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

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 Cx3cr1(CreER/+); Rosa26(zsGreen/+) mice to fate map retinal surface macrophages. Using confocal immunofluorescence of retinal sections and flatmounts, we quantified IBA1(+)Tmem119(+)CD169(neg) microglia, IBA1(+)Tmem119(neg)CD169(neg) perivascular macrophages, and IBA1(+)Tmem119(neg)CD169(+) 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(+)CD169(neg) microglia, 9% IBA1(+)Tmem119(neg)CD169(+) vitreal hyalocytes, and 9% IBA1(+)Tmem119(neg)CD169(neg) 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.

Automated summary

It has been demonstrated that macrophages within close proximity to the vitreoretinal interface are predominantly self-renewing microglia, with minor populations of perivascular macrophages and vitreal hyalocytes. In the context of neuroinflammation, monocytes and monocyte-derived macrophages also play a significant role in the composition of retinal surface macrophages. Furthermore, human OCT-based imaging of retinal surface macrophages has the potential to serve as a biomarker for inflammation during retinal vascular disease.