ABSTRACT The immunoregulatory metabolite itaconate accumulates in innate immune cells upon Toll-like receptor stimulation. In response to macrophage activation by lipopolysaccharide,

itaconate inhibits inflammasome activation and boosts type I interferon signalling; however, the molecular mechanism of this immunoregulation remains unclear. Here, we show that the

enhancement of type I interferon secretion by itaconate depends on the inhibition of peroxiredoxin 5 and on mitochondrial reactive oxygen species. We find that itaconate non-covalently

inhibits peroxiredoxin 5, leading to the modulation of mitochondrial peroxide in activating macrophages. Through genetic manipulation, we confirm that peroxiredoxin 5 modulates type I

actions and biological rationale for the predominantly immune specification of itaconate. Access through your institution Buy or subscribe This is a preview of subscription content, access

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INFLAMMATORY RESPONSE IN MURINE MACROPHAGES Article Open access 18 October 2024 MESACONATE IS SYNTHESIZED FROM ITACONATE AND EXERTS IMMUNOMODULATORY EFFECTS IN MACROPHAGES Article 02 June

2022 ITACONATE INHIBITS TET DNA DIOXYGENASES TO DAMPEN INFLAMMATORY RESPONSES Article 07 March 2022 DATA AVAILABILITY The bulk redox mass spectrometry proteomics data have been deposited to

the MassIVE repository under dataset accession no. MSV000093232. The protein itaconation mass spectrometry proteomics data have been deposited to the ProteomeXchange repository under dataset

accession no. PXD047348 or MassIVE repository under accession no. MSV000093522. The RNA sequencing data published in this paper are available from the Gene Expression Omnibus (GEO) public

repository under GEO accession number GSE277689. The data that support the plots within this paper are included in source data files for each figure. Source data are provided with this

paper. CODE AVAILABILITY No new code has been generated for this work. Sources for the code used are cited in the Methods. REFERENCES * Cordes, T. et al. Immunoresponsive gene 1 and

itaconate inhibit succinate dehydrogenase to modulate intracellular succinate levels. _J. Biol. Chem._ 291, 14274–14284 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  *

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). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Lampropoulou, V. et al. Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation.

_Cell Metab._ 24, 158–166 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bambouskova, M. et al. Itaconate confers tolerance to late NLRP3 inflammasome activation. _Cell

Rep._ 34, 108756 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hooftman, A. et al. The immunomodulatory metabolite itaconate modifies NLRP3 and inhibits inflammasome

activation. _Cell Metab._ 32, 468–478.e7 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mills, E. L. et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2

via alkylation of KEAP1. _Nature_ 556, 113–117 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ryan, T. A. J. et al. Dimethyl fumarate and 4-octyl itaconate are

anticoagulants that suppress tissue factor in macrophages via inhibition of type I interferon. _Nat. Commun._ 14, 3513 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * 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). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Kwai, B. X. C. et al. Itaconate is a covalent inhibitor of the _Mycobacterium tuberculosis_ isocitrate lyase. _RSC Med. Chem._ 12, 57–61

(2021). Article  CAS  PubMed  Google Scholar  * McKinney, J. D. et al. Persistence of _Mycobacterium tuberculosis_ in macrophages and mice requires the glyoxylate shunt enzyme isocitrate

lyase. _Nature_ 406, 735–738 (2000). Article  CAS  PubMed  Google Scholar  * Van Nguyen, T. et al. Itaconic acid inhibits growth of a pathogenic marine _Vibrio_ strain: a metabolomics

approach. _Sci. Rep._ 9, 5937 (2019). Article  PubMed  PubMed Central  Google Scholar  * McFadden, B. A., Williams, J. O. & Roche, T. E. Mechanism of action of isocitrate lyase from

_Pseudomonas indigofera_. _Biochemistry_ 10, 1384–1390 (1971). Article  CAS  PubMed  Google Scholar  * Bambouskova, M. et al. Electrophilic properties of itaconate and derivatives regulate

the IκBζ–ATF3 inflammatory axis. _Nature_ 556, 501 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Runtsch, M. C. et al. Itaconate and itaconate derivatives target JAK1 to

suppress alternative activation of macrophages. _Cell Metab._ 34, 487–501.e8 (2022). Article  CAS  PubMed  Google Scholar  * Qin, W. et al. Chemoproteomic profiling of itaconation by

bioorthogonal probes in inflammatory macrophages. _J. Am. Chem. Soc._ 142, 10894–10898 (2020). Article  CAS  PubMed  Google Scholar  * Ryan, D. G. et al. Nrf2 activation reprograms

macrophage intermediary metabolism and suppresses the type I interferon response. _iScience_ 25, 103827 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Olagnier, D. et al.

Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming. _Nat. Commun._ 9, 3506 (2018). Article  PubMed  PubMed Central  Google Scholar  *

Gunderstofte, C. et al. Nrf2 negatively regulates type I interferon responses and increases susceptibility to herpes genital infection in mice. _Front. Immunol._ 10, 2101 (2019). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Wood, Z. A., Poole, L. B. & Karplus, P. A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. _Science_ 300,

650–653 (2003). Article  CAS  PubMed  Google Scholar  * Hanley, P. J., Mickel, M., Löffler, M., Brandt, U. & Daut, J. KATP channel‐independent targets of diazoxide and 5‐hydroxydecanoate

in the heart. _J. Physiol._ 542, 735–741 (2002). Article  CAS  PubMed  PubMed Central  Google Scholar  * Brault, M., Olsen, T. M., Martinez, J., Stetson, D. B. & Oberst, A.

Intracellular nucleic acid sensing triggers necroptosis through synergistic type I IFN and TNF signaling. _J. Immunol._ 200, 2748–2756 (2018). Article  CAS  PubMed  Google Scholar  * Su, C.,

Cheng, T., Huang, J., Zhang, T. & Yin, H. 4-Octyl itaconate restricts STING activation by blocking its palmitoylation. _Cell Rep._ 42, 113040 (2023). Article  CAS  PubMed  Google

Scholar  * Onomoto, K., Onoguchi, K. & Yoneyama, M. Regulation of RIG-I-like receptor-mediated signaling: interaction between host and viral factors. _Cell Mol. Immunol._ 18, 539–555

(2021). Article  CAS  PubMed  Google Scholar  * Xu, H. et al. Structural basis for the prion-like MAVS filaments in antiviral innate immunity. _Elife_ 3, e01489 (2014). Article  PubMed 

PubMed Central  Google Scholar  * Widdrington, J. D. et al. Mitochondrial DNA depletion induces innate immune dysfunction rescued by IFN-γ. _J. Allergy Clin. Immunol._ 140, 1461–1464.e8

(2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Siebels, I. & Dröse, S. Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle

dicarboxylates. _Biochim. Biophys. Acta_ 1827, 1156–1164 (2013). Article  CAS  PubMed  Google Scholar  * Robb, E. L. et al. Selective superoxide generation within mitochondria by the

targeted redox cycler MitoParaquat. _Free Radic. Biol. Med._ 89, 883–894 (2015). Article  CAS  PubMed  Google Scholar  * Murphy, M. P. et al. Guidelines for measuring reactive oxygen species

and oxidative damage in cells and in vivo. _Nat. Metab._ 4, 651–662 (2022). Article  PubMed  PubMed Central  Google Scholar  * Pak, V. V. et al. Ultrasensitive genetically encoded indicator

for hydrogen peroxide identifies roles for the oxidant in cell migration and mitochondrial function. _Cell Metab._ 31, 642–653.e6 (2020). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Dikalova, A. E. et al. Therapeutic targeting of mitochondrial superoxide in hypertension. _Circ. Res._ 107, 106–116 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Ramalho, T. et al. Itaconate impairs immune control of _Plasmodium_ by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells. _Cell Metab._ 36, 484–497 (2024).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Yan, S. et al. Biochemical characterization of human peroxiredoxin 2, an antioxidative protein. _Acta Biochim. Biophys. Sin.

(Shanghai)_ 44, 759–764 (2012). Article  CAS  PubMed  Google Scholar  * Baković, J. et al. A key metabolic integrator, coenzyme A, modulates the activity of peroxiredoxin 5 via covalent

modification. _Mol. Cell. Biochem._ 461, 91–102 (2019). Article  PubMed  PubMed Central  Google Scholar  * Coppo, L., Montano, S. J., Padilla, A. C. & Holmgren, A. Determination of

glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione. _Anal. Biochem._ 499, 24–33 (2016). Article  CAS  PubMed  Google Scholar  * Holmgren, A.

Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. _J. Biol. Chem._ 254, 9627–9632 (1979). Article  CAS  PubMed  Google Scholar  * Knoops, B.,

Goemaere, J., Van der Eecken, V. & Declercq, J.-P. Peroxiredoxin 5: structure, mechanism, and function of the mammalian atypical 2-Cys peroxiredoxin. _Antioxid. Redox Signal_ 15, 817–829

(2011). Article  CAS  PubMed  Google Scholar  * Aguirre, C., Brink, T., ten, Guichou, J.-F., Cala, O. & Krimm, I. Comparing binding modes of analogous fragments using NMR in

fragment-based drug design: application to PRDX5. _PLoS ONE_ 9, e102300 (2014). Article  PubMed  PubMed Central  Google Scholar  * Declercq, J.-P. et al. Crystal structure of human

peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 Å resolution. _J. Mol. Biol._ 311, 751–759 (2001). Article  CAS  PubMed  Google Scholar  * Shekhova, E. Mitochondrial reactive

oxygen species as major effectors of antimicrobial immunity. _PLoS Pathog._ 16, e1008470 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Dubuisson, M. et al. Human

peroxiredoxin 5 is a peroxynitrite reductase. _FEBS Lett._ 571, 161–165 (2004). Article  CAS  PubMed  Google Scholar  * Daniels, B. P. et al. The nucleotide sensor ZBP1 and kinase RIPK3

induce the enzyme IRG1 to promote an antiviral metabolic state in neurons. _Immunity_ 50, 64–76.e4 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sohail, A. et al. Itaconate

and derivatives reduce interferon responses and inflammation in influenza A virus infection. _PLoS Pathog._ 18, e1010219 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cho,

H. et al. Differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses. _Nat. Med._ 19, 458–464

(2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Szeligowski, R. V. et al. Molecular evolution of IRG1 shapes itaconate production in metazoans and alleviates the

“double-edged dilemma” of innate immune defense. Preprint at _bioRxiv_ https://doi.org/10.1101/2022.06.17.496652 (2022). * Buchmann, K. Evolution of innate immunity: clues from invertebrates

via fish to mammals. _Front. Immunol._ 5, 459 (2014). Article  PubMed  PubMed Central  Google Scholar  * Chen, F. et al. Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon

responses and oxidative stress, and modulates inflammation and cell metabolism. _Nat. Metab._ 4, 534–546 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Waqas, S. F.-H. et

al. ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives. _Clin. Transl. Med._ 12, e931 (2022). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Yue, Y.-X. et al. 4-Octyl itaconate inhibits poly(I:C)-induced interferon-β secretion in mouse bone marrow-derived macrophages partially by activating Nrf2.

_Heliyon_ 9, e23001 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * He, W. et al. Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages.

_Nat. Metab._ 4, 524–533 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Graham, D. B. et al. Nitric oxide engages an anti-inflammatory feedback loop mediated by

peroxiredoxin 5 in phagocytes. _Cell Rep._ 24, 838–850 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Evavold, C. L. et al. Control of gasdermin D oligomerization and

pyroptosis by the Ragulator–Rag–mTORC1 pathway. _Cell_ 184, 4495–4511.e19 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Borin, B. N. et al. Murine norovirus protein NS1/2

aspartate to glutamate mutation, sufficient for persistence, reorients side chain of surface exposed tryptophan within a novel structured domain. _Proteins_ 82, 1200–1209 (2014). Article 

CAS  PubMed  Google Scholar  * Slaby, I. & Holmgren, A. Thioredoxin reductase-dependent insulin disulfide reduction by phage T7 DNA polymerase reflects dissociation of the enzyme into

subunits. _J. Biol. Chem._ 264, 16502–16506 (1989). Article  CAS  PubMed  Google Scholar  * Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. _Bioinformatics_ 29, 15–21 (2013).

