Abstract
Since its discovery in inflammatory macrophages, itaconate has attracted much attention due to its antimicrobial and immunomodulatory activity1,2,3. However, instead of investigating itaconate itself, most studies used derivatized forms of itaconate and thus the role of non-derivatized itaconate needs to be scrutinized. Mesaconate, a metabolite structurally very close to itaconate, has never been implicated in mammalian cells. Here we show that mesaconate is synthesized in inflammatory macrophages from itaconate. We find that both, non-derivatized itaconate and mesaconate dampen the glycolytic activity to a similar extent, whereas only itaconate is able to repress tricarboxylic acid cycle activity and cellular respiration. In contrast to itaconate, mesaconate does not inhibit succinate dehydrogenase. Despite their distinct impact on metabolism, both metabolites exert similar immunomodulatory effects in pro-inflammatory macrophages, specifically a reduction of interleukin (IL)-6 and IL-12 secretion and an increase of CXCL10 production in a manner that is independent of NRF2 and ATF3. We show that a treatment with neither mesaconate nor itaconate impairs IL-1β secretion and inflammasome activation. In summary, our results identify mesaconate as an immunomodulatory metabolite in macrophages, which interferes to a lesser extent with cellular metabolism than itaconate.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Source data for all figures and extended data figures as well as RNA-seq data are deposited in the repository platform of Technische Universität Braunschweig at https://doi.org/10.24355/dbbs.084-202203091309-0. The uncropped images of all blots for Fig. 3f are provided in Supplementary Fig. 2. All other information is available from the corresponding author on reasonable request.
References
Michelucci, A. et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc. Natl Acad. Sci. USA 110, 7820–7825 (2013).
Mills, E. L. et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature 556, 113–117 (2018).
Bambouskova, M. et al. Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis. Nature 556, 501–504 (2018).
Wynn, T. A., Chawla, A. & Pollard, J. W. Macrophage biology in development, homeostasis and disease. Nature 496, 445–455 (2013).
He, W., Heinz, A., Jahn, D. & Hiller, K. Complexity of macrophage metabolism in infection. Curr. Opin. Biotechnol. 68, 231–239 (2021).
Cordes, T. et al. Immunoresponsive gene 1 and itaconate inhibit succinate dehydrogenase to modulate intracellular succinate levels. J. Biol. Chem. 291, 14274–14284 (2016).
Nemeth, B. et al. Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage. FASEB J. 30, 286–300 (2016).
Cordes, T. & Metallo, C. M. Itaconate alters succinate and coenzyme a metabolism via Inhibition of mitochondrial complex II and methylmalonyl-CoA mutase. Metabolites 11, 117 (2021).
Lampropoulou, V. et al. Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of Inflammation. Cell Metab. 24, 158–166 (2016).
Liao, S. T. et al. 4-Octyl itaconate inhibits aerobic glycolysis by targeting GAPDH to exert anti-inflammatory effects. Nat. Commun. 10, 5091 (2019).
Qin, W. et al. S-glycosylation-based cysteine profiling reveals regulation of glycolysis by itaconate. Nat. Chem. Biol. 15, 983–991 (2019).
ElAzzouny, M. et al. Dimethyl itaconate is not metabolized into itaconate intracellularly. J. Biol. Chem. 292, 4766–4769 (2017).
Swain, A. et al. Comparative evaluation of itaconate and its derivatives reveals divergent inflammasome and type I interferon regulation in macrophages. Nat. Metab. 2, 594–602 (2020).
Bambouskova, M. et al. Itaconate confers tolerance to late NLRP3 inflammasome activation. Cell Rep. 34, 108756 (2021).
Ghosn, E. E. et al. Two physically, functionally, and developmentally distinct peritoneal macrophage subsets. Proc. Natl Acad. Sci. USA 107, 2568–2573 (2010).
Wang, J. & Zhang, K. Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway. Metab. Eng. 30, 190–196 (2015).
Meiser, J. et al. Pro-inflammatory macrophages sustain pyruvate oxidation through pyruvate dehydrogenase for the synthesis of itaconate and to enable cytokine expression. J. Biol. Chem. 291, 3932–3946 (2016).
Hooftman, A. et al. The immunomodulatory metabolite itaconate modifies NLRP3 and inhibits inflammasome activation. Cell Metab. 32, 468–478 (2020).
Broz, P. & Dixit, V. M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).
Mangan, M. S. J. et al. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat. Rev. Drug Disco. 17, 688 (2018).
Chen, F. et al. Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism. Nat. Metab.
Kornberg, M. D. et al. Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity. Science 360, 449–453 (2018).
