부산대학교 도서관

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis. Author summary: Dormancy enables human opportunistic pathogens to persist in the host in a limited replicative state, establishing a latent infection. Upon immunosuppression, dormant cells can reactivate and resume growth, leading to an active infection. Cryptococcus neoformans is the causative agent of cryptococcosis, a deadly yeast infection characterized by a latent phase. Building upon previous evidence that macrophages serve as a cellular reservoir for inducing and hosting dormant C. neoformans cells, we demonstrated that these phagocytes not only support dormancy, but can also paradoxically promote reactivation. Specifically, non-activated and anti-inflammatory macrophages promote the reactivation of a subset of dormant cells by the release of extracellular vesicles, as well as by yeast expulsion through a phenomenon known as non-lytic exocytosis. Importantly, dormant C. neoformans infection maintains macrophages in a non-inflammatory state required for reactivation. Our findings establish a determining role of macrophages, which is dependent on macrophage phenotype, in the maintenance or reactivation of dormant C. neoformans infection. In addition, we have uncovered a pro-pathogen role of non-lytic exocytosis and extracellular vesicle release in facilitating the reactivation of this model dormant yeast. Our study contributes to the understanding of the pathophysiology of cryptococcosis and potentially other latent infections. [ABSTRACT FROM AUTHOR]