Cytokine-like Roles for Metabolites in Immunity

Metabolites have functions in the immune system independent of their conventional roles as sources or intermediates in biosynthesis and bioenergetics. We are still in the pioneering phase of gathering information about the functions of specific metabolites in immunoregulation. In this review, we cover succinate, itaconate, α-ketoglutarate, and lactate as examples. Each of these metabolites has a different story of how their immunoregulatory functions were discovered and how their roles in the complex process of inflammation were revealed. Parallels and interactions are emerging between metabolites and cytokines, well-known immunoregulators. We depict molecular mechanisms by which metabolites prime cellular and often physiological changes focusing on intra- and extra-cellular activities and signaling pathways. Possible therapeutic opportunities for immune and inflammatory diseases are emerging. Metabolites have functions in the immune system independent of their conventional roles as sources or intermediates in biosynthesis and bioenergetics. We are still in the pioneering phase of gathering information about the functions of specific metabolites in immunoregulation. In this review, we cover succinate, itaconate, α-ketoglutarate, and lactate as examples. Each of these metabolites has a different story of how their immunoregulatory functions were discovered and how their roles in the complex process of inflammation were revealed. Parallels and interactions are emerging between metabolites and cytokines, well-known immunoregulators. We depict molecular mechanisms by which metabolites prime cellular and often physiological changes focusing on intra- and extra-cellular activities and signaling pathways. Possible therapeutic opportunities for immune and inflammatory diseases are emerging. We are currently witnessing an unprecedented rise in our understanding of how intracellular metabolic changes in leukocytes promote effector mechanisms in the immune response. Specific emphasis in the field of immunometabolism has been on the modulation of the Krebs cycle (also called the tricarboxylic acid [TCA] cycle) and glycolysis. It is well established that, upon activation, leukocytes such as inflammatory macrophages or Th17 T cells switch from the TCA cycle as the primary source of energy and rely on more catabolic pathways, such as aerobic glycolysis. Glycolysis is a less efficient and more ancient pathway used when cells require a rapid boost in energy levels. Remarkably, TCA-cycle-and glycolysis-derived metabolites are not just components for ATP production but have been recently demonstrated to possess signaling functions that are critical for leukocyte activation and differentiation. What is emerging is a new concept whereby specific metabolites are having a profound immunoregulatory role as cytokines, with metabolites either regulating cytokine production or exerting effects similar to those of cytokines. Our understanding of cytokines in immunology was greatly enhanced by techniques such as the ELISA. Similarly, studies in immunometabolism have been greatly enhanced by highly sensitive techniques in mass spectrometry, which have helped to define the alterations in metabolites that occur during leukocyte activation and in disease states. The extent of extracellular activities of immune metabolites is still a work in progress. We have learned that a single metabolite can possess multiple biological activities (O’Neill et al., 2016O’Neill L.A. Kishton R.J. Rathmell J. A guide to immunometabolism for immunologists.Nat. Rev. Immunol. 2016; 16: 553-565Crossref PubMed Scopus (669) Google Scholar). Immune metabolites (metabolites, which have roles outside of metabolism that promote specific events during immune cell activation) can modulate the activity of transcription factors, inhibit specific enzymes, or drive covalent modifications of proteins to alter their functions, resulting in altered gene expression or even changes in the epigenome. The overall focus of this review is to provide an update on the immunomodulatory targets for 4 metabolites for which substantial information has been obtained: succinate, itaconate, α-ketoglutarate (α-KG), and lactate. We now discuss our current knowledge of how these immune metabolites affect leukocytes at the molecular level, draw parallels with cytokines, and describe the immunological consequences of their actions. A major mechanistic insight into the role of succinate as a modulator of immune responses was shown in lipopolysaccharide (LPS)-activated macrophages, where succinate was shown to stabilize hypoxia-inducible factor 1-α (HIF-1α) (Tannahill et al., 2013Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. et al.Succinate is an inflammatory signal that induces IL-1β through HIF-1α.Nature. 