Imaging the dynamic recruitment of monocytes to the blood–brain barrier and specific brain regions during<i>Toxoplasma gondii</i>infection

Abstract

Brain infection by the parasite <i>Toxoplasma gondii</i> in mice is thought to generate vulnerability to predation by mechanisms that remain elusive. Monocytes play a key role in host defense and inflammation and are critical for controlling <i>T. gondii</i> However, the dynamic and regional relationship between brain-infiltrating monocytes and parasites is unknown. We report the mobilization of inflammatory (CCR2⁺Ly6C^hi) and patrolling (CX3CR1⁺Ly6C^lo) monocytes into the blood and brain during <i>T. gondii</i> infection of C57BL/6J and CCR2^RFP/+CX3CR1^GFP/+ mice. Longitudinal analysis of mice using 2-photon intravital imaging of the brain through cranial windows revealed that CCR2-RFP monocytes were recruited to the blood-brain barrier (BBB) within 2 wk of <i>T. gondii</i> infection, exhibited distinct rolling and crawling behavior, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact <i>T. gondii</i>-infected brains using iDISCO⁺ and light-sheet microscopy enabled global 3D detection of monocytes. Clusters of <i>T. gondii</i> and individual monocytes across the brain were identified using an automated cell segmentation pipeline, and monocytes were found to be significantly correlated with sites of <i>T. gondii</i> clusters. Computational alignment of brains to the Allen annotated reference atlas [E. S. Lein et al., <i>Nature</i> 445:168-176 (2007)] indicated a consistent pattern of monocyte infiltration during <i>T. gondii</i> infection to the olfactory tubercle, in contrast to LPS treatment of mice, which resulted in a diffuse distribution of monocytes across multiple brain regions. These data provide insights into the dynamics of monocyte recruitment to the BBB and the highly regionalized localization of monocytes in the brain during <i>T. gondii</i> CNS infection.