Nitroalkene inhibition of pro-inflammatory macrophage effector function via modulation of signaling metabolite levels

Classically activated innate immune cells undergo a metabolic switch to aerobic glycolysis to support effector function. We report that the small-molecule nitroalkene 10-n-octadec-9-enoic acid (NO₂-OA) attenuates the Warburg- like phenotype of aerobic glycolysis in lipopolysaccharide (LPS)-activated macrophages, thus inhibiting pro-inflammatory signaling.

RAW264.7 and bone marrow derived macrophage were treated with LPS with and without NO₂-OA or 1400W. Pro-inflammatory cytokines were measured by ELISA and protein expression was determined by immunoblot. Central carbon metabolites with and without ¹³C stable isotope tracing were measured using liquid chromatography-high resolution mass spectrometry.

Overall, the present observations indicate that nitroalkene-induced changes in central carbon metabolism contribute to the anti-inflammatory actions of this class of multi-target lipid signaling mediators. Comparison of macrophage responses to NO₂-OA with the inducible nitric oxide synthase (NOS2 and iNOS) inhibitor 1400W affirms that NO₂-OA inhibition of NOS₂ expression and activity alone was not sufficient to account for the decreases in pro-inflammatory cytokine expression. NO₂-OA treatment reduced intracellular succinate levels, which may be attributed to a concomitant reduction in intracellular itaconate and reliance on glutamine, thereby contributing to hypoxia-inducible factor 1α (HIF1α) destabilization observed in LPS-activated macrophages.

The current data provide additional perspective on the actions of this small-molecule electrophile, which is currently in a Phase 2 clinical trial for the treatment of obesity-related chronic pulmonary inflammation and associated airway dysfunction.