Analysis of Cell Cycle and Replication of Mouse Macrophages after In Vivo and In Vitro Cryptococcus neoformans Infection Using Laser Scanning Cytometry

We investigated the outcome of the interaction of Cryptococcus neoformanswith murine macrophages using laser scanning cy-tometry (LSC). Previous results in our lab had shown that phagocytosis of C. neoformans promoted 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, reli-able, and high-throughput method for quantifying phagocytosis. Phagocytosis of C. neoformans promoted cell cycle progres-sion, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associ-ated with intracellular C. neoformans residence 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 replication in vivo and demonstrated that these cells are capable of low levels of cell division in the presence or absence of C. neoformans infection. 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 of C. neo-formans infection onmurine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macro-phage proliferation in vivo. The interaction of the human pathogenic fungus Cryptococcusneoformans with macrophages is thought to be a key event in the outcome of cryptococcal infection (10, 13, 17, 28, 29). C. neo