Real-time in vivo imaging of interactions of phagocytes with disseminating Cryptococcus neoformans in the brain vasculature

Cryptococcus neoformans is an encapsulated pathogenic fungus, accounting for 180,000 deaths worldwide annually. The infection starts in the lung and the yeast cells can disseminate to the bloodstream, particularly in HIV patients due to impaired cellular immunity. Once the organisms enter the blood, they can invade the brain across the blood-brain barrier, causing fatal meningoencephalitis. The role of phagocytes during the invasion of C. neoformans into the brain remains incompletely understood. Here, with the use of intravital microscopy, we study the dynamic interactions of neutrophils and monocytes with disseminating C. neoformans in the brain vasculature of infected mice. We observed that neutrophils migrate to the arrested C. neoformans in the luminal surface of brain vasculature. Following interactions with C. neoformans, neutrophils were seen to internalize the organism and then circulate back into the bloodstream, resulting in a direct removal of the organism from the endothelial surface before its transmigration into the brain parenchyma. Complement C3 was critically involved in the recognition of C. neoformans by neutrophils and subsequent clearance of the organism from the brain. We also observed a substantial recruitment of monocytes, predominantly Ly6Clow monocytes in the brain vasculature during infection. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. Furthermore, TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Interestingly, the recruited Ly6Clow monocytes internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. These data are the first to characterize directly the dynamic interactions of leukocytes with a microbe in the brain of a living animal and demonstrate a different role of neutrophils and monocytes during brain invasion of C. neoformans.