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Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages

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.

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Fig. 1: Mesaconate is endogenously synthesized from itaconate in macrophages.
Fig. 2: Impact of mesaconate and itaconate on macrophage metabolism.
Fig. 3: Both non-derivatized itaconate and mesaconate are immunomodulatory in macrophages.
Fig. 4: Itaconate and mesaconate modulate cytokine production in NRF2- and ATF3-deficient macrophages, and protect mice from LPS-induced sepsis.

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.

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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).

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Authors

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.

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Correspondence to Karsten Hiller.

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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.

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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.

Extended Data Table 1 Overview of the immunometabolic effects of itaconate and mesaconate in mouse macrophages. SUCLG, succinate-CoA ligase; AUH, AU RNA-binding protein/enoyl-coenzyme A hydratase, also known as methylglutaconyl-CoA hydratase

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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

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