Article  CAS  PubMed  Google Scholar  * Wang, L., Wang, S. & Li, W. RSeQC: quality control of RNA-seq experiments. _Bioinformatics_ 28, 2184–2185 (2012). Article  CAS  PubMed  Google

Scholar  * Ewels, P., Magnusson, M., Lundin, S. & Käller, M. MultiQC: summarize analysis results for multiple tools and samples in a single report. _Bioinformatics_ 32, 3047–3048 (2016).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic

features. _Bioinformatics_ 30, 923–930 (2014). Article  CAS  PubMed  Google Scholar  * Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of

genomic datasets with the R/Bioconductor package biomaRt. _Nat. Protoc._ 4, 1184–1191 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ritchie, M. E. et al. limma powers

differential expression analyses for RNA-sequencing and microarray studies. _Nucleic Acids Res._ 43, e47 (2015). Article  PubMed  PubMed Central  Google Scholar  * Kleverov, M. et al.

Phantasus, a web application for visual and interactive gene expression analysis. _eLife_ 13, e85722 (2024). Article  PubMed  PubMed Central  Google Scholar  * Korotkevich, G. et al. Fast

gene set enrichment analysis. Preprint at _bioRxiv_ https://doi.org/10.1101/060012 (2021). * Mölder, F. et al. Sustainable data analysis with Snakemake. _F1000Res_ 10, 33 (2021). Article 

PubMed  PubMed Central  Google Scholar  * Mertins, P. et al. Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid

chromatography–mass spectrometry. _Nat. Protoc._ 13, 1632–1661 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Meier, F. et al. Online parallel accumulation–serial

fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer. _Mol. Cell. Proteom._ 17, 2534–2545 (2018). Article  CAS  Google Scholar  * Perkins, D. N., Pappin, D. J. C.,

Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. _Electrophoresis_ 20, 3551–3567 (1999). Article 

CAS  PubMed  Google Scholar  * Day, N. J. et al. A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne muscular dystrophy model. _Free Radic. Biol.

Med_ 193, 373–384 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gaffrey, M. J., Day, N. J., Li, X. & Qian, W. Resin-assisted capture coupled with isobaric tandem mass

tag labeling for multiplexed quantification of protein thiol oxidation. _J. Vis. Exp._ 172, e62671 (2021). Google Scholar  * Guo, J. et al. Resin-assisted enrichment of thiols as a general

strategy for proteomic profiling of cysteine-based reversible modifications. _Nat. Protoc._ 9, 64–75 (2014). Article  CAS  PubMed  Google Scholar  * Li, X. et al. Mass spectrometry-based

direct detection of multiple types of protein thiol modifications in pancreatic beta cells under endoplasmic reticulum stress. _Redox Biol._ 46, 102111 (2021). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Wang, Y. et al. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. _Proteomics_ 11,

2019–2026 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Duan, J. et al. Quantitative profiling of protein S-glutathionylation reveals redox-cependent regulation of

macrophage function during nanoparticle-induced oxidative stress. _ACS Nano_ 10, 524–538 (2016). Article  CAS  PubMed  Google Scholar  * Kim, S. & Pevzner, P. A. MS-GF+ makes progress

towards a universal database search tool for proteomics. _Nat. Commun._ 5, 5277 (2014). Article  CAS  PubMed  Google Scholar  * Fuhrer, T., Heer, D., Begemann, B. & Zamboni, N.

High-throughput, accurate mass metabolome profiling of cellular extracts by flow injection–time-of-flight mass spectrometry. _Anal. Chem._ 83, 7074–7080 (2011). Article  CAS  PubMed  Google

Scholar  * Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. _J. Comput. Chem._ 30, 2785–2791 (2009). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Forli, S. et al. Computational protein–ligand docking and virtual drug screening with the AutoDock suite. _Nat. Protoc._ 11, 905–919 (2016). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. _J. Chem. Inf. Model_ 51, 2778–2786

(2011). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank C. Evavold for providing iBMDMs; E. Aladyeva, B. S. Andhey for computational support; P. Bohacova

for helping with experiments; R. Xavier and E. A. Creasey for providing _Prdx5_−/− mice; M. S. Diamond for critical reading of the manuscript; R. Sprung, P. Erdmann Gilmore and R. Townsend

from the WashU Proteomics Core for itaconate peptide MS/MS analysis. Images in some of the figures were created in BioRender.com. The study was supported in part by NIAID grant R01-A1125618

