Only IL‐1β release is inflammasome‐dependent upon ultraviolet B irradiation although IL‐18 is also secreted
Open Access
- 19 March 2020
- journal article
- research article
- Published by Wiley in The FASEB Journal
- Vol. 34 (5), 6437-6448
- https://doi.org/10.1096/fj.201902355rr
Abstract
DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1β mRNAs were detected with qRT-PCR and secreted amounts of IL-1β, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1β was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1β but NLRP3 contributed only to the secretion of IL-1β. At the mechanistic level, the release of IL-1β was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1β.Keywords
Funding Information
- Päivikki ja Sakari Sohlbergin Säätiö
- Pohjois-Savon Rahasto
- Emil Aaltosen Säätiö
- Academy of Finland (AK297267,AK307341 and KK5503743)
- Sigrid Juséliuksen Säätiö
- Itä-Suomen Yliopisto
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