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

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: CCR2Ly6C "inflammatory" monocytes involved in acute inflammation, and CX3CR1Ly6C "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2Ly6C inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1Ly6C "repair" macrophages in the brain.

Methods: CX3CR1CCR2 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 CX3CR1Ly6C monocytes, was also performed.

Results: Brain mapping of CX3CR1 and CCR2 cells 3 days after MCAo showed absence of CX3CR1 Mo/MΦ but accumulation of CCR2 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 CCR2 accrued. Whereas the amoeboid morphology of CCR2 Mo/MΦ remained unchanged over time, CX3CR1 cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1 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 CX3CR1Ly6C Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2Ly6C Mo/MΦ recruitment, but not in NR4A1 chimeric mice, which lack of circulating CX3CR1Ly6C monocytes, our data suggest a local transition of CCR2Ly6C Mo/MΦ into CX3CR1Ly6C Mo/MΦ phenotype.

Conclusions: CX3CR1Ly6C arise in the brain parenchyma from CCR2Ly6C 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.