Only IL‐1β release is inflammasome‐dependent upon ultraviolet B irradiation although IL‐18 is also secreted
Eveliina Korhonen, Niina Piippo, Maria Hytti, Juha M. T. Hyttinen, Kai Kaarniranta, Anu Kauppinen
Abstract
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 (H 2 DCFDA) 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.