Exploring the Mitochondrial Metabolite Itaconate and Mitochondrial Glutathione in Inflammation

Abstract

Immunometabolism is a new branch of immunology that studies the interplay between metabolism and immune cell activation. Mitochondria play a pivotal role in immunometabolism as they host key metabolic processes such as the Krebs cycle and the electron transport chain (ETC) operating oxidative phosphorylation (OXPHOS). This project concerns two aspects of the role played by the mitochondria in metabolism – the anti- inflammatory effect of the mitochondrial metabolite itaconate on eosinophils and the role of mitochondrial glutathione (mtGSH) on the inflammatory responses in macrophages. Itaconate is a metabolite produced from the Krebs cycle through cis-aconitate decarboxylation by the enzyme aconitate decarboxylase (ACOD1) encoded by immunoresponsive gene 1 (IRG1). It is important in the context of immunometabolism due to its wide array of immunomodulatory actions. 4-octyl itaconate (4-OI) is a cell-permeable derivative of itaconate and similarly displays a variety of immunomodulatory roles. Through studies from itaconate derivatives or from Irg1-deficient macrophages, several targets of itaconate have been uncovered, such as NRF2, ATF3, and NLRP3. 4-OI and itaconate are also effective in dampening inflammation in various animal disease models, including sepsis, psoriasis, and ischemia-reperfusion injury (IRI). In asthma studies, itaconate and 4-OI have shown promise in mitigating inflammation induced by allergens like ovalbumin (OVA) and house dust mite (HDM). In my PhD project, I have found that 4-OI significantly reduces the production of eosinophil-targeted chemokines in a variety of cell types, including lipopolysaccharide (LPS)- and IL-4 stimulated macrophages, Th2 cells, and A549 respiratory epithelial cells. Notably, NRF2-dependent mechanisms underlie the suppression of these chemokines in LPS-stimulated macrophages. Furthermore, 4-OI interferes with IL-5 signaling, directly impacting eosinophil differentiation. In a murine model of eosinophilic airway inflammation, 4-OI alleviates airway resistance and reduces lung eosinophil recruitment, highlighting its therapeutic potential in asthma management. The ETC is situated in the mitochondrial inner membrane and consists of four major iron-sulfur clusters (ISC)-containing protein complexes. Besides its role in ATP generation, the ETC also influences immune responses through reactive oxygen species (ROS). Studies have linked ETC-generated ROS to IL-1b and IL-10, and OXPHOS-produced ATP to NLRP3-mediated IL-1b release. Itaconate has been shown to limit ROS via its inhibitory effect of succinate dehydrogenase (SDH), which might also impact on GSH, or mtGSH translocated by SLC25A39 and SLC25A40. mtGSH neutralizes ROS and participates in ISC synthesis and is likely linked to cytokine production. In the second part of this study, I investigated the role of SLC25A40 in bone marrow-derived macrophages (BMDMs) activation. I found that SLC25A40 is expressed in BMDMs and it stabilized ISC-containing proteins in the ETC and protected cells from oxidative damage. Downregulation of SLC25A40 upregulated Gclc and Gclm, two genes encoding enzymes responsible for GSH synthesis, implicating a compensatory mechanism. Furthermore, I linked SLC25A39 and SLC25A40 to macrophage cytokine production. I showed that although the two proteins are functional homologs, their knockdowns resulted in distinct cytokine effects. Specifically, SLC25A39 knockdown increased IL-10 and decreased IL-6, while SLC25A40 knockdown resulted in decreases in IL-1b and IL-10. Taken together, these findings add to the understanding of the complex role of mitochondria in immunometabolism, providing evidence of itaconate derivatives targeting eosinophils in asthma-like lung inflammation and a regulatory role for mtGSH in macrophage responses. My results therefore underscore the therapeutic potential of targeting mitochondrial metabolites in inflammatory diseases.