2013; 496: 238-242Crossref PubMed Scopus (1247) Google Scholar). Enhanced HIF-1α activity resulted in the transcription of a number of inflammatory genes, including that encoding IL-1β and was essential for glycolytic reprograming. A role for succinate in HIF-1α stabilization had been demonstrated previously in the context of cancer (Guzy et al., 2008Guzy R.D. Sharma B. Bell E. Chandel N.S. Schumacker P.T. Loss of the SdhB, but Not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis.Mol. Cell. Biol. 2008; 28: 718-731Crossref PubMed Scopus (269) Google Scholar). Mutations in succinate dehydrogenase (SDH) in such tumors as paraganglioma caused inefficient SDH activity, resulting in the accumulation of succinate, the inhibition of prolyl hydroxylases (PHDs), and subsequent HIF-1α activation (Selak et al., 2005Selak M.A. Armour S.M. MacKenzie E.D. Boulahbel H. Watson D.G. Mansfield K.D. Pan Y. Simon M.C. Thompson C.B. Gottlieb E. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase.Cancer Cell. 2005; 7: 77-85Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar). IN LPS-activated macrophages, accumulated succinate can also undergo oxidation by SDH and induce mitochondrial reactive oxygen species (ROS), which can also activate HIF-1α and separately inhibit the production of anti-inflammatory mediators such as interleukin (IL)-10 and IL-1RA (Mills et al., 2016Mills E.L. Kelly B. Logan A. Costa A.S.H. Varma M. Bryant C.E. Tourlomousis P. Dabritz J.H.M. Gottlieb E. Latorre I. et al.Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages.Cell. 2016; 167: 457-470.e413Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Succinate was also shown to accumulate in hypoxia during ischemia-reperfusion injury, where its accumulation and oxidation controlled ROS production and contributed to the severity of tissue injury (Chouchani et al., 2014Chouchani E.T. Pell V.R. Gaude E. Aksentijević D. Sundier S.Y. Robb E.L. Logan A. Nadtochiy S.M. Ord E.N.J. Smith A.C. et al.Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.Nature. 2014; 515: 431-435Crossref PubMed Scopus (858) Google Scholar). The buildup of succinate upon LPS stimulation is accompanied by an accumulation of succinyl-coenzyme A (CoA), which can directly modify proteins by succinylation (Tannahill et al., 2013Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. et al.Succinate is an inflammatory signal that induces IL-1β through HIF-1α.Nature. 2013; 496: 238-242Crossref PubMed Scopus (1247) Google Scholar). A number of metabolic enzymes have been shown to be modified, including carbamoyl phosphate synthase 1 (Nakagawa et al., 2009Nakagawa T. Lomb D.J. Haigis M.C. Guarente L. SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle.Cell. 2009; 137: 560-570Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar), SDH itself, pyruvate dehydrogenase (Park et al., 2013Park J. Chen Y. Tishkoff D.X. Peng C. Tan M. Dai L. Xie Z. Zhang Y. Zwaans B.M. Skinner M.E. et al.SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.Mol. Cell. 2013; 50: 919-930Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), acyl-CoA oxidase 1 (Chen et al., 2018Chen X.F. Tian M.X. Sun R.Q. Zhang M.L. Zhou L.S. Jin L. Chen L.L. Zhou W.J. Duan K.L. Chen Y.J. et al.SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer.EMBO Rep. 2018; 19: e45124Crossref PubMed Scopus (24) Google Scholar), 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Rardin et al., 2013Rardin M.J. He W. Nishida Y. Newman J.C. Carrico C. Danielson S.R. Guo A. Gut P. Sahu A.K. Li B. et al.SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.Cell Metab. 