Division of Immunology, Virginia-Maryland College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, USA.
Cell Microbiol. 2021 Jun;23(6):e13330. doi: 10.1111/cmi.13330. Epub 2021 Apr 7.
Migration of Cryptococcus neoformans from the blood to the brain parenchyma is crucial to cause fatal meningoencephalitis. Although mechanisms involved in brain migration of C. neoformans have been widely studied in vitro, less is known about how the fungus crosses the blood-brain barrier (BBB) in vivo. This is in part because of the lack of an approach to quantitatively analyse the dynamics of fungal transmigration into the brain across the BBB in vivo. In this study, we report a novel approach to quantitatively analyse the interactions between C. neoformans and brain endothelial cells in a mouse model using flow cytometry. Using this system, we show that C. neoformans was internalised by brain endothelial cells in vivo and that mice infected with acapsular or heat-killed C. neoformans yeast cells displayed a lower frequency of brain endothelial cells containing the yeast cell compared to mice infected with wild-type or viable yeast cells, respectively. We further demonstrate that brain endothelial cells were invaded by serotype A strain (H99 strain) at a higher rate compared to serotype D strain (52D strain). Our experiments established that internalisation of C. neoformans by brain endothelial cells occurred in vivo and offered a powerful approach to quantitatively analyse fungal migration into the brain.
新生隐球菌从血液向脑组织的迁移对于引起致命性脑膜脑炎至关重要。尽管已经在体外广泛研究了新生隐球菌向大脑迁移的机制，但对于真菌如何在体内穿过血脑屏障（BBB）知之甚少。这在一定程度上是因为缺乏一种方法来定量分析真菌穿过 BBB 向大脑迁移的动力学。在这项研究中，我们报告了一种使用流式细胞术在小鼠模型中定量分析新生隐球菌与脑内皮细胞相互作用的新方法。使用该系统，我们表明新生隐球菌在体内被脑内皮细胞内化，与感染野生型或活酵母细胞的小鼠相比，感染无荚膜或热杀死的新生隐球菌酵母细胞的小鼠大脑内皮细胞中包含酵母细胞的频率较低。我们进一步证明，与血清型 D 株（52D 株）相比，脑内皮细胞被血清型 A 株（H99 株）以更高的速率入侵。我们的实验证实了新生隐球菌被脑内皮细胞内化发生在体内，并提供了一种强大的方法来定量分析真菌向大脑的迁移。
