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Oct 16, 2024

Itaconate drives mtRNA-mediated type I interferon production through inhibition of succinate dehydrogenase | Nature Metabolism

Nature Metabolism (2024)Cite this article Metrics details Itaconate is one of the most highly upregulated metabolites in inflammatory macrophages and has been shown to have immunomodulatory

Nature Metabolism (2024)Cite this article

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Itaconate is one of the most highly upregulated metabolites in inflammatory macrophages and has been shown to have immunomodulatory properties. Here, we show that itaconate promotes type I interferon production through inhibition of succinate dehydrogenase (SDH). Using pharmacological and genetic approaches, we show that SDH inhibition by endogenous or exogenous itaconate leads to double-stranded mitochondrial RNA (mtRNA) release, which is dependent on the mitochondrial pore formed by VDAC1. In addition, the double-stranded RNA sensors MDA5 and RIG-I are required for IFNβ production in response to SDH inhibition by itaconate. Collectively, our data indicate that inhibition of SDH by itaconate links TCA cycle modulation to type I interferon production through mtRNA release.

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The RNA-seq data in this manuscript have been deposited at Dryad (https://doi.org/10.5061/dryad.vhhmgqp36). The IREA data in this manuscript can be found in Supplementary Table 1. Source data are provided with this paper.

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We acknowledge the Irish Blood Transfusion Service for supporting our research by approving us to use anonymized untransfused blood components in our research. L.A.J.O. was funded by the European Research Council (Metabinnate 834370) and the Science Foundation Ireland (20/SPP/3685). The Simarro lab is funded by (1) Agencia Estatal de Investigación, Grant Number PID2020-118517RB-I00, and (2) Programa Estratégico Instituto de Biomedicina y Genética Molecular (IBGM), Junta de Castilla y León, Award Number CCVC8485. We thank all members of the O’Neill laboratory for helpful discussions.

These authors contributed equally: Shane M. O’Carroll, Christian G. Peace.

School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

Shane M. O’Carroll, Christian G. Peace, Juliana E. Toller-Kawahisa, Yukun Min, Alexander Hooftman, Sara Charki, Aline Zoller, Anne F. Mcgettrick, Emily A. Day & Luke A. J. O’Neill

Histology Lab, Children’s Health Ireland at Crumlin, Dublin, Ireland

Louise Kehoe & Maureen J. O’Sullivan

Departments of Histopathology and Paediatrics, Trinity College Dublin, Dublin, Ireland

Maureen J. O’Sullivan

Department of Cell Biology, Genetics, Histology and Pharmacology, Faculty of Medicine, University of Valladolid, Valladolid, Spain

Maria Simarro

Unit of Excellence Institute of Biomedicine and Molecular Genetics (IBGM), University of Valladolid and Spanish National Research Council (CSIC), Valladolid, Spain

Maria Simarro

Department of Medicine, Division of Endocrinology, Stanford School of Medicine, Stanford, CA, USA

Neali Armstrong & Justin P. Annes

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S.M.O., C.G.P. and L.A.J.O. conceptualized the project. S.M.O. and C.G.P. were experimentalists, provided intellectual input, designed all experiments, analyzed and visualized the data and co-wrote the paper with input from all authors. J.E.T.-K., Y.M., A.H., S.C., A.Z., A.F.M., E.A.D. and L.K. performed in vitro experiments. M.J.O., M.S., N.A. and J.P.A. assisted with genetic models and provided intellectual input. L.A.J.O. obtained funding, provided intellectual input and oversaw the research program.

Correspondence to Luke A. J. O’Neill.

L.A.J.O. is a paid consultant for Sitryx Therapeutics, Sail Biomedicines and Montai Health. The other authors declare no competing interests.

Nature Metabolism thanks Ricardo Gazzinelli, Karsten Hiller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt, in collaboration with the Nature Metabolism editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

a, PCA plot of RNA-Seq analysis. b, Differential expression gene clustering heatmap with Z-score. c, MsigDB Hallmark pathway enrichment analysis of differentially expressed genes from itaconate pretreated BMDMs followed by 3 h LPS treatment. d, NRF2 pathway enrichment plot of itaconate-treated BMDMs. e) Western blot of IRG1 in Irg1+/+ and Irg1−/− BMDMs treated with LPS for 24 h and 48 h. f, qPCR of IRG1 (p = 1.017e-11) in human PBMCs that were harvested in the presence of Irg1 siRNA (n=3 donors; LPS 24 h). Data are presented as mean ± SEM. P values were calculated using two-way ANOVA for multiple comparisons.

