Background Diabetic retinopathy and retinal vein occlusion are vision threatening retinal vascular diseases. Current first-line therapy targets the vascular component, but many patients are treatment-resistant due to unchecked inflammation. Non-invasive inflammatory imaging biomarkers are a significant unmet clinical need for patients. Imaging of macrophage-like cells on the surface of the retina using clinical optical coherence tomography (OCT) is an emerging field. These cells are increased in patients with retinal vascular disease, and could be a potential inflammatory biomarker. However, since OCT is limited by an axial resolution of 5–10 microns, the exact location and identity of these retinal cells is currently unknown. Methods We performed OCT followed by confocal immunofluorescence in wild-type mice to identify macrophages within 5–10 microns of the vitreoretinal interface. Next, we used Cx3cr1CreER/+; Rosa26zsGreen/+ mice to fate map retinal surface macrophages. Using confocal immunofluorescence of retinal sections and flatmounts, we quantified IBA1+Tmem119+CD169neg microglia, IBA1+Tmem119negCD169neg perivascular macrophages, and IBA1+Tmem119negCD169+ vitreal hyalocytes. Finally, we modeled neuroinflammation with CCL2 treatment and characterized retinal surface macrophages using flow cytometry, OCT, and confocal immunofluorescence. Results We were able to detect IBA1+ macrophages within 5–10 microns of the vitreoretinal interface in wild-type mice using OCT followed by confirmatory confocal immunofluorescence. Retinal surface macrophages were 83.5% GFP+ at Week 1 and 82.4% GFP+ at Week 4 using fate mapping mice. At steady state, these macrophages included 82% IBA1+Tmem119+CD169neg microglia, 9% IBA1+Tmem119negCD169+ vitreal hyalocytes, and 9% IBA1+Tmem119negCD169neg perivascular macrophages. After CCL2-driven neuroinflammation, many Ly6C+ cells were detectable on the retinal surface using OCT followed by confocal immunofluorescence. Conclusions Macrophages within close proximity to the vitreoretinal interface are self-renewing cells, and predominantly microglia with minor populations of perivascular macrophages and vitreal hyalocytes at steady state. In the context of neuroinflammation, monocytes and monocyte-derived macrophages are a significant component of retinal surface macrophages. Human OCT-based imaging of retinal surface macrophages is a potential biomarker for inflammation during retinal vascular disease.

Patients with neovascular AMD (nAMD) suffer vision loss from destructive angiogenesis, termed choroidal neovascularization (CNV). Macrophages are found in CNV lesions from nAMD patients. Additionally, Ccr2 -/mice, which lack classical monocyte-derived macrophages, show reduced CNV size. However, macrophages are highly diverse cells that can perform multiple functions. We performed single-cell RNA-sequencing on immune cells from wildtype and Ccr2 -/eyes to uncover macrophage heterogeneity during the laser-induced CNV mouse model of nAMD. We identified 12 macrophage clusters, including Spp1 + macrophages. Spp1 + macrophages were enriched from wildtype lasered eyes and expressed a pro-angiogenic transcriptome via multiple pathways, including vascular endothelial growth factor signaling, endothelial cell sprouting, cytokine signaling, and fibrosis. Additionally, Spp1 + macrophages expressed the marker CD11c, and CD11c + macrophages were increased by laser and present in CNV lesions. Finally, CD11c + macrophage depletion reduced CNV size by 40%. These findings broaden our understanding of ocular macrophage heterogeneity and implicate CD11c + macrophages as a potential therapeutic target for treatment-resistant nAMD patients.