Metabolic shift towards pentose-phosphate-pathway sustains NOX activity in pro-inflammatory microglia

Elevated NADPH-oxidase (NOX) activity, particularly NOX2, is implicated in the persistent pro-inflammatory phenotype observed in microglia following traumatic brain injury. NOX activation requires metabolic support, especially within the pentose-phosphate-pathway (PPP), to fulfil the heightened NADPH demand. Our objective was to identify key metabolic changes associated with NOX activation during pro-inflammatory stimulation in microglia. Using an immortalized microglia (IMG) cell line and PPP inhibitor, 6-aminonicotinamide (6-AN), we stimulated IMGs with LPS (100 ng/mL) and/or 6-AN for 24 hours, followed by 10 minutes of ATP (1mM). NOX activity was assessed via lucigenin chemiluminescence, and cytokine release (TNF-α, IL-1β, and IL-10) in supernatants by ELISA. Tetramethylrhodamine-ester investigated mitochondrial membrane potential, while SeaHorse analysis explored mitochondrial oxygen consumption and glycolytic rate. Transcriptional regulation of Hk2, Cybb, and G6pd2 (genes encoding glycolysis and PPP pace-maker enzymes, and the NOX2 catalytic subunit, respectively) was assessed by RT-qPCR. Profound pro-glycolytic shift in microglia, including decreased mitochondrial membrane potential and oxygen consumption, and upregulation in Hk2 and G6pd2, were observed following LPS+ATP stimulation, paralleled by increased NOX activity, pro-inflammatory cytokine release, and Cybb expression. PPP inhibition mitigated G6pd2 and Cybbupregulation, halting NOX activity, restoring mitochondrial respiration, attenuating pro-glycolytic metabolism, and reducing pro-inflammatory indicators. A moderate negative correlation between mitochondrial membrane potential and pro-inflammatory cytokine levels suggested at a critical link between immune response and microglial metabolism. These findings offer insights on microglial metabolic landscape and highlight PPP's crucial role in supporting microglial pro-inflammatory activation and sustaining NOX2 activity, offering potential therapeutic targets to alter immunometabolism and regulate neuroinflammation.