Source data

a, IFNβ release and b, Ifnb1 expression in BMDMs pretreated with DMSO or 4-OI a (n=6 mice; LPS 4 h) b, (n=9; LPS 4 h). c, Ifnb1 expression of BMDMs pretreated with DMSO or DEM (n=3 mice; LPS 4 h) from 3 independent experiments. d, Normalised counts from RNA-seq data in BMDMs pretreated with DMSO or DMF (n=3 mice; LPS 4 h) from 3 independent experiments Data are presented as mean ± SEM. P values were calculated using one-way ANOVA for multiple comparisons or two-tailed student’s t-test for pairwise comparisons.

Source data

a, Sdhb and b, Il1b expression in Sdhbfl/fl and Sdhb−/− BMDMs (n=2 mice; LPS 4 h) from 2 independent experiments. c, IFNB expression in IRG1+/+ and IRG1−/− THP-1 cell lines pretreated with DMSO or TTFA (n=4 replicates; LPS/IFN𝛄 4 h) from 2 independent experiments d, IFNB expression in THP-1 derived macrophages pretreated with PBS or Mesaconate (n=4 replicates; LPS/IFNγ 4 h) from 2 independent experiments. Data are presented as mean ± SEM. P values were calculated using two-way ANOVA for multiple comparisons or two-tailed student’s t-test for pairwise comparisons.

Source data

a-d, Expression of a, Ddx58, b, Ifih1, c, Mb21d1, and d, Tlr9 in BMDMs in the presence of a,c, Mb21d1, Ddx58, b,d, Tlr9 and Ifih1 siRNA pretreated with DMSO or TTFA (n=3 mice; LPS 4 h) from 3 independent experiments. e-g, Expression of e, Mb21d1 f, Ifih1, and g, Ddx58 in the presence of Mb21d1, Ifih1, and Ddx58 siRNA in BMDMs pretreated with PBS or ITA (n=2-3 mice; LPS 4 h) from 2 or 3 independent experiments. h-j IFNβ release in BMDMs transfected with h, G3-YSD, i, Poly(I:C), or j, 5′PPP-dsRNA in the presence of h, Mb21d1, i, Ifih1, or j, Ddx58 siRNA (n=3 mice; 4 h) from 3 independent experiments. k, Ifnb1 and l, Tmem173 expression in BMDMs pretreated with DMSO or TTFA in the presence of Tmem173 siRNA (n=3 mice; LPS 4 h). Data are presented as mean ± SEM. P values were calculated using two-way ANOVA for multiple comparisons.

Source data

a, qPCR of mitochondrial DNA-encoded genes in BMDMs after mtDNA depletion with EtBr (6 days) followed by TTFA pretreatment (0.5 mM, 3 h) and LPS stimulation (n = 3 mice, 4 h) from 3 independent experiments. b, qPCR of mitochondrial DNA-encoded gene in BMDMs after POLRMT inhibition with IMT1 followed by itaconate pretreatment (3 h) and LPS stimulation (n = 3 mice, 4 h) from 3 independent experiments. c, Western blot of organellar and cytosolic fractions of BMDMs after subcellular digitonin fractionation (n=2 mice); Images representative of two independent experiments. d, qPCR of Ifnb1, Bak1, and Bax, after Bak1 or Bax siRNA knockdown followed by TTFA pretreatment (3 h) and LPS stimulation (n = 3 mice, 4 h) from 3 independent experiments. qPCR data of e, Vdac1 or f, Snx9 in BMDMs after Vdac1 or Snx9 siRNA knockdown followed by pretreated with TTFA (0.5 mM, 3 h) followed by LPS stimulation (n = 5 mice, 4 h) from 3 independent experiments. g, Immunofluorescence images of BMDMs pretreated with DMSO or TTFA (0.5 mM, 3 h) followed by LPS (n = 2 mice, 4 h). Images representative of 2 independent experiments. Data are presented as mean ± SEM. P values were calculated using two-way ANOVA for multiple comparisons.

Source data

Sequence of primers IREA raw data.

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Source data for Extended Data Fig. 1.

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Uncropped blots.

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O’Carroll, S.M., Peace, C.G., Toller-Kawahisa, J.E. et al. Itaconate drives mtRNA-mediated type I interferon production through inhibition of succinate dehydrogenase. Nat Metab (2024). https://doi.org/10.1038/s42255-024-01145-1

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Received: 11 April 2024

Accepted: 19 September 2024

Published: 15 October 2024

DOI: https://doi.org/10.1038/s42255-024-01145-1

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