Mitochondrial Membrane Potential in Human Neutrophils Is Maintained by Complex III Activity in the Absence of Supercomplex Organisation

textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory chain complexes. We investigated the source of ΔΨm in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this ΔΨm for the generation of ATP. Methods and Principal Findings: Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but ΔΨm was still decreased byinhibition of complex III, confirming the role of the respiratory chain in maintaining ΔΨm. Complex V did not maintain ΔΨm by consumption of ATP, as has previously been suggested for eosinophils shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were ladding in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercimplex organisation while gaining increased aerobic glycolysis, just like neutrophils. Conclusions: We show that neutrophils can maintain ΔΨm via the glycerol-3-phosphate shuttle, wereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors

A Combination of Hypoxia and Lipopolysaccharide Activates Tristetraprolin to Destabilize Proinflammatory mRNAs such as Tumor Necrosis Factor-α

Inflammation is often accompanied by hypoxia because of the high oxygen consumption of invading bacteria and immune cells. During resolution of inflammation, the formation of inflammatory mediators such as tumor necrosis factor-α (TNF-α), which is produced by macrophages, needs to be terminated. We show in RAW264.7 macrophages that TNF-α mRNA as well as intracellular and secreted TNF-α protein levels are reduced after prolonged incubations with lipopolysaccharide (LPS) under hypoxic conditions. The decrease in TNF-α was mediated by destabilization of TNF-α mRNA via a 3′-untranslated region-dependent mechanism. Specifically, the RNA-binding protein tristetraprolin (TTP) increased at mRNA and protein levels after 16-hour incubations with LPS under hypoxia. Interestingly, TTP accumulated in a dephosphorylated and active form, and this accumulation was attributable to reduced p38 mitogen-activated protein kinase activity under these conditions. Knockdown of TTP by small interfering RNA abolished destabilization of TNF-α mRNA. Prolonged incubations with LPS under hypoxia also reduced mRNA amounts and stability of other proinflammatory mediators such as macrophage inflammatory protein-2, interleukin-6, and granulocyte macrophage colony-stimulating factor. Therefore, we propose that hypoxia plays a key role during resolution of inflammation by activating posttranscriptional, TTP-dependent regulatory mechanisms