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Jian Liu, David A Copland, Sofia Theodoropoulou, Miriam Barba, Lindsay B Nicholson, Andrew D Dick, ; The role of impaired retinal pigment epithelial function in modulating macrophage activation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3551.
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Progression of age-related macular degeneration (AMD) involves degeneration hallmarks of retinal pigment epithelium and photoreceptor loss is accompanied by unchecked immune response or pathological angiogenesis. Here we investigated whether RPE cells, in presence of impaired autophagy or under oxidative stress, differentially condition macrophage responses and thus drive further cell death or promote angiogenesis.
Wortmannin and chloroquine were used to suppress autophagy at upstream and downstream checkpoints, respectively, in murine RPE cells. Rotenone was used to induce RPE autophagy. Oxidative stress was induced by hydrogen peroxide. Beclin-1 gene silencing was performed using siRNAs, to inhibit autophagy. RPE cytotoxicity was assessed by LDH release. An in vitro phagocytosis assay was applied to elucidate the role of impaired RPE in modulating inflammatory cytokine and nitric oxide (NO) production from bone marrow derived macrophages (BMMΦ). Macrophage caspase-1 activation was examined by western blot and immunofluorescence microscopy. Macrophage derived angiogenesis-associated proteins were assessed by antibody array. The pro-angiogenic potential of RPE-macrophage conditioned medium (CM) was assessed in an explant choroidal sprouting model. To confirm in vivo, autophagy was inhibited by intravitreal injection of wortmannin. RPE damage and macrophage accumulation/activation were detected in whole-mounts of RPE/choroid.
Inhibition of basal activity or rotenone-induced autophagy in RPE cells using wortmannin or chloroquine led to increased cell death, mediated by caspase-3 activation. Beclin-1 knockdown also provoked RPE susceptibility to rotenone toxicity. After treatment with damaged RPE cells caused by impaired autophagy or oxidative stress, BMMΦ expressed markers of inflammasome activation and secreted higher levels of IL-1β, IL-6 and NO. Comparably, in vivo suppression of retinal autophagy led to RPE degeneration and focal accumulation of activated macrophages. In addition, impaired RPE cells differentially conditioned macrophage production of proteins involved in promulgating inflammation or driving angiogenesis. CM of macrophages treated with oxidative stressed RPE was more pro-angiogenic.
Our data suggest that the nature of RPE degeneration will differentially regulate macrophage phenotype and function and drive angiogenesis.
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