Macrophage origin influences heterogeneity during experimental choroidal neovascularization

Abstract

Purpose : Macrophages (Macs) are complex, heterogenous cells that are found in human choroidal neovascularization (CNV) and are critical for experimental CNV. Macs have diverse functions including inflammation, fibrosis, and angiogenesis, which are determined by both their origin and tissue microenvironment. We hypothesize that Mac origin influences heterogeneity, and determines function during experimental CNV.

Methods : We generated Cx3cr1^CreER/+;Rosa26^zsGFP/+ female mice (Mac^GFP) to fate map Mac origin. We used female Ccr2^-/- mice to identify classical monocyte-derived Macs. Mac^GFP mice were treated with tamoxifen at 4-6 weeks of age. Mice underwent laser-induced CNV at 10-12 weeks of age. Multi-parameter flow cytometry was performed on blood (3-5 days and 4-6 weeks post-tamoxifen) and eyes (Day 3 post-laser, the peak of Mac recruitment). We identified four CD64⁺ populations: microglia (CD45^dim), MHCII- (CD45^highMHCII^-), CD11c- (CD45^highMHCII⁺CD11c^-), and CD11c+ (CD45^highMHCII⁺CD11c⁺) Macs.

Results : In Mac^GFP mice, blood monocytes were 72.3% GFP+ on Day 3-5 and 1.4% GFP+ on Week 4-6 post-tamoxifen (N=4-5, p<0.0001), confirming that ocular GFP+ cells are tissue resident Macs. In wildtype (WT) and Ccr2^-/- mice, microglia numbers were unchanged by laser or genotype. In Mac^GFP mice, 97.2% of microglia were GFP+, and decreased to 83.7% with laser (N=8-9, p<0.01). MHCII-, CD11c-, and CD11c+ Macs were 30.2-61.1% GFP+ without laser and were reduced to 4.8-12.0% with laser treatment (N=8-9, p<0.001 for each). MHCII- Macs increased from 420 to 6126 cells (N=9, p<0.01) with laser in WT mice, but only expanded from 256 to 524 cells (N=8, p<0.05) in lasered Ccr2^-/- mice, which was 91.5% less than lasered WT mice (p<0.01). CD11c- Mac numbers grew 9.6-fold with laser in WT mice (p=0.001), but were unchanged in Ccr2^-/-mice. CD11c+ Macs increased 12.7-fold in WT mice (p<0.001) and 4.3 fold in Ccr2^-/- mice (p<0.01) with laser treatment, a 75% reduction between lasered WT and Ccr2^-/- mice (p<0.01).

Conclusions : Microglia are tissue resident Macs that are partially replenished after laser injury independent from classical monocytes. Mac infiltration after laser injury is 100% and >90% from classical monocyte-derived Macs in CD11c- and MHCII- populations, respectively. CD11c+ Mac recruitment is from 75% classical and 25% non-classical monocyte-derived Macs. These data demonstrate that origin affects ocular Mac heterogeneity.

This is a 2020 ARVO Annual Meeting abstract.