(to M.N.A.). Experiments on live-cell imaging were supported by Russian Science Foundation grant 23-75-30023 (to V.V.B.). Correspondence and requests for materials should be addressed to the

corresponding author. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Tomas Paulenda, 

Barbora Echalar, Lucie Potuckova, Veronika Vachova, Denis A. Kleverov, Devashish Sen, Chris Nelson, Rick Stegeman, Vladimir Sukhov, Cheryl F. Lichti, Kamila Husarcikova, Daved H. Fremont 

& Maxim N. Artyomov * Bruker Biosensors, Munich, Germany Johannes Mehringer * Kurt Schwabe Institute for Sensor Technologies, Waldheim, Germany Johannes Mehringer * Pirogov Russian

National Research Medical University, Moscow, Russia Ekaterina Potekhina & Vsevolod V. Belousov * Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological

Agency, Moscow, Russia Ekaterina Potekhina & Vsevolod V. Belousov * Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA Alex Jacoby, 

Danielle Kemper, Sergej Djuranovic & Slavica Pavlovic-Djuranovic * Bursky Center for Human Immunology and Immunotherapy Programs, St. Louis, MO, USA Cheryl F. Lichti * Biological

Sciences Division, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA Nicholas J. Day, Tong Zhang & Wei-jun Qian * Department of

Medicine, Washington University School of Medicine, St. Louis, MO, USA Monika Bambouskova * Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St.

Louis, MO, USA Daved H. Fremont & Andrzej M. Krezel * Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA Daved H. Fremont Authors * Tomas

Paulenda View author publications You can also search for this author inPubMed Google Scholar * Barbora Echalar View author publications You can also search for this author inPubMed Google

Scholar * Lucie Potuckova View author publications You can also search for this author inPubMed Google Scholar * Veronika Vachova View author publications You can also search for this author

inPubMed Google Scholar * Denis A. Kleverov View author publications You can also search for this author inPubMed Google Scholar * Johannes Mehringer View author publications You can also

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Vladimir Sukhov View author publications You can also search for this author inPubMed Google Scholar * Danielle Kemper View author publications You can also search for this author inPubMed 

Google Scholar * Cheryl F. Lichti View author publications You can also search for this author inPubMed Google Scholar * Nicholas J. Day View author publications You can also search for this

author inPubMed Google Scholar * Tong Zhang View author publications You can also search for this author inPubMed Google Scholar * Kamila Husarcikova View author publications You can also

search for this author inPubMed Google Scholar * Monika Bambouskova View author publications You can also search for this author inPubMed Google Scholar * Daved H. Fremont View author

publications You can also search for this author inPubMed Google Scholar * Wei-jun Qian View author publications You can also search for this author inPubMed Google Scholar * Sergej

Djuranovic View author publications You can also search for this author inPubMed Google Scholar * Slavica Pavlovic-Djuranovic View author publications You can also search for this author

inPubMed Google Scholar * Vsevolod V. Belousov View author publications You can also search for this author inPubMed Google Scholar * Andrzej M. Krezel View author publications You can also

search for this author inPubMed Google Scholar * Maxim N. Artyomov View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.P. and M.N.A.

conceived and designed the study and wrote the manuscript. T.P. performed biomass preparation for RNA sequencing, metabolomics and proteomics experiments, western blot, qPCR, cytometry,

cytokine assays, recombinant protein activity assays, lentiviral transduction, 3-nitrotyrosine ELISA and 3D protein visualization. B.E. performed western blot, cytometry and cytokine assays,

nitrite + nitrate assay, Griess assay and recombinant protein preparation, L.P. performed cytokine and western blot assays. V.V. performed in vivo poly(I:C) injection and cytokine

measurement. D.A.K. performed the docking analysis, RNA sequencing data processing and analysis. V.S. performed pathway enrichment analysis. E.P. and V.V.B. designed and performed the

mitoHyPer7 oxidation experiments. J.M. performed switchSENSE fluorescence proximity sensing analysis. A.M.K. designed and performed 15N-protein nuclear magnetic resonance analysis. A.J. and

