Analysis of Cell Cycle and Replication of Mouse Macrophages afterIn VivoandIn VitroCryptococcus neoformans Infection Using Laser Scanning Cytometry

Abstract

ABSTRACTWe investigated the outcome of the interaction ofCryptococcus neoformanswith murine macrophages using laser scanning cytometry (LSC). Previous results in our lab had shown that phagocytosis ofC. neoformanspromoted cell cycle progression. LSC allowed us to simultaneously measure the phagocytic index, macrophage DNA content, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation such that it was possible to study host cell division as a function of phagocytosis. LSC proved to be a robust, reliable, and high-throughput method for quantifying phagocytosis. Phagocytosis ofC. neoformanspromoted cell cycle progression, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associated with intracellularC. neoformansresidence that manifested itself in impaired cell cycle completion as a consequence of a block in the G2/M stage of the mitotic cell cycle. Cell cycle arrest was not due to increased cell membrane permeability or DNA damage. We investigated alveolar macrophage replicationin vivoand demonstrated that these cells are capable of low levels of cell division in the presence or absence ofC. neoformansinfection. In summary, we simultaneously studied phagocytosis, the cell cycle state of the host cell and pathogen-mediated cytotoxicity, and our results demonstrate a new cytotoxic effect ofC. neoformansinfection on murine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macrophage proliferationin vivo.