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 13Alvarez M. Casadevall A. Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.BMC Immunol. 2007; 8: 16Crossref PubMed Scopus (91) Google Scholar). Macrophages may even deliver the fungus directly into the meninges, helping the yeast to cross the blood brain barrier via the "Trojan horse" model (14Liu T.B. Perlin D.S. Xue C.Y. Molecular mechanisms of cryptococcal meningitis.Virulence. 2012; 3: 173-181Crossref PubMed Scopus (77) Google Scholar, 15McQuiston T.J. Williamson P.R. Paradoxical roles of alveolar macrophages in the host response to Cryptococcus neoformans.J. Infect. Chemother. 2012; 18: 1-9Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 16Garcia-Rodas R. Zaragoza O. Catch me if you can: Phagocytosis and killing avoidance by Cryptococcus neoformans.FEMS Immunol. Med. Microbiol. 2012; 64: 147-161Crossref PubMed Scopus (64) Google Scholar, 17Coelho C. Bocca A.L. Casadevall A. The intracellular life of Cryptococcus neoformans.Annu. Rev. Pathol. 2014; 9: 219-238Crossref PubMed Scopus (79) Google Scholar). Given that macrophages can determine the outcome of a cryptococcal infection, it is crucial to understand the factors that mediate phagocytosis of C. neoformans, a major function of macrophages. Phagocytosis is a process by which extracellular entities are internalized by phagocytic cells. It is a key weapon in the immune system's arsenal to defend against pathogens, but the process may often be subverted by pathogens to allow for internalization and dissemination throughout the body (18Sarantis H. Grinstein S. Subversion of phagocytosis for pathogen survival.Cell Host Microbe. 2012; 12: 419-431Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Phagocytosis is mediated by several signaling events that result in attachment and engulfment via rearrangements of the host cell's cytoskeleton. "Professional" phagocytes are able to recognize and bind to opsonins on the surface of the invading pathogen to signal for attachment and the formation of lamellipodia, which engulf the pathogen and form a phagosome (2Eisenman H.C. Casadevall A. McClelland E.E. New insights on the pathogenesis of invasive Cryptococcus neoformans infection.Curr. Infect. Dis. Rep. 2007; 9: 457-464Crossref PubMed Scopus (38) Google Scholar). Macrophages are highly specialized cells that carry out protective functions that include seeking out and eliminating disease-causing agents, repairing damaged tissues, and mediating inflammation, most of these through the process of phagocytosis (18Sarantis H. Grinstein S. Subversion of phagocytosis for pathogen survival.Cell Host Microbe. 2012; 12: 419-431Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Rougerie P. Miskolci V. Cox D. Generation of membrane structures during phagocytosis and chemotaxis of macrophages: Role and regulation of the actin cytoskeleton.Immunol. Rev. 2013; 256: 222-239Crossref PubMed Scopus (140) Google Scholar). Previous work in other pathogen systems point to lipid rafts formed by cholesterol and sphingomyelin (SM) as having an important role to play in phagocytosis (20Rao M. Peachman K.K. Alving C.R. Rothwell S.W. Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.Immunol. Cell Biol. 2003; 81: 415-423Crossref PubMed Scopus (11) Google Scholar, 21Sein K.K. Aikawa M. The prime role of plasma membrane cholesterol in the pathogenesis of immune evasion and clinical manifestations of falciparum malaria.Med. Hypotheses. 1998; 51: 105-110Crossref PubMed Scopus (15) Google Scholar, 22Pucadyil T.J. Tewary P. Madhubala R. Chattopadhyay A. Cholesterol is required for Leishmania donovani infection: Implications in leishmaniasis.Mol. Biochem. Parasitol. 2004; 133: 145-152Crossref PubMed Scopus (91) Google Scholar, 23Kannan S. Audet A. Huang H. Chen L.J. Wu M. Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection.J. Immunol. 2008; 180: 2396-2408Crossref PubMed Scopus (69) Google Scholar, 24Wang M. Hajishengallis G. Lipid raft-dependent uptake, signalling and intracellular fate of Porphyromonas gingivalis in mouse macrophages.Cell Microbiol. 2008; 10: 2029-2042Crossref PubMed Scopus (55) Google Scholar, 25Lee J.-H. Kim D.Y. Lee S.H. Song D.-K. Bae J.-H. Im S.-S. LPS-induced mTORC1 signaling activates lipid raft-actin cytoskeletal interactions for phagocytosis through SREBP-1a.. 2019; 33: lb318Google Scholar, 26Magenau A. Benzing C. Proschogo N. Don A.S. Hejazi L. Karunakaran D. Jessup W. Gaus K. Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order.Traffic. 