FUNCTIONAL SPECIALIZATION OF BORDER-ASSOCIATED MACROPHAGES IS DEFINED BY NICHE-SPECIFIC TRANSCRIPTOMIC AND ULTRASTRUCTURAL PROFILES IN MOUSE AND HUMAN

Border-associated macrophages (BAMs) are a specialized population of tissue-resident macrophages at interfaces between the central nervous system and the periphery, including the meninges, choroid plexus, and perivascular spaces. Meningeal macrophages are categorized into dural and leptomeningeal types, which differ both anatomically and transcriptomically. Differential expression of MHC Class II (MHCII) and Lyve1 is a hallmark of this heterogeneity. We conducted a multi-modal investigation to 1) validate these transcriptomic distinctions, 2) determine if they translate to protein and ultrastructural features, and 3) assess their conservation in humans. By integrating single-cell RNA sequencing datasets from mice and humans, we confirmed the regional heterogeneity of BAMs. Dural macrophages were predominantly MHCII-high, while leptomeningeal and perivascular macrophages were Lyve1-high. Within MHCII-high dural cells, we identified a subtype with monocyte-like transcriptomic features and low expression of canonical BAM genes, distinct from another subtype resembling differentiated, tissue-resident macrophages. Lyve1⁺ BAMs contained abundant phagocytic inclusions, autophagosomes, and lipid droplets, consistent with roles in phagocytosis and metabolic support. In contrast, MHCII⁺ BAMs exhibited a well-developed endoplasmic reticulum, typical of antigen-presenting cells. We also identified a novel BAM subpopulation with low MHCII and Lyve1 expression, enriched for interferon (IFN)-responsive genes. Human single-cell data showed a conserved specialization pattern, including segregation of MHCII⁺ and Lyve1⁺ BAMs and an IFN-responsive cluster. Histological analysis of postmortem human brain confirmed the spatial localization of these subtypes. Our study provides a multi-modal atlas of BAMs, demonstrating that their functional specialization is closely linked to anatomical niches and defined by distinct transcriptomic and ultrastructural features.