Perivascular Macrophages Promote Leukocyte Transendothelial Migration

Abstract

Purpose : Perivascular macrophages are a distinct CD206⁺ macrophage subtype, compared to microglia, that exist between the Collagen IV vascular sheath and endothelial cells of retinal vessels. Despite being the second most abundant macrophage in the retina, the function of perivascular macrophages remains unknown.

Methods : We reanalyzed previously published single-cell RNA-sequencing (scRNA-seq) data to find potential markers for perivascular macrophages and perform differential expression. Pf4^Cre mice were crossed with Rosa26^CAG-LSL-GFP mice to target perivascular macrophages and Rosa26^CAG-LSL-DTR to deplete perivascular macrophages. We performed intravitreal CCL2 injections to determine if Ly6C⁺ monocytes were associated with perivascular macrophages and if perivascular macrophage depletion reduced leukocyte chemotaxis.

Results : In previously published scRNA-seq studies of retina isolates, we found non-microglia retinal macrophages express significantly greater Mrc1/CD206 and Pf4/CXCL4 compared to microglia. In a separate previously published scRNA-seq data set, two Mrc1⁺Pf4⁺ macrophages clusters were identified. Differential gene expression followed by gene ontology enrichment found that both clusters were enriched for lymphocyte (8-13), monocyte (8-11 fold), and neutrophil (5-9 fold) chemotaxis. In Pf4^Cre :: Rosa26^CAG-LSL-GFP mice, perivascular macrophages and hyalocytes were nearly 100% GFP⁺ while microglia were less than 5% GFP⁺ (p<0.01). In Pf4^Cre :: Rosa26^CAG-LSL-DTR mice, diphtheria toxin (DT) treatment reduced perivascular macrophages by 32% (p<0.01) with no detectable changes in microglia or hyalocytes compared to vehicle (PBS) controls. After intravitreal CCL2 injection, Ly6C⁺ monocytes were detected in close proximity to perivascular macrophages, and DT treatment decreased extravascular Ly6C⁺ monocytes by 83.6% (p<0.05).

Conclusions : Perivascular macrophages express a pro-chemotactic transcriptional profile and are necessary for leukocyte transendothelial migration during acute inflammation. These data suggest that perivascular macrophages are crucial members of the blood-brain barrier that regulate immune cell extravasation. Targeting perivascular macrophages is a potential therapeutic area for uveitis, diabetic retinopathy, and retinal vein occlusion.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.