Abstract
Purpose :
Macrophages are heterogeneous cells that are important during neovascular age-related macular degeneration, and their function depends upon their origin. Cx3cr1-knockout mice show increased laser-induced choroidal neovascularization (CNV) but are deficient in both non-classical monocytes and display microglia alterations. NR4A1 is a transcription factor that is necessary for both differentiation of non-classical monocytes and regulation of macrophage function. Deficiency in the super enhancer 2 (se2) promoter region impairs non-classical monocyte differentiation without affecting NR4A1 function in macrophages. In this study, we investigated Nr4a1-/- and Nr4a1se2/se2 mice to determine the role of non-classical monocytes and NR4A1 during CNV.
Methods :
All experiments were performed on 10-12 week-old mice on the C57BL6/J background. Choroidal explants were cultured from wildtype (WT) and Nr4a1-/- mice for 4 days in Matrigel. WT, Nr4a1-/-, and Nr4a1se2/se2 mice underwent laser-induced CNV. Multi-parameter flow cytometry was performed on Day 3 to investigate macrophage infiltration. CNV area was measured by immunofluorescence on Day 7.
Results :
In the choroidal sprouting assay, WT and Nr4a1-/- mice showed identical angiogenenic growth, suggesting that NR4A1-deficiency does not affect endothelial cells. Nr4a1-/- and Nr4a1se2/se2 mice demonstrated a 63% (p<0.001) and 69% (p<0.001) reduction in non-classical monocytes numbers compared to wildtype mice, confirming the fidelity of each model. Alternatively, macrophages were significantly increased after laser by 2.7-fold in wildtype mice (p<0.001), 3.8-fold in Nr4a1-/- mice (p<0.001) and by 4.2-fold in Nr4a1se2/se2 mice (p<0.01) with no significant differences between groups. Importantly, Nr4a1-/- mice demonstrated a 1.7-fold increase (p<0.01, N=21-22 mice per group) in CNV area without sex-specific effects compared to wildtype mice, while Nr4a1se2/se2 mice showed no change in CNV size compared to wildtype mice (p=0.92, N=32-35 mice).
Conclusions :
While Nr4a1-/- and Nr4a1se2/se2 mice are both deficient in non-classical monocytes, no change in total macrophage recruitment was detected compared to wildtype mice. These data suggest that non-classical monocyte-derived macrophages are dispensable during laser-induced CNV. Since only Nr4a1-/- mice have increased laser-induced CNV area, NR4A1 expression in macrophages reduces pro-angiogenic function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.