Ocular Macrophage Origin and Heterogeneity During Steady State and Experimental Choroidal Neovascularization

Background: Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored.

Methods: We performed multi-parameter flow cytometry on wildtype (WT) and Ccr2 mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1 ; Rosa26 mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function.

Results: We identified 4 macrophage subsets in mice: microglia, MHCIICD11c, MHCIICD11c, and MHCII. Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII macrophages are long lived, tissue resident macrophages, while MHCIICD11c and MHCIICD11c macrophages are partially replenished from blood monocytes. After laser injury, MHCIICD11c macrophages are entirely derived from classical monocytes, MHCII macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCIICD11c macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCIICD11C and three MHCIICD11C populations. One MHCIICD11C subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCIICD11C and two MHCIICD11C subsets, including the disease-associated cluster. The upregulated MHCIICD11C pro-angiogenic genes were unique compared to the increased MHCIICD11C angiogenesis genes.

Conclusions: Macrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.