S.P.D. performed cell culture experiments. D.S. performed western blot experiments. K.H. performed human monocyte isolation D.K. and S.D. prepared the recombinant proteins. M.B. performed

biomass preparation for RNA sequencing and MS/MS protein itaconation experiment. C.F.L. assisted in the analysis of the MS/MS protein itaconation results. N.J.D., T.Z. and W.J.Q. performed

and analysed the bulk redox proteomics experiment. C.N., R.S. and D.H.F performed recombinant protein generation. CORRESPONDING AUTHOR Correspondence to Maxim N. Artyomov. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Metabolism_ thanks 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_ 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 EXOGENOUS AND

ENDOGENOUS ITACONATE REGULATE IFNΒ RELATED PHENOTYPES. A IFNβ release by activated BMDMs pretreated with itaconic acid (IA, 5 mM), or sodium itaconate (NaI, 10 mM or 20 mM) (16 h) or media

(M) and stimulated with LPS (0.1 μg/ml) for 4 h (n = 8 (M, IA), n = 7 (NaI 10), n = 5 (NaI 20) biological replicates). B pH of cRPMI supplemented with 5 mM IA or malonic acid (MA) (n = 3

independent experiments). C Survival of BMDMs pretreated with IA (5 mM, 16 h), MA (5 mM, 16 h) or acidified media (HCl, 16 h) measured by LDH activity in the supernatant (left) or ATP levels

in cell lysates and supernatants (right) (n = 4 (left) n = 6 (right) independent cultures) D IFNβ release from WT or Irg1-/- BMDMs stimulated with LPS (0.1 μg/ml, 24 h) (n = 6 individual

cultures). E Survival of WT or IFNAR-/- mice upon lethal dose of DMXAA i.p. (n = 3 mice/group). F Serum levels of IFNβ in WT or Irg1-/- repeated experiment to Fig. 1i (n = 4 WT and n = 3

Irg1-/-). G Survival of WT or Irg1-/- female mice treated with increasing doses of DMXAA i.p. (n = 15 (WT 40 mg/kg), n = 4 (WT 50 mg/kg) n = 6 (WT 60 mg/kg), n = 5 (Irg1-/- 40 mg/kg)

mice/group). H Survival of WT or Irg1-/- female mice injected with 60 mg/kg lethal dose of DMXAA i.p. (n = 3 mice/group). Data are presented as mean ±SEM. Statistical analysis: A Two sided

Brown-Forsythe and Welch ANOVA test. C Two sided One sample t-test. D, F Two sided Welch’s t-test. Source data EXTENDED DATA FIG. 2 ITACONIC ACID REGULATES IFNΒ PRODUCTION THROUGH

MTDNA/CGAS/STING PATHWAY. A Representative immunoblot of STAT1 and STAT2 protein levels in BMDMs pretreated with itaconic acid (IA) (5 mM, 16 h) and co-treated with αIFNAR blocking antibody

or isotype control (representative of n = 2-4). GAPDH serves as loading control and corresponding statistics. B IFNβ release by iBMDMs pretreated with media (M), IA or malonic acid (MA) or

acidified media (HCl) in the presence of a gradient of RU.521 (1, 5, 10 μg/ml) or C176 (0.5, 1, 2 μM) inhibitors followed by poly(I:C) (20 μg/ml, 4 h) stimulation (n = 6 M, IA, RU.521 1 and

5; n = 4 MA, HCl, all C176; n = 2 RU.521 10 μg/ml). C Representative immunoblot of STAT1 expression in iBMDMs treated with inhibitors as in c (middle concentration) in presence of IA and

corresponding statistics (n = 5). D IFNβ release from WT or MAVS-/- BMDMs pretreated as in a and stimulated with poly(I:C) as in b (n = 3 biological replicates). E mtDNA in cytosolic

fraction of iBMDMs pretreated with itaconic acid as in b (n = 3 individual experiments). F IFNβ release from WT or mtDNA depleted (EtBr) iBMDMs pretreated with media or IA as in b and

stimulated with DMXAA (5 μg/ml, 4 h) (n = 5 independent experiments). G-H Representative immunoblot of STAT1 and STAT2 protein levels in iBMDMs pretreated as in e and corresponding

statistics (representative of n = 4 independent experiments). I-J IFNβ release from BMDMs pretreated with 3NPA (200 μM, 16 h) or media and co-treated or not with C176 (j, 1 μM, 16 h) or