2011; 12: 1730-1743Crossref PubMed Scopus (26) Google Scholar). In fact, a recent study implicated lipid rafts in the phagocytic response to Aspergillus fumigatus, another opportunistic fungal pathogen (27Schmidt F. Thywissen A. Goldmann M. Cunha C. Cseresnyes Z. Schmidt H. Rafiq M. Galiani S. Graler M.H. Chamilos G. Lacerda J.F. Campos Jr., A. Eggeling C. Figge M.T. Heinekamp T. et al.Flotillin-dependent membrane microdomains are required for functional phagolysosomes against fungal infections.Cell Rep. 2020; 32: 108017Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Cholesterol is the single most abundant lipid species in mammalian plasma membranes, comprising 25 to 50% of the cell membrane lipid content (28Crane J.M. Tamm L.K. Role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes.Biophysical J. 2004; 86: 2965-2979Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). It has been found to play a role in modulating the biophysical properties of membranes by changing their rigidity (29Brown D.A. London E. Functions of lipid rafts in biological membranes.Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2530) Google Scholar). Cholesterol and sphingolipids together form lipid microdomains within the membrane known as lipid rafts. Lipid rafts have been found to be involved in the formation of caveolae as well as activation of various signaling cascades, whereby domains favor specific protein–protein interactions (28Crane J.M. Tamm L.K. Role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes.Biophysical J. 2004; 86: 2965-2979Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 29Brown D.A. London E. Functions of lipid rafts in biological membranes.Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2530) Google Scholar, 30Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5059) Google Scholar). In fact, previous studies have implicated the role of rafts in immune receptor signaling (31Rajendran L. Simons K. Lipid rafts and membrane dynamics.J. Cell Sci. 2005; 118: 1099-1102Crossref PubMed Scopus (464) Google Scholar). Given their small size, it is difficult to study lipid rafts in vivo. One useful way to study the role of lipid rafts is to alter their constituents. Cyclodextrins and sphingomyelinase have been found to deplete, replace, or alter lipids from host mammalian membranes and are commonly used to study the role of lipid rafts (30Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5059) Google Scholar, 32Li G.T. Kim J. Huang Z. St Clair J.R. Brown D.A. London E. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 14025-14030Crossref PubMed Scopus (42) Google Scholar, 33Kim J. Singh A. Del Poeta M. Brown D.A. London E. The effect of sterol structure upon clathrin-mediated and clathrin-independent endocytosis.J. Cell Sci. 2017; 130: 2682-2695Crossref PubMed Scopus (32) Google Scholar, 34Diaz O. Mebarek-Azzam S. Benzaria A. Dubois M. Lagarde M. Nemoz G. Prigent A.F. Disruption of lipid rafts stimulates phospholipase d activity in human lymphocytes: Implication in the regulation of immune function.J. Immunol. 2005; 175: 8077-8086Crossref PubMed Scopus (41) Google Scholar, 35Audi A. Soudani N. Dbaibo G. Zaraket H. Depletion of host and viral sphingomyelin impairs influenza virus infection.Front. Microbiol. 2020; 11: 612Crossref PubMed Scopus (15) Google Scholar, 36Li G. Wang Q. Kakuda S. London E. Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids.J. Lipid Res. 2020; 61: 758-766Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 37Suresh P. Miller W.T. London E. Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered raft domains.J. Biol. Chem. 2021; 297Google Scholar). In this study, several approaches were employed to alter cholesterol or SM on the outer leaflet of the plasma membrane to examine how lipid rafts may play a role in phagocytosis of C. neoformans. We found that cholesterol and SM are critical for immunoglobulin G (IgG)-mediated phagocytosis of C. neoformans. Moreover, repletion with domain-promoting sterols like cholesterol or 7-dehydrocholesterol promoted efficient antibody-mediated phagocytosis of the fungal cells, whereas domain-inhibiting sterols like coprostanol significantly reduced it. Using a photoswitchable SM (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. Mechanistically, we found that cholesterol and SM-enriched ordered domains may be important for the function of Fcγ receptors (FcγRs), the class of immune receptors activated by IgG immune complexes (IgGICs). 