Mitochondrial reactive oxygen species regulate HIF-1α stabilization and methylglyoxal accumulation in classically activated mouse macrophages.

BACKGROUND: The economic and medical burden of sepsis worldwide underscores the need for novel therapeutics. Early sepsis involves dramatic metabolic changes. Classically activated macrophages, stimulated with lipopolysaccharide and interferon-γ, shift their metabolism to glycolysis. The reactive glycolytic metabolite, methylglyoxal, accumulates and has been associated with adverse outcomes in sepsis. We previously demonstrated that hypoxia-inducing factor-1α (HIF-1α) contributes to methylglyoxal accumulation. Treatment with lipopolysaccharide or interferon-γ individually stabilized HIF-1α protein; however, co-stimulation with both lipopolysaccharide and interferon-γ accelerated HIF-1α stabilization, implying a shared upstream mediator. Therefore, we sought to characterize mechanisms underlying methylglyoxal accumulation. METHODS: Quantitative polymerase chain reaction and immunoblotting were used to analyze HIF-1α expression in classically activated primary mouse macrophages. RESULTS: Nos2 expression was induced by lipopolysaccharide or interferon-γ and markedly enhanced by combined treatment, possibly linking inducible nitric oxide synthase (iNOS) activity to HIF-1α stabilization. Inhibiting iNOS with l-NG-Nitro arginine methyl ester (l-NAME) reduced HIF-1α stabilization in a dose-dependent manner. Mitochondrial reactive oxygen species (mROS), generated following nitric oxide inhibition of cytochrome oxidase, similarly contributed to HIF-1α stabilization, as shown by the effects of suppressors of ROS production by mitochondrial complex I (S1QEL1.1) and III (S3QEL1.2) and the compartment-specific antioxidant Mito-TEMPO. Blocking mROS also decreased Il1b, Il6, and Cxcl10 expression in activated macrophages, supporting a broader impact on inflammation. Additionally, S1QEL1.1 treatment reduced accumulation of methylglyoxal. CONCLUSIONS: These data support a model in which nitric oxide-mediated mitochondrial dysfunction increases mROS, promoting HIF-1α stabilization and methylglyoxal accumulation, thereby shaping macrophage inflammatory responses. Thus, targeting mROS may offer a therapeutic strategy to improve sepsis outcomes.