Montes Diaz, G., Hupperts, R., Fraussen, J. & Somers, V. Dimethyl fumarate treatment in multiple sclerosis: recent advances in clinical and immunological studies. Autoimmun. Rev. 17, 1240–1250 (2018).
Hartman, M. G. et al. Role for activating transcription factor 3 in stress-induced beta-cell apoptosis. Mol. Cell. Biol. 24, 5721–5732 (2004).
Chen, Y. et al. Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure. Hepatology 45, 1118–1128 (2007).
Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R. & Forster, I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 8, 265–277 (1999).
Weindl, D. et al. Bridging the gap between non-targeted stable isotope labeling and metabolic flux analysis. Cancer Metab. 4, 10 (2016).
Sapcariu, S. C. et al. Simultaneous extraction of proteins and metabolites from cells in culture. MethodsX 1, 74–80 (2014).
He, W. et al. TLR4 triggered complex inflammation in human pancreatic islets. Biochim. Biophys. Acta Mol. Basis Dis. 1865, 86–97 (2019).
Battello, N. et al. The role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells. Cancer Metab. 4, 3 (2016).
Hiller, K. et al. MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal. Chem. 81, 3429–3439 (2009).
Nonnenmacher, Y. et al. Analysis of mitochondrial metabolism in situ: combining stable isotope labeling with selective permeabilization. Metab. Eng. 43, 147–155 (2017).
Nonnenmacher, Y., Palorini, R. & Hiller, K. Determining compartment-specific metabolic fluxes. Methods Mol. Biol. 1862, 137–149 (2019).
Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537–W544 (2018).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).
Acknowledgements
This work is funded by the Deutsche Forschungsgemeinschaft (German Research Foundation) project HI1400/3-1 (to K.H.), SFB-1403 project 414786233 (to E.L.), SFB-1454 project 432325352 (to Z.A., E.L. and K.H.) and ImmunoSensation2 EXC2151 project 390873048 (to Z.A. and E.L.). D.B. is supported by the FNR-ATTRACT programme (A14/BM/7632103), and by the FNR-CORE (C18/BM/12691266). D.B. and C.D. receive funding through the FNRS-Televie programme (7.4597.19). We thank the NIH Common Fund Metabolite Standards Synthesis Core (NHLBI contract no. HHSN268201300022C) for providing isotopic labelled itaconate ([U-13C5]itaconate).
Author information
Authors and Affiliations
Contributions
W.H. and K.H. conceived and designed the study and wrote the manuscript, with contributions from M.L., E.G., C.D., T.C., R.G., C.M.M., Z.A., E.L. and D.B. W.H., A. Henne, M.L., E.G., F.N., A. Heinz, C.K., C.D., M.G., T.C., J.H., O.G., A.E., C.V., J.B.-C. and Z.A. performed experiments. W.H., A. Henne, M.L., E.G., F.N., A. Heinz, C.K., C.D., M.G., T.C., J.H., R.G., Z.A., E.L., D.B. and K.H. analysed the data. W.H., M.L., E.G., C.D., T.C., C.K.H., C.M.M., E.M., Z.A., E.L., D.B. and K.H. contributed to the discussion. C.D., O.G., H.G., M.K., C.K.H., E.M. and D.B. contributed to vital material.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Metabolism thanks Ping-Chih Ho, Luke O’Neill and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson, in collaboration with the Nature Metabolism team.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Mesaconate is endogenously synthesized from itaconate and cellularly permeable in mouse macrophages.
a. Intracellular itaconate and mesaconate of BMDMs from wildtype (WT) or IRG1-deficient (KO) mice stimulated with or without LPS for 24 h. b. Intracellular itaconate and mesaconate of BMDMs incubated with exogenous itaconate or mesaconate for the indicated time points. c. Intracellular itaconate and mesaconate of RAW264.7 cells transfected with indicated siRNA, followed with LPS stimulation for 24 h. d. Silencing efficiency of indicated genes from experiments shown in (c). Data are presented as mean ± SEM: a,b. calculated from n = 3 mice from a representative experiment of 2 independent experiments; c,d. n = 6 biological replicates pooled from 2 independent experiments. P values were calculated by one-way ANOVA with Dunnet post-test and overlayed on respective comparisons.
Extended Data Fig. 2 Mesaconate and itaconate exhibit different metabolic impacts in macrophages.
a. Intracellular metabolite levels of glycolysis and TCA cycle in human monocyte-derived macrophages pre-treated with 10 mM itaconate or mesaconate for 4 h prior to LPS stimulation for 3 h. b,c. Unnormalized oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) from the experiment shown in Fig. 2e. Data are presented as mean ± SEM, and figures are representative of 3 independent experiments.
