Analysis of how the TCA cycle enzymes Fumarate hydratase and Succinate dehydrogenase regulate cytokine production

Abstract

Mitochondria are organelles which release metabolites and nucleic acids that act as signals to regulate immune cell activation and cytokine production. Tricarboxylic acid (TCA) cycle metabolites and enzymes have particularly important functions as regulators of macrophage function. To discover roles for novel immunometabolites, I performed unbiased metabolomics in LPS activated macrophages and found that fumarate was one the most highly upregulated metabolites. I then determined the pathway by which fumarate accumulates, which is the inflammatory aspartate-argininosuccinate shunt whereby TCA- cycle derived oxaloacetate is converted to aspartate and further processed to argininosuccinate and fumarate in the cytosol. I then aimed the elucidate mechanistically what the physiological role of fumarate in inflammatory macrophages may be. To probe the mechanisms of action of fumarate I use pharmacological and genetic ablation of Fumarate hydratase (FH) to increase endogenous fumarate as well as the esterified derivative of fumarate, dimethyl fumarate (DMF), to mimic the cysteine thiol-reactive properties of fumarate. With RNAseq in BMDMs treated with a pharmacological inhibitor of FH, FHIN1 and DMF, I found that two pathways that were strongly regulated by FHIN1 and DMF were a downregulation of IL-10 signaling and an upregulation of TNF signaling. Mechanistically this was found to be through impaired cFos activity which is required for transcription of IL-10. Additionally, I found that decreased IL-10 production was responsible for the upregulation of TNF. Surprisingly, I also found that FH inhibition leads to increased type I IFN production which was independent of fumarate accumulation as DMF had no effect on IFNβ expression. This was confirmed in human PBMCs and in vivo in a murine model of LPS-induced inflammation. Mechanistically, FH inhibition was found to increase IFNβ through mtRNA release from mitochondria, resulting in subsequent activation of the RNA sensors RIG-I, MDA5, and TLR7, while DNA sensing pathways were not required for the induction of type I IFN. Additionally, I found that the whole blood samples patients with systemic lupus erythematosus (SLE) exhibited decreased levels of FH expression, suggesting 22 that this pathway may contribute to the pathogenesis of human interferon-driven diseases. SDH is another important regulator of macrophage cytokine production, so I next examined the effect of pharmacological and genetic SDH ablation on type I IFN production and found that this resulted in increased IFNβ production. I also found that this was dependent on the release of mtRNA and the activation of MDA5. Additionally, in this system I expanded on the previously described mechanism and identified that SDH inhibition causes the VDAC1dependent release of mtRNA which can be inhibited by genetically or pharmacologically targeting VDAC1. In summary, this work provides novel insight into the mechanisms of action of fumarate as an immunometabolite as well how TCA cycle disruption leads to a mtRNA-dependent activation of a type I IFN response, a pathway which may contribute to human interferon-driven disease.