FIG. 5.

Intracellular survival of C. neoformans var. neoformans spores is dependent on alveolar macrophage activation state. (A) C. neoformans var. neoformans spores were cocultured with untreated alveolar macrophages, and intracellular germination was assessed 24 h postinfection. Numerous budding yeast (white arrowheads) enveloped in capsule (black arrowheads) were observed inside alveolar macrophages. (B) Within 24 h, intracellular C. neoformans var. neoformans spores germinated into yeast (white arrowheads). Confocal microscopy of calcofluor labeled yeast inside actin stained alveolar macrophages showed yeast exiting intact alveolar macrophages (yellow arrow). (C) Three-dimensional reconstruction of a deconvolved confocal image of calcofluor labeled yeast inside actin-stained alveolar macrophages. The three-dimensional image confirmed the intracellular localization of yeast (white arrowheads) and exit of yeast from the alveolar macrophages (yellow arrowhead). The images are representative of three independent experiments. (D) C. neoformans var. neoformans spores were cocultured with alveolar macrophages (AM) treated with LPS and IFN-γ (Stim.) or left untreated as a control. After 4 h of coculture the wells were washed to remove extracellular spores and yeast, and cultures were allowed to continue for 24 h. Phagocytosis (time point [TP] 0 h) and inhibitory activity (TP 24 h) were assessed by CFU analysis. The results demonstrate that activation state affects alveolar macrophage (AM) antifungal activity. (E) An identical experiment was performed with spores and yeast opsonized with anti-GXM IgG. Activated macrophages inhibited the growth of spores, but not yeast. The bars represent means plus the standard errors.