2013; 18: 920-933Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), and pyruvate kinase M2 (Wang et al., 2017Wang F. Wang K. Xu W. Zhao S. Ye D. Wang Y. Xu Y. Zhou L. Chu Y. Zhang C. et al.SIRT5 Desuccinylates and Activates Pyruvate Kinase M2 to Block Macrophage IL-1β Production and to Prevent DSS-Induced Colitis in Mice.Cell Rep. 2017; 19: 2331-2344Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), suggesting an important role for succinylation in the regulation of metabolic processes. LPS induces succinylation and a decrease in Sirt5 expression (Tannahill et al., 2013Tannahill G.M. Curtis A.M. Adamik J. Palsson-McDermott E.M. McGettrick A.F. Goel G. Frezza C. Bernard N.J. Kelly B. Foley N.H. et al.Succinate is an inflammatory signal that induces IL-1β through HIF-1α.Nature. 2013; 496: 238-242Crossref PubMed Scopus (1247) Google Scholar), which can act as a desuccinylase. The importance of this mechanism for the consequent immune response is highlighted by elevated levels of IL-1β in SIRT5-deficient mice examined in a colitis model (Wang et al., 2017Wang F. Wang K. Xu W. Zhao S. Ye D. Wang Y. Xu Y. Zhou L. Chu Y. Zhang C. et al.SIRT5 Desuccinylates and Activates Pyruvate Kinase M2 to Block Macrophage IL-1β Production and to Prevent DSS-Induced Colitis in Mice.Cell Rep. 2017; 19: 2331-2344Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). An extracellular role for succinate has also been identified, where it acts via its receptor succinate receptor 1 (SUCNR1) (He et al., 2004He W. Miao F.J. Lin D.C. Schwandner R.T. Wang Z. Gao J. Chen J.L. Tian H. Ling L. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.Nature. 2004; 429: 188-193Crossref PubMed Scopus (517) Google Scholar). This is a G-protein-coupled receptor (GPCR), and its activation by succinate triggers at least two signaling pathways: a pertussis-toxin (PTX)-sensitive Gi/Go pathway and a PTX-insensitive Gq pathway (He et al., 2004He W. Miao F.J. Lin D.C. Schwandner R.T. Wang Z. Gao J. Chen J.L. Tian H. Ling L. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.Nature. 2004; 429: 188-193Crossref PubMed Scopus (517) Google Scholar). Since these are well-known signaling pathways, many molecular biology tools were immediately available to explore extracellular properties of succinate. Although originally SUCNR1 was shown to be predominantly expressed in the kidney, successive studies demonstrated the importance of SUCNR1 in immune cells. In dendritic cells (DCs), succinate triggers intracellular calcium mobilization, induces migratory responses, and acts in synergy with Toll-like receptor ligands to induce production of pro-inflammatory cytokines (Rubic et al., 2008Rubic T. Lametschwandtner G. Jost S. Hinteregger S. Kund J. Carballido-Perrig N. Schwärzler C. Junt T. Voshol H. Meingassner J.G. et al.Triggering the succinate receptor GPR91 on dendritic cells enhances immunity.Nat. Immunol. 2008; 9: 1261-1269Crossref PubMed Scopus (201) Google Scholar). By acting through SUCNR1, succinate was also effective in enhancing antigen-specific activation of T cells (Rubic et al., 2008Rubic T. Lametschwandtner G. Jost S. Hinteregger S. Kund J. Carballido-Perrig N. Schwärzler C. Junt T. Voshol H. Meingassner J.G. et al.Triggering the succinate receptor GPR91 on dendritic cells enhances immunity.Nat. Immunol. 2008; 9: 1261-1269Crossref PubMed Scopus (201) Google Scholar). In vivo immunomodulatory properties of succinate were determined in SUCNR1-deficient allografts, which elicited weaker transplant rejection compared to the corresponding grafts from wild-type mice, suggesting that SUCNR1 is involved in sensing immunological danger (Rubic et al., 2008Rubic T. Lametschwandtner G. Jost S. Hinteregger S. Kund J. Carballido-Perrig N. Schwärzler C. Junt T. Voshol H. Meingassner J.G. et al.Triggering the succinate receptor GPR91 on dendritic cells enhances immunity.Nat. Immunol. 2008; 9: 1261-1269Crossref PubMed Scopus (201) Google Scholar). Succinate was also shown to promote hematopoietic cell proliferation by phosphorylation of the ERK1/2 mitogen-activated protein kinase (MAPK) pathway and inositol phosphate accumulation in a PTX-sensitive manner (Hakak et al., 2009Hakak Y. Lehmann-Bruinsma K. Phillips S. Le T. Liaw C. Connolly D.T. Behan D.P. The role of the GPR91 ligand succinate in hematopoiesis.J. Leukoc. Biol. 2009; 85: 837-843Crossref PubMed Scopus (46) Google Scholar). The significance of growth-factor-like pro-proliferative SUCNR1 signaling was later indicated by the ability of succinate to stimulate osteoclastogenesis (Guo et al., 2017Guo Y. Xie C. Li X. Yang J. Yu T. Zhang R. Zhang T. Saxena D. Snyder M. Wu Y. Li X. Succinate and its G-protein-coupled receptor stimulates osteoclastogenesis.Nat. Commun. 