Extended Data Fig. 3 Both non-derivatized itaconate and mesaconate are immunomodulatory in mouse macrophages.
a. Cytokine expression of RAW264.7 cells pre-treated with 10 mM itaconate, 0.25 mM DMI or 0.125 mM 4-OI for 4 h prior to LPS stimulation for 3 h. b. Viability of BMDMs pre-treated with indicated concentrations of mesaconate, itaconate, DMI and 4-OI for 4 h prior to LPS stimulation overnight. c. Non-targeted intracellular metabolome of RAW264.7 cells treated with 1 mM itaconate, DMI or 4-OI for 6 h. d. IL-1β in supernatants of BMDMs treated with indicated concentrations of mesaconate, itaconate, DMI or 4-OI, LPS stimulation as well as NLRP3 or NLRC4 inflammasome activation with nigericin or a mixture of BsaK and protective antigen, respectively, following a post-treatment protocol as indicated at top. Data are shown as: a,b,d. mean ± SEM calculated from (a) n = 3 biological replicates from one representative experiment, (b) n = 6 biological replicates from 3 mice, (d) n = 8 mice from 3 independent experiments; c. a representative heatmap showing z-scores of the data by row, from 2 independent experiments; P values were calculated by one-way ANOVA with Dunnet post-test (a) or paired t test (d, paired by each mouse) and overlayed on respective comparisons.
Extended Data Fig. 4 Itaconate derivatives impairs IL-1β secretion in human macrophages.
a,b. IL-1β in supernatants of human monocyte-derived macrophages treated with mesaconate, itaconate, DMI or 4-OI of indicated concentrations, LPS stimulation as well as NLRP3 or NLRC4 inflammasome activation by nigericin or a mixture of BsaK and protective antigen, respectively, following the pre- (a) or post-treatment protocol (b) as shown at the top. c. IL-1β in supernatants of human whole blood pre-treated with mesaconate, itaconate, DMI or 4-OI of indicated concentrations for 4 h prior to LPS stimulation overnight. Data are shown as mean ± SEM calculated from n = 5 donors. P values were calculated by one-way ANOVA with Dunnet post-test and overlayed on respective comparisons.
Extended Data Fig. 5 Dependency of DMI and 4-OI on NRF2 and ATF3 for their anti-inflammatory effects in mouse BMDMs.
Cytokine secretion of BMDMs from wildtype (WT) and Nrf2-KO (a) or Atf3-KO (b) mice pre-treated with 0.25 mM DMI or 0.125 mM 4-OI for 4 h prior to LPS stimulation for 21 h. Data are shown as mean ± SEM calculated from n = 5 (a) or 3 (b) mice. P values were calculated by multiple unpaired t-test and overlayed on respective comparisons.
Extended Data Fig. 6 Effects of itaconate and mesaconate are independent of GSH.
a. Cytokine secretion of BMDMs from wildtype mice pre-treated with 10 mM itaconate or mesaconate, or 0.25 mM DMI, in the presence or absence of 1 mM cellular permeable GSH (EtGSH) for 4 h prior to LPS stimulation for 21 h. b,c. Cytokine secretion (b), glucose uptake and lactate secretion (c) of Gclc-deficient (Gclc-KO) BMDMs pre-treated with 10 mM itaconate or mesaconate for 4 h prior to LPS stimulation for 21 h. Data are shown as: a. mean ± SEM calculated from n = 4 mice; b,c. mean with individual mouse data pooled from n = 6 (b) or 3 (c) mice, and conditions from individual mouse are connected with a line. P values were calculated by multiple unpaired t-test (a) or paired t -test (b,c, paired by each mouse) and overlayed on respective comparisons.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2
Rights and permissions
About this article
Cite this article
He, W., Henne, A., Lauterbach, M. et al. Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages. Nat Metab 4, 524–533 (2022). https://doi.org/10.1038/s42255-022-00565-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s42255-022-00565-1
This article is cited by
-
Itaconate inhibits corticosterone-induced necroptosis and neuroinflammation via up-regulating menin in HT22 cells
Journal of Physiology and Biochemistry (2024)
-
Metabolite itaconate in host immunoregulation and defense
Cellular & Molecular Biology Letters (2023)
-
Itaconate: A Potent Macrophage Immunomodulator
Inflammation (2023)
-
Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism
Nature Metabolism (2022)
-
A genetically encoded fluorescent biosensor for detecting itaconate with subcellular resolution in living macrophages
Nature Communications (2022)