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Aram Asatryan, Nicolas G Bazan; Stereoselective transcriptional modulation of inflammasome in hRPE cells by the docosanoid Neuroprotectin D1 (NPD1). Invest. Ophthalmol. Vis. Sci. 2016;57(12):6550.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) shares pathological features, including uncompensated oxidative stress stimuli and inflammation with other neurodegenerative diseases such as Alzheimer’s disease. It has been shown that drusen isolated from human eyes with AMD activates the NLRP3 inflammasome. We found that the lipid mediator NPD1, produced in response to oxidative stress, down-regulates inflammasome-related genes, including AIM2, NLRC4, NOD-2, and Il-1β. NOD-2 is of particular interest since mutations of it are associated with Blau syndrome, which results in uveitis and retinal damage in childhood. We hypothesized that NPD1 regulates the expression of inflammasome components at a transcriptional level and that this modulatory bioactivity is stereospecific.
To define the effect of NPD1 on inflammasomes, we used human retinal pigment epithelial (hRPE) cells as an in vitro model. We have shown previously that oxidative stress triggers activation of certain inflammasome-related genes, including NOD-2. To assess whether this effect is stereoselective, we induced oxidative stress in hRPE cells with 600µM of H2O2/TNFα (10ng/ml) and co-treated cells with NPD1, its stereoisomers, resolvin D1, resolvin E1 and lipoxin A4 at 200nM concentration each. Extracted RNA was analyzed using RT-PCR. To understand the scope of NPD1 action, NOD-2 promoter was subcloned into PGL4 luciferase vector and co-transfected into hRPE cells with GFP vector to analyze NOD-2 promoter activity in response to oxidative stress.
As observed previously, oxidative stress triggered the expression of NOD-2 gene. NPD1 had the greatest effect and down-regulated NOD-2 expression more than 2.5 fold. The results of the luciferase assay demonstrate that NPD1 suppressed the transcriptional activation of NOD-2 gene two fold 2h after introduction of oxidative stress.
Our results demonstrate that NPD1 suppresses expression of the pro-inflammatory gene NOD-2 at the transcriptional level and that this effect is stereoselective. NOD-2 is an intracellular pattern-recognition receptor and has a synergistic effect on NLRP3 inflammasome activation. It forms so called "nodosome" that results in the activation of pro-inflammatory NFkB. NOD-2 is also involved in activation of caspase 1 and Il-1β, however this requires the presence of NLRP3.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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