2017; 8: 15621Crossref PubMed Scopus (22) Google Scholar). A SUCNR1/succinate-dependent feed-forward loop has been demonstrated in leukocyte activation. LPS induces SUCNR1 in macrophages and promotes the release of succinate, which, in an autocrine manner, further induces IL-1β production (Littlewood-Evans et al., 2016Littlewood-Evans A. Sarret S. Apfel V. Loesle P. Dawson J. Zhang J. Muller A. Tigani B. Kneuer R. Patel S. et al.GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis.J. Exp. Med. 2016; 213: 1655-1662Crossref PubMed Scopus (135) Google Scholar). Succinate has been shown to be abundant in synovial fluid from rheumatoid arthritis (RA) patients. This fluid, in turn, has been shown to induce IL-1β released from macrophages in a SUCNR1-dependent manner, implicating succinate in the fluid as a determining factor for IL-1β production and implying SUCNR1 antagonism as a novel RA treatment (Littlewood-Evans et al., 2016Littlewood-Evans A. Sarret S. Apfel V. Loesle P. Dawson J. Zhang J. Muller A. Tigani B. Kneuer R. Patel S. et al.GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis.J. Exp. Med. 2016; 213: 1655-1662Crossref PubMed Scopus (135) Google Scholar). Chemokine-like properties of succinate-SUCNR1 signaling have been demonstrated by the enhanced infiltration of inflammatory macrophages in a model of type 2 diabetes (van Diepen et al., 2017van Diepen J.A. Robben J.H. Hooiveld G.J. Carmone C. Alsady M. Boutens L. Bekkenkamp-Grovenstein M. Hijmans A. Engelke U.F.H. Wevers R.A. et al.SUCNR1-mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes.Diabetologia. 2017; 60: 1304-1313Crossref PubMed Scopus (35) Google Scholar). In this model, hypoxia and hyperglycemia independently drove the release of succinate from adipose tissue. Patient plasma levels of succinate were higher in patients with type 2 diabetes compared with non-diabetic individuals, suggesting that SUCNR1 is a promising therapeutic target in obesity-induced type 2 diabetes (van Diepen et al., 2017van Diepen J.A. Robben J.H. Hooiveld G.J. Carmone C. Alsady M. Boutens L. Bekkenkamp-Grovenstein M. Hijmans A. Engelke U.F.H. Wevers R.A. et al.SUCNR1-mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes.Diabetologia. 2017; 60: 1304-1313Crossref PubMed Scopus (35) Google Scholar). Finally, SUCNR1 was shown to be required for microbiota-triggered type 2 immunity (Lei et al., 2018Lei W. Ren W. Ohmoto M. Urban Jr., J.F. Matsumoto I. Margolskee R.F. Jiang P. Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine.Proc. Natl. Acad. Sci. USA. 2018; 115: 5552-5557Crossref PubMed Scopus (49) Google Scholar) and promote immunoglobulin secretion by naive B cells (Caielli et al., 2019Caielli S. Veiga D.T. Balasubramanian P. Athale S. Domic B. Murat E. Banchereau R. Xu Z. Chandra M. Chung C.H. et al.A CD4+ T cell population expanded in lupus blood provides B cell help through interleukin-10 and succinate.Nat. Med. 2019; 25: 75-81Crossref PubMed Scopus (42) Google Scholar), demonstrating that the biological activities of succinate are not limited to classical inflammatory responses. Succinate might, therefore, be an important link between innate and adaptive immunity. More recent studies have indicated an even more complex role for succinate in immunity. Macrophage-derived succinate released during central nervous system inflammation activates SUCNR1 on neural stem cells, leading them to secrete prostaglandin E2 and, furthermore, scavenge extracellular succinate with consequential anti-inflammatory effects (Peruzzotti-Jametti et al., 2018Peruzzotti-Jametti L. Bernstock J.D. Vicario N. Costa A.S.H. Kwok C.K. Leonardi T. Booty L.M. Bicci I. Balzarotti B. Volpe G. et al.Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.Cell Stem Cell. 2018; 22: 355-368.e13Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This work revealed a suppressive role for the succinate-SUCNR1 axis in the chronically inflamed brain. Another study examined the contribution of SUCNR1 to macrophage polarization. Mice with SUCNR1-deficient myeloid cells exhibited a pro-inflammatory phenotype and disrupted glucose homeostasis (Keiran et al., 2019Keiran N. Ceperuelo-Mallafré V. Calvo E. Hernández-Alvarez M.I. Ejarque M. Núñez-Roa C. Horrillo D. Maymó-Masip E. Rodríguez M.M. Fradera R. et al.SUCNR1 controls an anti-inflammatory program in macrophages to regulate the metabolic response to obesity.Nat. Immunol. 