Abstract
Purpose :
Despite monthly intravitreal injection therapy, 15% of neovascular age-related macular degeneration (nAMD) patients lose vision. Interleukin-6 (IL6) is a pro-inflammatory and pro-angiogenic cytokine that is correlated with AMD progression and nAMD activity. We hypothesize that anti-IL6 therapy is a potential therapeutic for nAMD.
Methods :
All experiments were performed on 10-12 week-old C57BL6/J mice. Wildtype (WT) and Il6-/- mice underwent laser injury. Choroidal neovascularization (CNV) area was measured on choroidal flatmounts by ICAM-2 immunofluorescence on Day 14. Choroidal explants were cultured from WT mice for 7 days in Matrigel with and without exogenous IL6 (10 ng/ml) addition on Day 2, 4, and 6. IL6 levels were measured from posterior eye cups via ELISA on Day 3 after laser in WT and Ccr2-/- mice. IL6 receptor expression was detected by immunofluorescence staining of whole eye frozen sections with IBA1 and lectin. Wildtype and Il6-/- mice underwent laser injury followed by multiparameter flow cytometry on Day 3 to investigate macrophage infiltration.
Results :
Male and female Il6-/- mice demonstrated a 42% reduction (p<0.01, N=24-25 mice per group) in CNV area without sex-specific effects. Exogenous IL6 stimulated ex vivo choroidal angiogenesis by 131% (p<0.001, N=5 per group). Since IL6 is a pro-inflammatory cytokine produced by macrophages, which are essential for laser-induced CNV, we measured IL6 levels in WT and Ccr2-/- mice. IL6 levels increased 1.15-fold (p<0.001, N=4-5 per group) in both genotypes, suggesting that classical monocyte-derived macrophages are dispensable for the increased IL6 production after laser. Next, we found that the IL6 receptor is expressed in choroidal endothelial cells and macrophages after laser injury. Due to IL6 receptor expression in macrophages, we investigated the effects of IL6-deficiency on macrophage recruitment using multiparameter flow cytometry. We found that laser injury increased the number of MHCII- (13.0 to 15.0-fold, p<0.05), MHCII+CD11c- (4.9 to 8.4-fold, p<0.05), and MHCII+CD11c+ (5.9 to 6.5-fold, p<0.01) macrophages (N=5-10 per group), with no differences between wildtype and Il6-/- mice.
Conclusions :
IL6 is both necessary and sufficient for choroidal angiogenesis. IL6 is not produced by classical monocyte-derived macrophages and signals to endothelial cells and/or macrophages to stimulate choroidal angiogenesis without affecting macrophage numbers.
This is a 2021 ARVO Annual Meeting abstract.