Macrophages in close proximity to the vitreoretinal interface are potential biomarkers of inflammation during retinal vascular disease

Abstract

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.

Changes in macrophage-like cells characterized by en face optical coherence tomography after retinal stroke

“…Recently, MLCs on the inner limiting membrane in mice were confirmed to be self-renewing cells and predominantly microglia with minor populations of perivascular macrophages and vitreal hyalocytes at steady state, but predominantly monocytes and monocyte-derived macrophages under neuroinflammation ( 30 ). We and others have demonstrated signs of aggregation and activation of MLCs following retinal stroke, retinal vein occlusion, diabetic retinopathy and Behçet’s uveitis ( 17 , 18 , 31 ).…”

“…Thus, the MLCs characterized by en face OCT could also comprise other immune cells recruited from blood circulation. During neuroinflammation in mouse model, Ly6C+ monocytes and monocyte-derived macrophages are significant additional components of the heterogenous vitreoretinal interface macrophage population ( 30 ). Recruited MLCs might migrate to ischemic retina, observed in a scattered distribution without obvious perivascular clustering or gathering.…”

Macrophage-like Cells Are Increased in Patients with Vision-Threatening Diabetic Retinopathy and Correlate with Macular Edema

“…We recently investigated the identity of MLCs in mice. During steady state, MLCs include microglia, perivascular macrophages, and vitreal hyalocytes [ 11 ]. After monocyte chemoattract protein-1 (MCP-1)-driven neuroinflammation, which promotes monocyte infiltration into tissue, MLCs also include classical monocytes and monocyte-derived macrophages [ 11 ].…”

“…During steady state, MLCs include microglia, perivascular macrophages, and vitreal hyalocytes [ 11 ]. After monocyte chemoattract protein-1 (MCP-1)-driven neuroinflammation, which promotes monocyte infiltration into tissue, MLCs also include classical monocytes and monocyte-derived macrophages [ 11 ]. MCP-1 is consistently increased in aqueous and vitreous samples from patients with DR, and MCP-1 correlates with macular edema [ 20 ].…”

“…MLCs possess properties associated with macrophages including mobility, a ramified morphology [ 9 ], and correspondence to Cx3cr1 + cells (a macrophage marker) in mice [ 10 ]. Furthermore, we recently showed that clinical MLCs correspond to microglia, vitreal hyalocytes, and perivascular macrophages at steady state, and additionally include classical monocytes and monocyte-derived macrophages during neuroinflammation [ 11 ]. Furthermore, MLCs are increased in eyes with Behcet’s uveitis and are correlated with worsened fluorescein leakage, suggesting their role in retinal inflammation [ 12 ].…”