Analysis of the role of Krebs cycle rewiring in macrophage cytokine production

Abstract

A striking change has happened in the field of immunology whereby specific metabolic processes have been shown to be a critical determinant of immune cell activation. Multiple immune receptor types rewire metabolic pathways as a key part of how they promote effector functions. Perhaps surprisingly for immunologists, the Krebs cycle has emerged as the central immunometabolic hub of the macrophage. During pro-inflammatory macrophage activation, there is an accumulation of the Krebs cycle intermediates succinate, succinyl-CoA, fumarate and citrate, and the Krebs cycle-derived metabolite itaconate. The first aspect of this project investigated the role of the aspartate-argininosuccinate shunt and fumarate accumulation in regulating cytokine production in macrophages. Lipopolysaccharide (LPS) was shown to increase argininosuccinate synthethase (Ass1) expression and boost the production of argininosuccinate and fumarate, while decreasing fumarate hydratase (Fh) and aspartate levels. Using the aspartate aminotransferase inhibitor aminooxyacetic acid (AOAA), the argininosuccinate shunt was shown to regulate fumarate accumulation in the macrophage. Furthermore, AOAA was also shown to inhibit LPS-induced TNF, IL-6 and IL-1β production. Using two pharmacological agents, dimethyl fumarate (DMF) and monomethyl fumarate (MMF), and one genetic approach, Fh-deficiency, fumarate was shown to modulate LPS-induced cytokine production. Interestingly, elevated fumarate levels also resulted in an increase in the non-enzymatic and covalent modification of protein cysteine residues by fumarate, a process termed succination, as well as an increase in the metabolite S-(2)-succinocysteine (2SC), a biomarker of succination. This represents the first report of this metabolite and the understudied post-translational modification (PTM) in LPS-activated macrophages. The second aspect of this project examined the immunomodulatory role of itaconate, which is derived from cis-aconitate, in LPS-activated macrophages. Here it’s shown that LPS induces immunoresponsive gene 1 (IRG1), also known as cis-aconitate decarboxylase (CAD), expression and modulates glycolysis and glutaminolysis to support itaconate synthesis. Using a new cell-permeable itaconate derivative 4-octyl itaconate (4-OI), itaconic acid (ITA) and Irg1/Acod1-deficient macrophages, itaconate was found to be required for LPS-induced succinate accumulation, inhibition of complex II of the ETC and activation of the anti-inflammatory transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Interestingly, itaconate directly modifies proteins via alkylation of cysteine residues, a novel post-translational modification (PTM) termed 2, 3-dicarboxypropylation not previously known. This also results in an increase in the itaconate-cysteine adduct, 2, 3-dicarboxypropyl cysteine, akin to 2SC. Itaconate was exported from the mitochondria where it is proposed to alkylate cysteine residues 151, 257, 273, 288 and 297 on the anti-oxidant protein kelch like-ECH-associated protein 1 (KEAP1), as determined by direct alkylation of KEAP1 by 4-OI, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. Furthermore, the activation of Nrf2 was impaired in Irg1 -/- BMDMs and was essential for the inhibition of LPS-induced IL-1β by 4-OI. Overall, immunomodulatory roles for fumarate, itaconate and their derivatives have been characterised, whilst new insights into the anti-inflammatory effect of itaconate have been uncovered that may have therapeutic potential for the treatment of inflammatory diseases.