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Congxiao Zhang, Arvydas Maminishkis, Guangpu Shi, Sheldon S Miller; MICRORNA-155 MEDIATES HUMAN RETINAL PIGMENT EPITHELIUM INFLAMMASOME ACTIVITY. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4223. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammasome activation is a well-established response of the innate immune system and has been implicated in the pathophysiology of AMD. It has been shown that miR-155 plays a key role in inflammatory disease and cancer. We have shown that miR-155 regulates RPE physiology, for example by increasing CFTR mediated fluid absorption across the RPE. In the present study, we investigated the role of miR-155 in RPE inflammasome activation.
Inflammasome activation was assessed using primary cultures of human fetal RPE (hfRPE) grown on transwells. miR-155 mimic and anti-miR oligonucleotides were transfected into hfRPE by Dharmarfect reagent. Taqman qRT-PCR was used for miRNA and mRNA measurements, as previously reported. Cytokine levels were quantified using a Milliplex multi-analysis detection kits. Western blots were performed from total protein extracts by conventional methods. Immunocytochemistry was carried out to detect IL-18R1 protein localization in the hfRPE monolayer.
In hfRPE, addition of a pro-inflammatory cytokine cocktail leads to a significant elevation of miR-155 which, in turn, increases expression of multiple inflammasome pathway genes. This gene set includes caspase-1, IL-18 and IL-18 binding protein (IL-18BP), but not IL-1beta. Addition of miR-155 mimic for 40 hr slightly increased IL-18 and IL-18BP secretion, but not gene expression. At 3.5 days, miR-155 significantly increased the level of caspase-1 active fragment and induced the secretion of IL-18 and IL-18BP to the apical but not basolateral side of polarized hfRPE cultures. IL-1beta secretion was unaltered. Transfection with anti-miR155 partially blocks the generation of capase-1 active fragment. Immunocytochemistry shows that IL-18R1 is localized specifically to the RPE apical membrane.
These data indicate that miR-155 plays a role in determining inflammasome formation and activity by hfRPE. The apical localization of IL-18R1 and the high affinity of IL-18 for its binding protein leads us to hypothesize that the subretinal space may be a crucial locus of inflammasome action and miR-155 regulation of RPE physiology.
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