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

Emily Rose Stevenson¹James P O`Brien<sup>1</sup>Allison M Manuel<sup>2</sup>Crystal Uvalle<sup>1</sup>Gregory J Buchan<sup>1</sup>Steven J Mullett<sup>1</sup>Karina C Lockwood<sup>1</sup>Tomeka Suber`³Bruce Alan Freeman¹Stacy L. Gelhaus^1*

• ¹University of Pittsburgh, Pittsburgh, Pennsylvania, United States
• ²The University of Utah, Salt Lake City, Utah, United States
• ³University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States

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. Previously, it was observed that NO2-OA and related endogenous and non-natural synthetic nitroalkenes broadly attenuate inflammation by covalently and reversibly modifying functionally significant reactive cysteines. This includes key thiolates in pro-inflammatory enzymes and redox-sensitive transcription factors that orchestrate both the propagation, inhibition, and repair of inflammatory injury. The current data adds perspective to the actions of this small molecule electrophile, currently in a Phase 2 clinical trial for the treatment of obesity-related chronic pulmonary inflammation and related airway dysfunction. 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 and the inducible nitric oxide synthase (NOS2, iNOS) inhibitor 1400W affirm that NO2-OA inhibition of NOS2 expression and activity alone was not sufficient to account for the decreases in pro-inflammatory cytokine expression. For example, reduction in intracellular succinate levels may be attributed to a concomitant reduction in intracellular itaconate and reliance on glutamine thereby accounting for HIF1α destabilization observed in LPS-activated macrophages treated with NO2-OA.