Photo-Oxidative Blue-Light Stimulation in Retinal Pigment Epithelium Cells Promotes Exosome Secretion and Increases the Activity of the NLRP3 Inflammasome

ABSTRACT

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:

• Retinal pigment epithelium cells
• exosomes
• NLRP3 inflammasome
• photooxidative
• oxidative stress

Disclosure

The authors state that they do not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article. The authors also do not discuss the use of off-label products, including unlabelled, unapproved, or investigative products or devices.

Additional information

Funding

This work was supported by Tianjin Science and Technology Project of China (No. 14JCYBJC27400) and the National Natural Science Foundation of China (No. 81700846).

Notes on contributors

Song Chen

Song Chen and Wei Zhang conceived and designed the experiments. Wei Zhang, Yingxue Ma, and Jing Yang performed the experiments and prepared the tables and figures. Guanghui He and Yue Zhang collected the sample data. Wei Zhang and Yue Zhang contributed to the writing of the manuscript. Song Chen reviewed the manuscript. All authors contributed to the writing of the manuscript and discussed the manuscript at various stages.