BACKGROUND Cryptococcus neoformans (C. neoformans) is an encapsulated budding yeast that causes life-threatening meningoencephalitis in immunocompromised individuals, especially those with acquired immunodeficiency syndrome (AIDS). To cause meningoencephalitis, C. neoformans circulating in the bloodstream must first be arrested in the brain microvasculature. Neutrophils, the most abundant phagocytes in the bloodstream and the first leukocytes to be recruited to an infection site, can ingest C. neoformans. Little is known about how neutrophils interact with arrested fungal cells in the brain microvasculature. METHODS A blood-brain barrier (BBB) in vitro model was established. The interactions between neutrophils adhering to brain endothelial cells and fungi were observed under a live cell imaging microscope. A flow cytometry assay was developed to explore the mechanisms. Immunofluorescence staining of brain tissues was utilized to validate the in vitro phenomena. RESULTS Using real-time imaging, we observed that neutrophils adhered to a monolayer of mouse brain endothelial cells could expel ingested C. neoformans without lysis of the neutrophils or fungi in vitro, demonstrating nonlytic exocytosis of fungal cells from neutrophils. Furthermore, nonlytic exocytosis of C. neoformans from neutrophils was influenced by either the fungus (capsule and viability) or the neutrophil (phagosomal pH and actin polymerization). Moreover, nonlytic exocytosis of C. neoformans from neutrophils was recorded in brain tissue. CONCLUSION These results highlight a novel function by which neutrophils extrude C. neoformans in the brain vasculature.

中文翻译：

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来自脑血管系统中性粒细胞的新隐球菌的非溶胞作用。

背景技术新型隐球菌（C.neoformans）是一种封装的发芽酵母，其在免疫受损的个体，特别是患有获得性免疫缺陷综合症（AIDS）的个体中引起危及生命的脑膜脑炎。为了引起脑膜脑炎，必须首先将在血液中循环的新孢子虫停滞在脑微血管中。中性粒细胞是血液中最丰富的吞噬细胞，也是最早募集到感染部位的白细胞，可以摄入新形成梭状芽胞杆菌。关于中性粒细胞如何与大脑微脉管系统中停滞的真菌细胞相互作用，人们知之甚少。方法建立血脑屏障（BBB）体外模型。在活细胞成像显微镜下观察到粘附于脑内皮细胞的嗜中性粒细胞与真菌之间的相互作用。开发了流式细胞术分析以探索其机制。脑组织的免疫荧光染色被用来验证体外现象。结果使用实时成像，我们观察到粘附在单层小鼠脑内皮细胞上的嗜中性粒细胞可以在不裂解嗜中性粒细胞或真菌体外的情况下将被摄入的新孢子虫排出，这表明嗜中性粒细胞对真菌细胞的不溶性胞吐作用。此外，真菌（胶囊和生存力）或嗜中性粒细胞（吞噬体pH和肌动蛋白聚合）都影响嗜中性粒细胞新孢梭菌的非溶胞作用。而且，在脑组织中记录了来自嗜中性粒细胞的新形成的梭状芽胞杆菌的非溶解性胞吐作用。结论这些结果突出了嗜中性粒细胞通过其在脑血管系统中挤出新形成梭状芽胞杆菌的新功能。脑组织的免疫荧光染色被用来验证体外现象。结果使用实时成像，我们观察到粘附在单层小鼠脑内皮细胞上的嗜中性粒细胞可以在不裂解嗜中性粒细胞或真菌体外的情况下将被摄入的新孢子虫排出，这表明嗜中性粒细胞对真菌细胞的不溶性胞吐作用。此外，来自嗜中性粒细胞的新孢梭菌的非溶胞作用受真菌（胶囊和生存力）或嗜中性粒细胞（吞噬体pH和肌动蛋白聚合）的影响。而且，在脑组织中记录了来自嗜中性粒细胞的新形成的梭状芽胞杆菌的非溶解性胞吐作用。结论这些结果突出了嗜中性粒细胞通过其在脑血管系统中挤出新形成梭状芽胞杆菌的新功能。脑组织的免疫荧光染色被用来验证体外现象。结果使用实时成像，我们观察到粘附在单层小鼠脑内皮细胞上的嗜中性粒细胞可以在不裂解嗜中性粒细胞或真菌体外的情况下将被摄入的新孢子虫排出，这表明嗜中性粒细胞对真菌细胞的不溶性胞吐作用。此外，来自嗜中性粒细胞的新孢梭菌的非溶胞作用受真菌（胶囊和生存力）或嗜中性粒细胞（吞噬体pH和肌动蛋白聚合）的影响。而且，在脑组织中记录了来自嗜中性粒细胞的新形成的梭状芽胞杆菌的非溶解性胞吐作用。结论这些结果突出了嗜中性粒细胞通过其在脑血管系统中挤出新形成梭状芽胞杆菌的新功能。