Abstract
Purpose :
Inflammasome activation is pivotal in maintaining epithelial homeostasis and has been implicated in the pathogenesis of age-related macular degeneration (AMD). This activation mechanism involves the formation of a multimeric protein complex (inflammasome) that activates caspase-1, and consequently cleaves precursors of pro-inflammatory cytokines IL-1β and IL-18 leading to the secretion of mature cytokines. In addition, a noncanonical pathway has been identified in multiple systems that utilizes caspase-4 (caspase-11 in mice) to activate the inflammasome and IFN-β1secretion.
Previously, we showed that miR-155 mediates the immune regulation of physiological function in hRPE via a canonical inflammasome activation pathway. In the present study, we examined a noncanonical pathway active in hRPE immune responses and the possible role of miR-155 in regulating this process.
Methods :
Inflammasome activation was assessed in fully differentiated hfRPE grown on transwells. miR mimic was transfected into hfRPE by Dharmarfect reagent. Taqman qRT-PCR was used for miRNA and mRNA measurement as previously published. The levels of cytokines were quantified using ELISA kits. Western blots were performed from total protein extracts as described previously.
Results :
Lipopolysaccharides (LPS) and a mixture of inflammatory cytokines consisting of TNF-α, IL-1β, and IFN-γ, separately increased miR-155 and mRNA of inflammasome components for both canonical and non-canonical inflammasome activation pathways. However, they did not increase the secretion of IL-18, IFN-β1 and IL-18 BP. In contrast, high levels of miR-155, induced by the transfection of miR-155 mimic, are sufficient to increase inflammasome component levels in both pathways and, in particular, the secretion of IFN-β1, indicating a specific role for miR-155 in the activation of noncanonical pathway mediated inflammasome activity.
Conclusions :
These data show that acute inflammatory responses in RPE did not involve a fully-executed noncanonical inflammasome activity that secrets IFN-β1. Only high levels of miR-155 drive noncanonical inflammasome activation. This result suggests that miR-155 accumulation in RPE, accompanied by IFN-β1 secretion, and subsequent continuous inflammatory responses, may mediate a significant departure from homeostasis and contribute to chronic degenerative disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.