2019; 20: 581-592Crossref PubMed Scopus (10) Google Scholar). Peritoneal macrophages activated with LPS decreased SUCNR1 expression, and SUCNR1 activation promoted the response to type 2 cytokines and an anti-inflammatory phenotype (Keiran et al., 2019Keiran N. Ceperuelo-Mallafré V. Calvo E. Hernández-Alvarez M.I. Ejarque M. Núñez-Roa C. Horrillo D. Maymó-Masip E. Rodríguez M.M. Fradera R. et al.SUCNR1 controls an anti-inflammatory program in macrophages to regulate the metabolic response to obesity.Nat. Immunol. 2019; 20: 581-592Crossref PubMed Scopus (10) Google Scholar). Succinate decreased the expression of inflammatory markers in adipose tissue from lean human subjects but not that from obese subjects who had lower expression of SUCNR1 in adipose-tissue-resident macrophages (Keiran et al., 2019Keiran N. Ceperuelo-Mallafré V. Calvo E. Hernández-Alvarez M.I. Ejarque M. Núñez-Roa C. Horrillo D. Maymó-Masip E. Rodríguez M.M. Fradera R. et al.SUCNR1 controls an anti-inflammatory program in macrophages to regulate the metabolic response to obesity.Nat. Immunol. 2019; 20: 581-592Crossref PubMed Scopus (10) Google Scholar). In a certain context, therefore, succinate might be anti-inflammatory. Conceivably, the intracellular and extracellular roles of succinate might be distinct and context dependent. Pharmacological modulators of SDH activity and the use of succinate analogs (Geubelle et al., 2017Geubelle P. Gilissen J. Dilly S. Poma L. Dupuis N. Laschet C. Abboud D. Inoue A. Jouret F. Pirotte B. Hanson J. Identification and pharmacological characterization of succinate receptor agonists.Br. J. Pharmacol. 2017; 174: 796-808Crossref PubMed Scopus (0) Google Scholar) may shed new light on the role of succinate in various diseases. Similarities in signaling between SUCNR1 and chemokine receptors, as well as structural similarities between SUCNR1 and the P2Y receptor (Gilissen et al., 2016Gilissen J. Jouret F. Pirotte B. Hanson J. Insight into SUCNR1 (GPR91) structure and function.Pharmacol. Ther. 2016; 159: 56-65Crossref PubMed Scopus (41) Google Scholar), suggest that the succinate receptor might be a promising drug target too. Although its modulation would need to be context dependent, blocking SUCNR1 might be useful in the treatment of RA, or in type-2-immune-related immune diseases such as asthma/allergy or fibrosis (Figure 1). Immune-responsive gene 1 (IRG1)/cis-aconitase decarboxylase is an enzyme responsible for itaconate synthesis (Michelucci et al., 2013Michelucci A. Cordes T. Ghelfi J. Pailot A. Reiling N. Goldmann O. Binz T. Wegner A. Tallam A. Rausell A. et al.Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.Proc. Natl. Acad. Sci. USA. 2013; 110: 7820-7825Crossref PubMed Scopus (317) Google Scholar). Itaconate is an offshoot from the TCA cycle being made from aconitate by IRG1/aconitate decarboxylase 1(ACOD1). Initially, itaconate was shown to be induced upon bacterial infection both in vitro (Strelko et al., 2011Strelko C.L. Lu W. Dufort F.J. Seyfried T.N. Chiles T.C. Rabinowitz J.D. Roberts M.F. Itaconic acid is a mammalian metabolite induced during macrophage activation.J. Am. Chem. Soc. 2011; 133: 16386-16389Crossref PubMed Scopus (116) Google Scholar) and in vivo (Shin et al., 2011Shin J.H. Yang J.Y. Jeon B.Y. Yoon Y.J. Cho S.N. Kang Y.H. Ryu D.H. Hwang G.S. (1)H NMR-based metabolomic profiling in mice infected with Mycobacterium tuberculosis.J. 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Chem. 2013; 288: 16225-16234Crossref PubMed Scopus (72) Google Scholar). An anti-inflammatory mechanism for itaconate was shown with the help of a cell-permeable methyl ester derivative of itaconate, dimethyl itaconate (DI) (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Google Scholar). DI inhibited IL-6, IL-12, and IL-1β induced in macrophages by LPS, while IRG1-deficient macrophages had enhanced pro-inflammatory cytokines, demonstrating the anti-inflammatory role of endogenous itaconate (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Google Scholar). Itaconate was shown to inhibit SDH both in vitro and in ischemia reperfusion, verifying a previously proposed hypothesis (Lampropoulou et al., 2016Lampropoulou V. Sergushichev A. Bambouskova M. Nair S. Vincent E.E. Loginicheva E. Cervantes-Barragan L. Ma X. Huang S.C. Griss T. et al.Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation.Cell Metab. 