Investigating the function and mechanism of interleukin 7 receptor-expressing pro-angiogenic macrophages during experimental choroidal neovascularization - PROJECT SUMMARY: Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world, and is treated solely by inhibiting vascular endothelial growth factor (VEGF). Although highly effective, 15% of patients still lose vision despite maximal anti-VEGF therapy. Thus, a critical need exists for novel non-VEGF therapies. The complement pathway is genetically associated with AMD, macrophages express complement proteins and receptors, and macrophages are found in surgically excised choroidal neovascularization (CNV). Additionally, choroidal macrophage depletion results in choroidal atrophy, and both global macrophage depletion and inhibition of classical monocyte-derived macrophages reduce experimental CNV area. Therefore, macrophages are implicated in human CNV, steady state vascular homeostasis, and pathological choroidal angiogenesis. However, macrophages are known to be highly heterogeneous cells that can perform many diverse functions. Therefore, we performed single-cell RNA-sequencing on wildtype and classical monocyte- depleted mice to investigate macrophage origin, heterogeneity, and function during laser-induced CNV. We identified that interleukin-7 receptor (IL7R)-expressing macrophages are likely derived from classical monocytes and display a pro-angiogenic transcriptome. Based upon these data, our central hypothesis is that IL7R+ macrophages are critical pro-angiogenic cells during CNV pathogenesis. To test this hypothesis, we formulated the following specific aims: 1) Demonstrate that IL7R+ macrophages are necessary for CNV. In this aim, we will use Il7rCre x Rosa26GFP mice to fate map IL7R+ macrophages, Il7rCre x Cx3cr1iDTR mice to deplete IL7R+ macrophages, and Cx3cr1CrER x Il7rflox mice to knockout the IL7R. We will test the function of IL7R+ macrophages and the IL7R using the laser-induced CNV model. 2) Determine that IL7R+ macrophages are derived from classical monocytes and are sufficient to stimulate CNV. In this aim, we will use Ccr2CreER x Rosa26GFP mice to fate map classical monocytes and determine if classical monocytes can differentiate into IL7R+ macrophages. Furthermore, we will show that IL7R+ macrophages are sufficient to stimulate angiogenesis by isolating IL7R+ macrophages from mouse eyes after laser injury. We will then test the ability of IL7R+ vs IL7Rneg macrophages to rescue reduced CNV area in Ccr2-/- mice. Completion of these aims will determine that IL7R+ macrophages are necessary for CNV, derived from classical monocytes, and sufficient to stimulate angiogenesis. These data will set the stage for cell-based anti-CNV therapies. Cell-based therapies, targeting IL7R+ macrophages, have the potential to inhibit multiple pro- angiogenic pathways simultaneously, rather than inhibiting VEGF alone, and change the paradigm for nAMD therapy.
