Müller cell (MC) inflammation is an early pathogenic step in the vision-threatening disease diabetic retinopathy (DR). Recent evidence indicates that diabetes-induced free fatty acid (FFA) dysregulation can elicit the release of inflammatory cytokines from MC, promoting DR progression. Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including interleukin-1β (IL-1β), IL-6, IL-8 and tumor necrosis factor-α (TNFα). IL-1β is elevated in DR and is purported to be a “master regulator” of cytokine-induced inflammation. IL-1β activates MC to produce itself (IL-1β auto-amplification) and other inflammatory cytokines in the retina. Consequently, a modality that blocks both FFA- and IL-1β-induced MC inflammation has therapeutic potential in DR. The CYP-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), offer one such therapeutic strategy. In this study we propose that experimental elevation of EET and EDP will reduce PA- and IL-1β-induced MC inflammation.
Human MC (hMC) were treated with vehicle, broad-spectrum CYP inhibitor SFK-525a, or PA or IL-1β in the presence or absence of 11,12-EET, 19,20-EDP or GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor. Relative expression of TNFα, IL-1β, IL-6, and IL-8 was assayed via qRT-PCR and NFκB-dependent transcription was tested using a luciferase assay. Data were analyzed using ANOVA with Tukey’s multiple comparisons post-hoc test.
CYP inhibition by SKF-525a increased hMC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP both significantly decreased PA- and IL-1β-induced hMC expression of IL-1β and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1β-induced hMC and TNFα in PA-induced hMC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1β-treated hMC. GSK2256294 significantly reduced PA- and IL-1β-stimulated hMC cytokine expression, demonstrating efficacy over a range of concentrations. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1β-induced NFκB-dependent transcriptional activity.
In summary, these data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation occurring under diabetes-relevant conditions and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR. Our data suggest that these epoxygenated fatty acids exert their anti-inflammatory activities, at least in part, by blocking NFκB-dependent transcription.