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. To investigate the role of cholesterol in phagocytosis of the fungal pathogen C. neoformans, we utilized methyl-beta-cyclodextrin (MβCD) to deplete cholesterol from the membrane of murine macrophages prior to infection with antibody-opsonized C. neoformans (38Bryan A.M. Farnoud A.M. Mor V. Del Poeta M. Macrophage cholesterol depletion and its effect on the phagocytosis of Cryptococcus neoformans.J. Vis. Exp. 2014; https://doi.org/10.3791/52432Crossref Scopus (13) Google Scholar). We previously showed that treatment with 10 mM or 30 mM MβCD depletes approximately 50% or 75% of the total cholesterol in the cells, respectively (38Bryan A.M. Farnoud A.M. Mor V. Del Poeta M. Macrophage cholesterol depletion and its effect on the phagocytosis of Cryptococcus neoformans.J. Vis. Exp. 2014; https://doi.org/10.3791/52432Crossref Scopus (13) Google Scholar). Furthermore, MβCD treatment did not significantly alter cell attachment or viability (38Bryan A.M. Farnoud A.M. Mor V. Del Poeta M. Macrophage cholesterol depletion and its effect on the phagocytosis of Cryptococcus neoformans.J. Vis. Exp. 2014; https://doi.org/10.3791/52432Crossref Scopus (13) Google Scholar). Most importantly, cholesterol depletion resulted in a significant decrease in antibody-mediated phagocytosis of C. neoformans (38Bryan A.M. Farnoud A.M. Mor V. Del Poeta M. Macrophage cholesterol depletion and its effect on the phagocytosis of Cryptococcus neoformans.J. Vis. Exp. 2014; https://doi.org/10.3791/52432Crossref Scopus (13) Google Scholar). This finding was recapitulated across two murine macrophage cell lines. Both alveolar (Fig. 1A) and peritoneally derived macrophages (J774.1; Fig. S1A) showed significant deficiency in phagocytosing C. neoformans cells opsonized with an anti-glucuronoxylomannan (GXM) IgG, an antibody specific to the cryptococcal capsule (39Casadevall A. Cleare W. Feldmesser M. Glatman-Freedman A. Goldman D.L. Kozel T.R. Lendvai N. Mukherjee J. Pirofski L.A. Rivera J. Rosas A.L. Scharff M.D. Valadon P. Westin K. Zhong Z.J. Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies.Antimicrob. Agents Chemother. 1998; 42: 1437-1446Crossref PubMed Google Scholar). When C. neoformans cells were instead opsonized with complement serum, phagocytosis was not affected (Fig. 1C). Given the collaborative role of cholesterol and SM in lipid rafts, we examined the effect of depleting SM on the plasma membrane of macrophages. One tool available for the study of SM is recombinant bacterial sphingomyelinase (bSMase) which directly probes for the role of SM on the plasma membrane, as the enzyme is too large to pass through the membrane (40Canals D. Jenkins R.W. Roddy P. Hernandez-Corbacho M.J. Obeid L.M. Hannun Y.A. Differential effects of ceramide and sphingosine 1-phosphate on ERM phosphorylation PROBING sphingolipid signaling at the outer plasma membrane.J. Biol. Chem. 2010; 285: 32476-32485Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). bSMase catalyzes the transformation of SM into ceramide (Fig. S2) and phosphorylcholine (40Canals D. Jenkins R.W. Roddy P. Hernandez-Corbacho M.J. Obeid L.M. Hannun Y.A. Differential effects of ceramide and sphingosine 1-phosphate on ERM phosphorylation PROBING sphingolipid signaling at the outer plasma membrane.J. Biol. Chem. 2010; 285: 32476-32485Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To confirm SM depletion, cellular lipids were analyzed following treatment with 250 mU/ml or 500 mU/ml bSMase for 20 min. We found that treatment of macrophages with bSMase resulted in a significant decrease in C16 SM, the most abundant SM species detected in the cells, and a corresponding increase in C16 ceramide (Fig. 2). To assess the effect of bSMase treatment on phagocytosis, cells were coincubated with C. neoformans cells opsonized with anti-GXM IgG after bSMase treatment. We found a significant decrease in phagocytosis after bSMase treatment with both alveolar (MH-S; Fig. 1B) and peritoneally derived macrophages (J774.1; Fig. S1B), albeit in a non–dose-dependent fashion. This effect was not observed when complement serum was used as an opsonin (Fig. 1C). Together, these data suggest that cholesterol and SM are critical for IgG-mediated phagocytosis of C. neoformans. Moreover, our data suggest that this phenomenon is not cell line dependent. Given that multiple factors confound the use of MβCD to deplete cholesterol from cell membranes, various sterols were added back into the cholesterol-depleted macrophages to investigate the role of cholesterol in IgG-mediated phagocytosis of C. neoformans. MβCD may remove cholesterol from both raft and nonraft domains, alter the distribution of cholesterol between plasma and intracellular membranes, and nonspecifically extract phospholipids (41Zidovetzki R. Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies.Biochim. Biophys. Acta. 2007; 1768: 1311-1324Crossref PubMed Scopus (778) Google Scholar). To ascertain whether cholesterol sensitivity of IgG-dependent phagocytosis of C. neoformans could be attributed to lipid rafts, cholesterol-depleted macrophages were repleted with cholesterol, 7-dehydrocholesterol, or coprostanol (Fig. S2). 7-dehydrocholesterol has been shown to be significantly more domain promoting than cholesterol, whereas coprostanol strongly inhibits domain formation (36Li G. Wang Q. Kakuda S. London E. Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids.J. Lipid Res. 2020; 61: 758-766Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 42Xu X. London E. The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation.Biochemistry. 