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

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.

Quantitative polymerase chain reaction and immunoblotting were used to analyze HIF-1α expression in classically activated primary mouse macrophages.

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.

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.