Spatio-temporal profile, phenotypic diversity, and fate of recruited monocytes into the post-ischemic brain

Abstract

Background: A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2+Ly6Chi "inflammatory" monocytes involved in acute inflammation, and CX3CR1+Ly6Clo "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2+Ly6Chi inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1+Ly6Clo "repair" macrophages in the brain. Methods: CX3CR1GFP/+CCR2RFP/+ bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2-/- mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1-/- BM chimeric mice, which lack circulating CX3CR1+Ly6Clo monocytes, was also performed. Results: Brain mapping of CX3CR1GFP/+ and CCR2RFP/+ cells 3 days after MCAo showed absence of CX3CR1GFP/+ Mo/MΦ but accumulation of CCR2RFP/+ Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1+ cells accumulated 14 days after MCAo at the border of the infarct core where CCR2RFP/+ accrued. Whereas the amoeboid morphology of CCR2RFP/+ Mo/MΦ remained unchanged over time, CX3CR1GFP/+ cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1GFP/+ cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1+Ly6Clo Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2+Ly6Chi Mo/MΦ recruitment, but not in NR4A1-/- chimeric mice, which lack of circulating CX3CR1+Ly6Clo monocytes, our data suggest a local transition of CCR2+Ly6Chi Mo/MΦ into CX3CR1+Ly6Clo Mo/MΦ phenotype. Conclusions: CX3CR1+Ly6Clo arise in the brain parenchyma from CCR2+Ly6Chi Mo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.