Elucidating the mechanism of mitochondrial superoxide production in pro-inflammatory macrophages
Pro-inflammatory macrophages produce reactive oxygen species (ROS) which act as bactericidal agents and redox signals. Recently, mitochondria were identified as an important source of ROS in pro-inflammatory macrophages and it has been proposed that lipopolysaccharide (LPS) stimulated macrophages produce mitochondria derived superoxide by reverse electron transport (RET) at complex I. However, this has not been demonstrated and the mechanism of mitochondrial superoxide production is unknown. Furthermore, the influence of LPS induced metabolic reprogramming on mitochondrial ROS (mtROS) production has not been elucidated. Therefore, the aim of this thesis was to determine the mechanism of mitochondrial superoxide production and how this is driven by LPS induced metabolic reprogramming. To address this question, I measured LPS induced mitochondrial superoxide production with a mitochondria targeted superoxide probe and performed a temporal analysis of LPS induced metabolic reprogramming. I also used bone marrow derived macrophages (BMDMs) expressing the alternative oxidase (AOX) to determine the influence of an oxidised CoQ pool on mitochondrial superoxide production. Using this approach, I demonstrated that LPS induced mitochondrial superoxide production is driven by an elevated proton motive force (∆p), measured as mitochondrial membrane potential (∆ψm), and a reduced CoQ pool. The key metabolic changes that give rise to these conditions are repurposing of ATP production from oxidative phosphorylation to glycolysis, which reduces the reliance on F1FO-ATP synthase activity resulting in an elevated ∆p, and succinate oxidation which maintains a reduced CoQ pool. Since a high ∆p and reduced CoQ pool could drive mitochondrial superoxide production at either complex I or complex III of the electron transport chain, I next set out to determine the site and mechanism of mtROS production. To do this, I used BMDMs from a mouse homoplasmic for the *ND6 G14600A* mtDNA mutation. This mutation results in a P25L substitution in the ND6 subunit of complex I which prevents RET but does not affect forward electron transport or complex III activity. Using BMDMs from these ND6P25L mice I demonstrated that LPS stimulated macrophages produce mitochondrial superoxide by RET at complex I. Finally, I used this genetic model to investigate the role of mtROS production by RET in the regulation of macrophage cytokine production and NLRP3 inflammasome activation to disentangle the distinct effects of mitochondrial metabolites and mtROS on macrophage effector functions. Overall, this study has elucidated key mechanistic details of mitochondrial superoxide production in pro-inflammatory macrophages and contributed to our understanding of how specific mitochondrial signals affect macrophage effector functions. This is crucial if we are to target these processes to modulate macrophage biology as a therapeutic strategy.
complex I, macrophage, mitochondria, reactive oxygen species, RET, reverse electron transport, ROS
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