Intracellular cryptococci suppress Fc-mediated cyclin D1 elevation

Abstract

Cryptococcus neoformans (Cn) is the only encapsulated fungal pathogen pathogenic to humans and macrophages play a vital role in the Cn infection pathway. Previously we documented that phagocytosis or crosslinking of FcγR on macrophage cell surface promoted macrophage cell cycle progression via the activation of MAPK pathway. However, we seldom observed sustained macrophage growth after they phagocytosed live Cn. In fact, many macrophages were finally observed to undergo apoptotic changes after phagocytosis of live Cn. Here we report that cyclin D1 elevation in macrophages promoted by phagocytosis was suppressed by intracellular Cn cells. This suggests a novel cytotoxic mechanism for host cells of intracellular cryptococci.

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Cryptococcus neoformans (Cn) is the only encapsulated fungal pathogen pathogenic to human and a major cause of life-threatening infections such as pulmonary cryptococosis and meningoencephalitis in patients with impaired immunity.1,2 Macrophages play a vital role in the Cn infection pathway.3–9 In previous studies using macrophage—Cn interaction as a model, we described that phagocytosis or crosslinking of FcγR on macrophage cell surface promoted macrophage cell cycle progression. Furthermore, we showed that the molecular mechanism underlying this phenomenon involved the activation of MAPK pathway in macrophages leading to the activation of major cell cycle machinery such as cyclin D1 expression.10,11 This phenomenon was commonly observed following phagocytosis of both dead Cn cells as well as inert polystyrene particles. However, we seldom observed sustained macrophage growth after they phagocytosed live Cn, even though transient mitotic figures in macrophages were observed after phagocytosis of live Cn. In fact, many macrophages were observed to undergo apoptotic changes after phagocytosis of live Cn after a relatively long period of co-incubation.

Given the fact that previous reports documented toxic effect of intracellular live Cn on macrophages,3,4,12,13 we suspected that there might be a underlying mechanism by which live Cn could induce the cytotoxic changes of macrophages intracellularly. To explore the mechanism for the relative paucity of cell cycle progression after phagocytosis live Cn, we probed cyclin D1 expression levels in bone marrow macrophages after Fc-mediated phagocytosis assay by western blots (Fig. 1). Bone marrow macrophages were obtained from mice and cultured with M-CSF. Before the phagocytosis assay, the cells were deprived of M-CSF for 2 d and the cells were synchronized in G₁ phase. Monoclonal antibody (mAb) 18B7 was used as opsonin for Fc-mediated phagocytosis as previously described.10,11 For comparison, we incubated macrophages with mAb 18B7 alone, live Cn opsonized with mAb 18B7 or polystyrene beads opsonized with mAb 18B7 at an Effector: Target (E:T) ratio of 1:1. In the control group, mAb 18B7 was added without the presence of live Cn or polystyrene beads. The cyclin D1 expression was not detectable after phagocytosis until 4 h post stimulation when a transient increase of cyclin D1 expression was noted. This might be due to spontaneous crosslinking of FcγR on macrophages through mAb 18B7. However, in the group that live Cn were phagocytosed, cyclin D1 expression increased as early as 0.5 h with a strong signal. These results were consistent with our previous findings that phagocytosis or crosslinking of FcγR on macrophage cell surface promoted macrophage cell cycle progression.10,11 However cyclin D1 levels decreased 6 h post ingestion of live Cn cells, possibly reflecting a fungal-mediated toxic effect on macrophage cell cycle. To test this possibility, phagocytosis assay was performed with inert polystyrene beads opsonized by mAb 18B7. Cyclin D1 expression was consistently increased after 0.5 h post stimulation and high levels were maintained at least until 6 h. No decrease of cyclin D1 expression was observed after 6 h. This result suggested that phagocytosed live Cn suppressed macrophage growth by decrease cyclin D1 expression which is vital in cell cycle progression of macrophages. This finding in turn suggests a novel cytotoxic mechanism for host cells of intracellular cryptococci. At this time we do not know if the suppression in cyclin D1 expression is a specific mechanism of fungal damage or a consequence of generalized cytotoxity mediated by intracellular cryptococci. The phenomenon is worthwhile to further explore given the importance of macrophage growth in host defense.

Abbreviations

Cn	=	Cryptococcus neoformans
mAb	=	monoclonal antibody

Figures and Tables

Figure 1 Western blots of Cyclin D1 in bone marrow macrophage cell extract after Fc-mediated phagocytosis of live Cn or polystyrene beads. Bone marrow macrophage cells were challenged with mAb 18B7-opsonized live Cn or polystyrene beads at an E:T ratio of 1:1. In the negative control group, mAb 18B7 without the presence of particles was added. Macrophage cell contents were extracted after 0, 0.5, 2, 4 and 6 h post challenge. Cyclin D1 expression levels in the cell extracts after phagocytosis assay were probed by western blots. The same loading amount for each sample was confirmed by β-tubulin western blots. The experiment has been independently repeated three times and produced similar results.

Acknowledgement

A. Casadevall is supported by NIH grants HL059842, AI033774, AI033142, and AI052733.

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