Abstract
Purpose :
Perivascular macrophages are an understudied population. In the central nervous system, perivascular macrophages play potential roles in blood brain barrier maintenance, vascular permeability, immune cell infiltration, and clearance of debris from the perivascular space. However, their function in the retina is unknown. The goal of this study was to identify perivascular macrophages and determine their function.
Methods :
All experiments were performed on Tmem119GFP/+ mice on the C57BL6/J background. Multi-parameter flow cytometry was performed to identify perivascular macrophages. Confocal immunofluorescence microscopy was performed to quantify IBA1+GFPneg perivascular macrophages by vascular plexus depth, vessel type, and vessel diameter. CCL2 intravitreal injections were performed followed by confocal immunofluorescence at 16 hours post-injection to determine if perivascular macrophages were associated with regions of Ly6C+ inflammatory cells infiltration.
Results :
Retinal macrophages were identified by multi-parameter flow cytometry as CD45+CD11b+CD64+ cells. Using Tmem119GFP and CD169 as markers for microglia and vitreal hyalocytes respectively, we delineated microglia as GFP+ macrophages, and defined perivascular macrophages as GFPnegCD169neg cells. Perivascular macrophages represented 25% of total retinal macrophages and expressed 5.7-fold more MHCII compared to microglia (p<0.0001, N=10). IBA1+GFPneg perivascular macrophages were adjacent to 97% venules, 3% arterioles, and 0% capillaries (p<0.0001, N=11) with an average venular diameter of 28 microns. After CCL2 injection, Ly6C+ inflammatory cells were detected in densities within venules; 83.9% of perivascular macrophages were associated with a Ly6C+ cluster (p<0.0001, N=31 perivascular macrophages from 3 mice).
Conclusions :
Retinal perivascular macrophages are MHCII+ and located on ~30 micron venules in the superficial capillary plexus. Inflammatory cell extravasation occurs in small to medium post-capillary venules in other tissues. These data suggest that perivascular macrophages are antigen-presenting cells at the blood retinal barrier and regulate retinal immune cell infiltration, which is supported by their association with Ly6C+ inflammatory cells. Perivascular macrophages are potential therapeutic targets for retinal inflammatory and vascular disorders.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.