The Early Innate Immune Response to, and Phagocyte-Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis

Abstract

The innate immune system is the primary defense against cryptococcal infection, but paradoxically it promotes infection of the central nervous system. We performed a detailed longitudinal study of neurocryptococcosis in normal, chimeric, green fluorescent protein phagocyte-positive mice and phagocyte-depleted mice and interrogated the central nervous system innate immune response to Cryptococcus neoformans H99 using confocal microscopy, histology, flow cytometry, and quantification of brain cytokine/chemokines and fungal burdens. C. neoformans was present in the perivascular space (PVS) of post-capillary venules. This was associated with a massive influx of blood-derived monocytes, neutrophils, and T lymphocytes into the PVS and a predominantly proinflammatory cytokine/chemokine response. Phagocytes containing cryptococci were present only in the lumen and corresponding PVS of post-capillary venules. Free cryptococci were observed breaching the glia limitans, the protective barrier between the PVS and the cerebral parenchyma. Parenchymal cryptococcomas were typically in direct contact with post-capillary venules and lacked surrounding immune cell infiltrates. Phagocyte depletion abrogated cryptococcoma formation and PVS infiltrates. Together, these observations suggest that cryptococcomas can originate via phagocyte-dependent transport across post-capillary venular endothelium into the PVS and thence via passage of free cryptococci into the brain. In conclusion, we demonstrate for the first time that the PVS of cortical post-capillary venules is the major site of the early innate immune response to, and phagocyte-dependent entry of, C. neoformans.

Intranasal inoculation ofCryptococcus neoformansin mice produces nasal infection with rapid brain dissemination

“…2B). It is possible that the detected yeast burden is arrested in the small capillaries (23–26), or in the post-capillary venules (27) and has not yet invaded the brain parenchyma. However, this is consistent with invasion of the brain since upon arrest in capillaries, C. neoformans quickly crosses endothelial barriers (24) and possibly the blood brain-barrier.…”

“…We detected no yeasts in the circulating blood of mice, which raises a question of how the bloodstream is so quickly invaded and quickly cleared. Other groups observed that in IV infection fungi are cleared from the bloodstream in hours or the day after infection (27, 35), with fungemia only resurfacing very late in the infection. These observations, together with ours, imply that yeast transit time in blood is very short (25).…”

“…These observations, together with ours, imply that yeast transit time in blood is very short (25). The short transit time is seemingly due to arrest of yeasts within small capillaries, as can occur in the ears of mice (24) and in the lumen of leptomeningeal capillaries (24, 27). Yeasts arrested in capillaries would not be detected by our methodology.…”

VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection

“…Although C. neoformans can enter the brain through direct invasion of the brain endothelial cells [21][22][23][24][25], evidence has been provided that mononuclear phagocytes promote brain invasion of the organism [21,26,27]. C. neoformans can survive in monocytes [28,29], and monocytes containing C. neoformans have been found present in the perivascular space of the brain [23,27]. Intravenous administration of C. neoformans-infected macrophages can enhance the brain fungal burden [26], while depletion of monocytes can reduce the brain fungal burden [26,27].…”

“…C. neoformans can survive in monocytes [28,29], and monocytes containing C. neoformans have been found present in the perivascular space of the brain [23,27]. Intravenous administration of C. neoformans-infected macrophages can enhance the brain fungal burden [26], while depletion of monocytes can reduce the brain fungal burden [26,27]. Notably, monocytes harboring C. neoformans have been directly seen to cross a monolayer of brain endothelial cells cultured in vitro [30,31].…”