Purpose: Age-related macular degeneration (AMD) is a major cause of blindness in the elderly, and the activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome is involved in AMD pathogenesis. We investigated whether photooxidative blue-light stimulation in retinal pigment epithelium (RPE) cells promotes exosome secretion and modulates the activity of the NLRP3 inflammasome in vitro.
Methods: Exosomes were isolated from ARPE-19 cultures stimulated or not with blue-light photostimulation (488 nm). Isolated exosomes were characterized by transmission electron microscope and Western blot analyses. The contents of the NLRP3 inflammasome (IL-1β, IL-18, and caspase-1 as markers of the inflammasome) in exosomes were analyzed by Western blotting. After culture, IL-1β, IL-18, and caspase-1 in RPE cells were analyzed by both immunofluorescence and Western blotting. RT-PCR and Western blotting were conducted to assess the contents of NLRP3 in RPE cells.
Results: Exosomes exhibited a typical characteristic morphology (cup-shaped) and size (diameter between 50 and 150 nm) in both groups. The exosome markers CD9, CD63, and CD81 were strongly present. After blue-light photostimulation, ARPE-19 cells were noted to release exosomes with higher levels of IL-1β, IL-18, and caspase-1 than those in the control group. The levels of IL-1β, IL-18, and caspase-1 in ARPE-19 cells were significantly enhanced when treated with stressed RPE exosomes. Additionally, the NLRP3 mRNA and protein levels were found to be markedly higher in the treated group than in the control group.
Conclusions: Under photooxidative blue-light stimulation, RPE-derived exosomes may aggravate a potentially harmful oxidative response through the upregulation of the NLRP3 inflammasome.
Keywords: NLRP3 inflammasome; Retinal pigment epithelium cells; exosomes; oxidative stress; photooxidative.