Evaluation of antioxidant treatments for the modulation of macrophage function in the context of retinal degeneration

PURPOSE: Oxidative stress and macrophages have been implicated in the pathogenesis of atrophic and neovascular age-related macular degeneration (aAMD and nvAMD). It is unclear whether oxidative injury mediates macrophage involvement in AMD. We aimed to investigate the effect of antioxidant treatments on human monocyte-derived macrophages (hMDMs) from patients with AMD in models for the disease. METHODS: Four antioxidant treatments were evaluated (G1: lutein + zeaxanthin, G2: lutein + zeaxanthin and zinc, G3: lutein + zeaxanthin, zinc, Lyc-O-Mato, and carnosic acid, G4: lutein + zeaxanthin, carnosic acid, and beta-carotene, G5: olive oil as vehicle control). The compounds were added to the culture medium of M1 (interferon-gamma [IFN-Ɣ] and lipopolysaccharide [LPS]) and M2a (interleukin-13 [IL-13] and IL-4) hMDMs from patients with AMD (n=7 and n=8, respectively). Mouse choroidal tissue was cultured with supernatants from treated M1/M2a hMDMs, to evaluate the effect of treatments on the angiogenic properties of macrophages with choroidal sprouting assay (CSA). Mouse retinal explants were cultured with treated hMDMs for 18 h, and evaluated for photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling. Adult BALB/c mice (n=8) were exposed to 8,000 lux bright light for 3 h, and treated orally with antioxidant supplements for 7 days that preceded light injury and following it. Oxidative stress was assessed using an anti-4 hydroxynonenal (4-HNE) antibody. Retinal function and the thickness of the outer nuclear layer were evaluated with electroretinography (ERG) and histological analysis, respectively. RESULTS: The G3 treatment reduced M2a hMDMs-associated sprouting in the CSA compared to the untreated group (n=7, −1.52-fold, p=0.05). Conversely, the G2 treatment was associated with an increased neurotoxic effect of M2a hMDMs in the retinal explant assay compared to the control group (n=7, 1.37-fold, p=0.047), as well as compared to the G3 treatment group (1.46-fold, p=0.01). The G4 treatment was also associated with increased cytotoxicity compared to the control group (1.48-fold, p=0.004), and compared to the G3 treatment group (1.58-fold, p=0.001). In the in vivo light damage model, mice (n=8) supplemented with G2, G3, and G4 had decreased levels of oxidative injury assessed using 4-HNE labeling (−2.32-fold, −2.17-fold, and −2.18-fold, respectively, p<0.05 for all comparisons). None of the treatments were associated with reduced photoreceptor cell loss, as shown with histology and ERG. CONCLUSIONS: Antioxidant treatment modulates M2a hMDMs at the functional level. In particular, we found that the G3 combination has a beneficial effect on M2a macrophages in reducing their angiogenic and neurotoxic capacity ex vivo. In addition, antioxidant treatments considerably reduced the oxidative stress level in light-damaged retinas. Further research is required to assess whether such therapies may curb macrophage-driven photoreceptor loss and neovascularization in AMD.