Perivascular Macrophages Are Associated With Permissive Sites of Immune Cell Extravasation in Retinal Venules

Purpose: Perivascular macrophages (pvMacs) exist on venules at the blood-retina barrier (BRB) and are important for leukocyte transendothelial migration during retinal inflammation. The venular basement membrane contains low expression regions (LERs), which act as permissive sites for extravasation in non-ocular tissues. We sought to identify LERs in retinal venules and determine their relationship with pvMacs at steady state and during inflammation. Methods: Retinal flatmounts from Pf4Cre:Rosa26zsGreen/+ mice were analyzed by confocal microscopy followed by three-dimensional reconstruction to visualize pvMacs, collagen IV, laminin, and pericytes. Qualitative and quantitative analysis of LERs was performed at steady state and after induction of the endotoxin-induced uveitis (EIU) model. The role of pvMacs and macrophages in EIU were investigated using diphtheria toxin (DT) in Pf4Cre:Rosa26DTR/+ mice or a colony-stimulating factor 1 receptor (CSF1R) antagonist, respectively. Results: Retinal venules contained discrete LERs characterized by reduced expression of both collagen IV and laminin with concurrent absence of pericyte coverage. pvMacs resided within LERs and adjacent to LERs. During EIU, neutrophils extravasated through pvMac-adjacent LERs, resulting in increased LER area and irregular morphology. Extravascular neutrophils co-localized with collagen IV fragments, suggesting basement-membrane degradation. Depletion of pvMacs or all macrophages did not alter neutrophil or monocyte infiltration, indicating that EIU-driven inflammation is macrophage independent. Conclusions: LERs are features of retinal venules that serve as permissive sites for immune cell extravasation and are closely associated with pvMacs. Although macrophages are dispensable for EIU, pvMac-LER complexes likely represent regulated portals for leukocyte trafficking and are potential loci of chronic BRB breakdown during retinal inflammation.