2016; 24: 158-166Abstract Full Text Full Text PDF PubMed Google Scholar, Ackermann and Potter, 1949Ackermann W.W. Potter V.R. Enzyme inhibition in relation to chemotherapy.Proc. Soc. Exp. Biol. Med. 1949; 72: 1-9Crossref PubMed Google Scholar). These studies on itaconate, therefore, built on the role of SDH in IL-1β production, indicating that SDH inhibition was likely to be an important aspect of the anti-inflammatory effect of itaconate. Further understanding of itaconate in immunity was achieved with the help of a new itaconate derivative, 4-octyl itaconate (4-OI) (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (274) Google Scholar). This study found a previously unknown post-translational modification, namely 2,3-dicarboxypropylation, providing an additional mechanism of the anti-inflammatory properties of itaconate (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (274) Google Scholar). 2,3-dicarboxypropylation is a form of alkylation induced by itaconate on target cysteine residues, including cysteine 151 (C151), on the redox-sensing protein kelch-like ECH-associated protein 1 (KEAP1). Alkylation of C151 inactivates KEAP1, which subsequently activates the anti-inflammatory transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Mutated C151 in KEAP1 impaired the ability of 4-OI to activate NRF2 (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (274) Google Scholar). Activated NRF2 initiated the transcription of genes driving an antioxidant program, reducing intracellular ROS, HIF-1α and IL-1β expression (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (274) Google Scholar). The in vivo anti-inflammatory actions of 4-OI were demonstrated in a mouse model of LPS-induced peritonitis. 4-OI reduced serum IL-1β and TNF-α in peritonitis and prolonged survival in a sepsis model (Mills et al., 2018Mills E.L. Ryan D.G. Prag H.A. Dikovskaya D. Menon D. Zaslona Z. Jedrychowski M.P. Costa A.S.H. Higgins M. Hams E. et al.Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.Nature. 2018; 556: 113-117Crossref PubMed Scopus (274) Google Scholar). Several proteins were shown to be modified by 4-OI and endogenous itaconate, including the glycolytic enzyme LDH-A. A specific thiol-reactive probe for quantitative profiling of cysteine modifications by itaconate was used in order to determine global proteome reactivity. Itaconate covalently modified key glycolytic enzymes and attenuated the inflammatory response in LPS-stimulated macrophages by impairing glycolysis (Qin et al., 2019Qin W. Qin K. Zhang Y. Jia W. Chen Y. Cheng B. Peng L. Chen N. Liu Y. Zhou W. et al.S-glycosylation-based cysteine profiling reveals regulation of glycolysis by itaconate.Nat. Chem. Biol. 2019; 15: 983-991Crossref PubMed Scopus (19) Google Scholar), demonstrating the ability of endogenous itaconate to alter metabolic pathways. The induction of NRF2 by itaconate was also observed in another study utilizing DI to increase expression of NRF2 (Bambouskova et al., 2018Bambouskova M. Gorvel L. Lampropoulou V. Sergushichev A. Loginicheva E. Johnson K. Korenfeld D. Mathyer M.E. Kim H. Huang L.H. et al.Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.Nature. 2018; 556: 501-504Crossref PubMed Scopus (121) Google Scholar). In this study, NRF2 expression was abrogated in LPS-stimulated IRG1-deficient macrophages (Bambouskova et al., 2018Bambouskova M. Gorvel L. Lampropoulou V. Sergushichev A. Loginicheva E. Johnson K. Korenfeld D. Mathyer M.E. Kim H. Huang L.H. et al.Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.Nature. 2018; 556: 501-504Crossref PubMed Scopus (121) Google Scholar). However, a novel mechanistic insight was proposed, and activation of NRF2 was explained here by the electrophilic nature of itaconate and the ability of DI to deplete glutathione (GSH) (Bambouskova et al., 2018Bambouskova M. Gorvel L. Lampropoulou V. Sergushichev A. Loginicheva E. Johnson K. Korenfeld D. Mathyer M.E. Kim H. Huang L.H. et al.Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.Nature. 2018; 556: 501-504Crossref PubMed Scopus (121) Google Scholar), as demonstrated previously (Kobayashi et al., 2006Kobayashi A. Kang M.I. Watai Y. Tong K.I. Shibata T. Uchida K. Yamamoto M. Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1.Mol. Cell. Biol. 2006; 26: 221-229Crossref PubMed Scopus (586) Google Scholar). Itaconate was al