Nonlytic exocytosis of Cryptococcus neoformans from neutrophils in the brain vasculature

Abstract

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. Graphical abstract:[Figure not available: See fulltext.]