Immune cells undergo metabolic reprogramming during activation which directly impacts on their phenotype. Glycolysis is a key feature of M1 macrophages and Th17 cells, whilst oxidative phosphorylation is more apparent in M2 macrophages and Treg cells. Signals regulated by the metabolites generated are still unclear. This is a complex area which could lead to new insights into the role of intracellular metabolism in immunoregulation. We will build on preliminary data on 3 aspects of immunometabolism. Extending previous findings on succinate we have found that succinate might also mediate reverse electron transport (RET) in mitochondria, generating reactive oxygen species (ROS). Another metabolite, termed malonylCoA (which is derived from citrate) might be a critical regulator in macrophages of a recently-described covalent modification termed malonylation, targeting several proteins, including GAPDH, with important consequences for mRNA translation. Finally we have evidence of gluathionylation, with proteins in TLR signaling being altered. Glutathione transferase omega (GSTO) could be important in this process. The current application builds on these highly innovative findings as follows:
1. We hypothesise that the directionality of electron flow in mitochondria governs cytokine production. We will explore the role of succinate here by examining reverse electron transport through complex I in the induction of ROS, leading to HIF1alpha activation and downstream activation of target genes. We will also explore data indicating that the forward flow of electrons is a signal for IL-10, electron flow therefore unexpectedly acting as an arbiter of inflammation versus homeostasis in macrophages.
2. We hypothesise that malonylation of specific proteins is critical for macrophage activation. We have identified proteins undergoing malonylation, notably GAPDH, which as well as being in glycolysis is a repressor of translation. We have identified mRNAs including COX2 and DAPK1 as possible GAPDH targets and will examine whether malonylation releases GAPDH allowing for translation, revealing a wholly novel signaling mechanism acting on mRNA translation. We will also determine the extent of the malonylome and characterise other malonylated proteins.
3. We hypothesise that a glutathionylation cycle is critical for macrophage activation. We have evidence that proteins in TLR signalling undergo glutathionylation/deglutathionylation, possibly following mitochondrial ROS generation, and that the enzyme GSTO, could be involved. Mice deficient in GSTO are completely resistant to LPS. This will also involve an analysis of global protein glutathionylation in macrophages.
In these three integrated hypotheses in immunometabolism, we aim to further open an important frontier area in immunology, providing new information on the immune system in health and disease.
