title: Macrophage cholesterol depletion and its effect on the phagocytosis of Cryptococcus neoformans.
abstract: Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University.
authors.forename: Arielle M
authors.initials: AM
authors.lastname: Bryan
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University.
authors.forename: Amir M
authors.initials: AM
authors.lastname: Farnoud
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University.
authors.forename: Visesato
authors.initials: V
authors.lastname: Mor
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University; maurizio.delpoeta@stonybrook.edu.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Del Poeta
mesh_terms: D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000172:deficiency* / Q000276:immunology / Q000378:metabolism; D003453:Cryptococcosis / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000276:immunology*; D054884:Host-Pathogen Interactions; D007371:Interferon-gamma / Q000276:immunology; D008168:Lung / Q000276:immunology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology*; D051379:Mice; D010587:Phagocytosis / Q000276:immunology*; D011134:Polysaccharides / Q000276:immunology
chemical_list: D011134:Polysaccharides; C027478:glucuronoxylomannan; D007371:Interferon-gamma; D002784:Cholesterol