Effect of small molecules modulators of Complex III of the electron transport chain on cytokine production in macrophages

Abstract

Innate immune cells promote inflammation when activated by Pathogen- associated molecular patterns (PAMPs) and initiate the immune response to stress and microbial invasion. When macrophages are primed with the Gram-negative bacterial product Lipopolysaccharide (LPS), they show an M1 pro-inflammatory phenotype and aberrant macrophage activation can contribute to the pathogenesis of inflammatory diseases. M1 macrophages also undergo profound metabolic changes, with glycolysis being elevated and a disturbance in mitochondrial metabolism, leading to an elevation in such metabolites as succinate and itaconate. The role of the electron transport chain (ETC) in shaping the macrophage immune response has been less studied. M1 macrophages release multiple cytokines including the pro-inflammatory cytokines Interleukin(IL)-1β and Tumour Necrosis Factor (TNF)-α and the immunomodulatory cytokine Interleukin (IL)-10. In this thesis, I have explored the link between the ETC and the innate immune response, with a focus on the mechanisms driving cytokine production. Novel specific inhibitors of Complex I and Complex III of the ETC termed Suppressors of site I Q and III QO Electron Leak (S1QELs and S3QELs respectively) have been assessed. These compounds inhibit Reactive oxygen species (ROS) production without affecting basal cellular respiration. S1QEL 1.1 and S3QEL 1.2 blocked IL-1β expression at the translational level in LPS-treated macrophages. They also suppressed Hypoxia- Inducible Factor (HIF)-1α activation but they failed to repress NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Moreover, S3QEL 1.2 slightly affected respiration and lowered ROS production in activated macrophages and importantly inhibited LPS and CpG-induced IL-10 release in macrophages in vitro but also in vivo. Macrophages from transgenic mice expressing Alternative Oxidase (AOX), which diverts electrons from Complex III lowering ROS, also had less IL-10 in response to LPS. Further analysis revealed that IL-10 downregulation might involve inhibition of Activator protein (AP)-1 activation but not CREB activation. The effect on AP-1 and AP-1-dependent genes was shown by RNAseq analysis, which also revealed that S3QEL 1.2 and Myxothiazol (which inhibits Complex III activity) also regulated the TNF-α pathway in a positive way likely via the negative regulation of IL-10. Subsequent studies in the B16F10 melanoma model showed that S3QEL 1.2 improved survival and decreased tumour growth in mice, particularly in combination with CpG DNA. These results provide evidence for ROS derived from Complex III in the regulation of cytokine production, particularly IL-10, in macrophages and possibly in tumour- mediated immune evasion. This work suggests the potential of S3QELs as mitochondria-targeted therapies in cancer.