Dimethyl fumarate blocks TNFα-driven inflammation and metabolic rewiring in retinal pigment epithelial cells

Abstract

Purpose : Oxidative damage and inflammatory activation of retinal pigment epithelial cells (RPE) are key contributors to age-related macular degeneration (AMD) pathogenesis. Metabolic reprogramming and oxidative stress are known to drive immune cell activation. However, the existence of such interplay between inflammation and metabolism in RPE remains elusive. This study examines the effects of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine involved in AMD, on RPE metabolism and investigates the metabolic drug, dimethyl fumarate, as a new regulator of RPE immunometabolism.

Methods : Matured primary human RPE cells were treated with TNFα (10 ng/ml) and/or pre-treated with dimethyl fumarate (DMFu, 80 μM) for 2 hours. Glycolytic and oxidative (OXPHOS) metabolic profiles were determined by Seahorse XFe96. Gene expression of metabolic markers was assessed using qPCR. IL-6 secretion was quantified by ELISA. Cellular ATP content was measured by bioluminescence. Reactive oxygen species (ROS) was measured using a fluorometric mitochondrial superoxide detection kit. Ultrastructural features of mitochondria were imaged by transmission electron microscopy (TEM).

Results : TNFα-induced upregulation of IL-6 secretion (p<0.0001, n=6) in RPE was accompanied by increased basal and maximal OXPHOS on Seahorse Mito Stress Test (p<0.0001, n=10). TNFα increased ATP production, mitochondrial ROS production (p<0.0001, n=8) and gene expression of the mitochondrial antioxidant SOD2 (p<0.0001, n=6). TEM revealed defects in mitochondrial morphology with engorged mitochondria and loss of cristae integrity with TNFα. DMFu suppressed TNFα-induced IL-6 secretion (p<0.0001, n=6) and maintained mitochondrial OXPHOS and ATP production at control levels (p>0.05, n=6). DMFu increased PFKFB3 gene expression (p<0.0001, n=6) and correspondingly, restored TNFα-induced suppression of glycolysis to control levels (p>0.05, n=6).

Conclusions : The pro-inflammatory effects of TNFα on RPE are accompanied by defects in mitochondrial morphology and a profound metabolic shift promoting OXPHOS and ROS accumulation. Treatment with the fumaric acid precursor, DMFu, efficiently blocks TNFα-induced pro-inflammatory activation and bioenergetic reprograming of RPE. These results reveal a critical interplay between inflammation and metabolic dysfunction in RPE, identifying DMFu as a potent immunoregulatory drug against AMD progression.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.