Defining molecular identity and fates of CNS-border associated macrophages after ischemic stroke in rodents and humans

Abstract

Central nervous system (CNS)-border associated macrophages (BAMs) maintain their steady-state population during adulthood and are not replaced by circulating monocytes under physiological conditions. Their roles in CNS integrity and functions under pathological conditions remain largely unknown. Until recently, BAMs and microglia could not be unequivocally distinguished due to expression of common macrophage markers. We investigated the transcriptional profiles of immunosorted BAMs from rat sham-operated and ischemic brains using RNA sequencing. We found that BAMs express the distinct transcriptional signature than microglia and infiltrating macrophages. The enrichment of functional groups associated with the cell cycle in CD163⁺ cells isolated 3 days after the ischemic injury indicates the proliferative capacity of these cells. The increased number of CD163⁺ cells 3 days post-ischemia was corroborated by flow cytometry and detecting the increased number of CD163⁺ cells positive for a proliferation marker Ki67 at perivascular spaces. CD163⁺ cells in the ischemic brains up-regulated many inflammatory genes and parenchymal CD163+ cells expressed iNOS, which indicates acquisition of a pro-inflammatory phenotype. In mice, BAMs typically express CD206 and we found a subset of these cells expressing CD169. Chimeric mice generated by transplanting bone marrow of donor Cx3cr1^gfpCCR2^rfp mice to wild type hosts showed an increased number of CX3CR1⁺CD169⁺ perivascular macrophages 3 days post-ischemia. Furthermore, these cells accumulated in the brain parenchyma and we detected replacement of perivascular macrophages by peripheral monocytic cells in the sub-acute phase of stroke. In line with the animal results, post-mortem brain samples from human ischemic stroke cases showed time-dependent accumulation of CD163⁺ cells in the ischemic parenchyma. Our findings indicate a unique transcriptional signature of BAMs, their local proliferation and migration of inflammatory BAMs to the brain parenchyma after stroke in animal models and humans.

Keywords: CNS border associated macrophages; Chimeric mice; Human ischemic stroke; Ischemia; RNA-sequencing.