2000; 39: 843-849Crossref PubMed Scopus (441) Google Scholar, 43Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). We found that repletion with 0.2 mM cholesterol in 2.5 mM MβCD resulted in a significant increase in total cellular cholesterol compared with the untreated control. On the other hand, repletion with 0.2 mM 7-dehydrocholesterol or 0.2 mM coprostanol in 2.5 mM MβCD resulted in a significant decrease in cellular cholesterol and a marked increase in substituted sterol comparable to the cellular cholesterol for the untreated control (Fig. 3A). 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 7-dehydrocholesterol repletion, whereas coprostanol repletion did not restore phagocytosis (Fig. 3B). To assess how sterol depletion/repletion may affect lipid raft stability in macrophages, we evaluated ordered nanodomains (rafts) in giant plasma membrane vesicles (GPMVs) derived from macrophages through FRET. When rafts are present, FRET donor and FRET acceptor become partially segregated in different (raft and nonraft) domains, and FRET decreases so the FRET donor is more fluorescent (i.e., F/F0 increases) As previously, we used the temperature at which the value of F/F0 is a minimum as an approximate temperature for the upper limit of when rafts were present, that is, a measure of their thermal stability (36Li G. Wang Q. Kakuda S. London E. Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids.J. Lipid Res. 2020; 61: 758-766Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We also used the total increase in F/F0 relative to the value at which F/F0 is a minimum in a sample as another rough measure of total raft formation over the entire temperature range. GPMVs prepared from cholesterol-depleted macrophages showed a significant shift in nanodomain stability (Fig. 4A and Table S1, raw unnormalized F/F0 values for FRET data are shown in Fig. S3A). The presence of detectable ordered nanodomains significantly decreased, with up to 20 °C decrease in Tend, the temperature at which ordered domains are completely melted (36Li G. Wang Q. Kakuda S. London E. Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids.J. Lipid Res. 2020; 61: 758-766Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Upon repletion with cholesterol, detectable ordered nanodomains significantly increased compared with the untreated and cholesterol-depleted macrophages with a corresponding recovery in Tend. As expected, repletion with 7-dehydrocholesterol resulted in significantly greater presence of detectable nanodomains as well as increased thermal stability (higher Tend compared with untreated control). On the other hand, repletion with coprostanol ablated both the presence of detectable ordered nanodomains and the GPMV thermal stability (Fig. 4B and Table S1; raw unnormalized F/F0 values for FRET data are shown in Fig. S3B). To ascertain whether SM depletion alters lipid raft stability in macrophages, we also evaluated nanodomain stability in GPMVs derived from bSMase-treated macrophages. We found that neither 250 nor 500 mU/ml bSMase treatment altered nanodomain stability, with Tend and GPMV levels comparable to the untreated control (Fig. S4 and Table S1; raw unnormalized F/F0 values for FRET data are shown in Fig. S4C). It should be noted that the lipid product of bSMase digestion, ceramide, is itself a raft-forming lipid but one that alters the lipid composition (e.g., resulting in displacement of cholesterol from rafts) and properties of lipid rafts (44Megha London E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): Implications for lipid raft structure and function.J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 45Megha Bakht O. London E. Cholesterol precursors stabilize ordinary and ceramide-rich ordered lipid domains (lipid rafts) to different degrees. Implications for the bloch hypothesis and sterol biosynthesis disorders.J. Biol. Chem. 2006; 281: 21903-21913Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Together, these results not only suggest that the presence of ordered lipid domains mediate IgG-dependent phagocytosis of C. neoformans but that lipid domain stability is cholesterol dependent. Enhanced GFP (EGFP)-nakanori is a protein that labels cell surface domains in an SM-dependent and cholesterol-dependent manner. More specifically, nakanori only binds pre-existing SM/cholesterol domains as it is unable to induce formation of SM–cholesterol complexes (46Makino A. Abe M. Ishitsuka R. Murate M. Kishimoto T. Sakai S. Hullin-Matsuda F. Shimada Y. Inaba T. Miyatake H. Tanaka H. Kurahashi A. Pack C.G. Kasai R.S. Kubo S. et al.A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann- Pick type C.FASEB J. 2017; 31: 1301-1322Crossref PubMed Scopus (22) Google Scholar). To exa
Referência(s)