MitoTempo (k, 250 μM, 16 h) (n = 5). Data are represented as mean ±SEM. Statistical analysis: A, C, F, I-J Brown-Forsythe and Welsh ANOVA with Dunnet’s T3 multiple comparison test. B

RM-Two-way ANOVA with Tukey’s multiple comparison test. E One sample t-test. H Ordinary One-way ANOVA with Tukey’s multiple comparison test. Source data EXTENDED DATA FIG. 3 ITACONIC

ENHANCES HYDROGEN PEROXIDE RETENTION. A IFNβ release from iBMDMs treated with media (M) or itaconic acid (IA) (5 mM, 16 h) in presence or absence of indicated concentrations of NAC (n = 3)

or MitoTempo (n = 6). B Statistics for Fig. 3d (n = 4 independent cultures). C Relative median fluorescent intensity fold change of CellROX green in iBMDMs pretreated M, IA or Malonic acid

(MA) (5 mM, 16 h) (n = 6). D Representative histograms of c and Fig. 3f, g. Dashed line represents median of media treated cells. E Oxidation status of mitoHyPer7 in Hela cells pretreated

with IA or media as in a for 20 min or 4 h (dots represent individual cells, n = 3 individual experiments). F Oxidation status of mitoHyPer7 in Hela cells pretreated with IA as in a and

treated with H2O2 (100 μM) followed by H2O2 removal and continued measurement for additional 30 min (representative experiment of n = 3). Data represent mean ±SEM. Statistical analysis: A

RM-Two-way ANOVA with Tukey’s multiple comparison test. B, E Brown-Forsythe and Welsh ANOVA with Dunnet’s T3 multiple comparison test. C One sample t-test. Source data EXTENDED DATA FIG. 4

ITACONATE NON-COVALENTLY INTERACTS WITH PRDX5. A Itaconated peptide MS/MS spectrum of Prdx5 in BMDMs treated with itaconic acid (IA, 5 mM, 16 h). B Observed occupancy of itaconated Cys in

the media (M) or IA treated samples. (n = 1) C Itaconated Prdx5 peptide with itaconate covalently bound to Cys200. D-E Activity assays of human Thioredoxin 1 (D) and human Glutaredoxin 1 (E,

n = 2 technical replicates in 2 individual experiments) F t-BOOH consumption by Prdx5 in activity assay in Fig. 4a. G Activity of Prdx5 pretreated with H2O2 (100 μM, 30 min) or DTT (10 mM,

30 min) assayed in buffer or in presence of DTT (1 mM) (n = 3 independent experiments). H Standard curve of t-BOOH in buffer or presence of itaconate or malonate with linear regression

equation (n = 5 independent experiments). I Activity of recombinant human Prdx5 performed as in Fig. 4c pretreated with indicated concentrations of Itaconic acid (n = 3 independent

experiments). J Immunoblot of recombinant human Prdx5 redox state in presence or absence of IA (5 mM) and corresponding statistics (n = 4 independent experiments). K Representative

immunoblot of Prdx5 in Ctrl or Prdx5-overexpressing iBMDMs (Prdx5-OE) (n = 3). L Representative immunoblot of Prdx5 expression in WT and Prdx5-/- BMDMs stimulated with DMXAA (5 μg/ml, 24 h)

(n = 3). M-N kCal/mol (M) and Ki (N) parameters towards Prdx5 derived for the strongest binding conformation for each chemical. Data represent mean ±SEM. Statistical analysis: F

Brown-Forsythe and Welsh ANOVA with Dunnet’s T3 multiple comparison test. J Two-sided One sample t-test. Source data EXTENDED DATA FIG. 5 ITACONATE NON-COVALENTLY INTERACTS WITH PRDX5. A-B

Sensorgrams for the interaction between Prdx5 and metabolites from Fig. 5b,c before bulk shift correction. C Three full superimposed spectra of 15N labeled Prdx5 free (black), with 20.52 mM

itaconate (red), and with 20.19 mM benzoate (blue). EXTENDED DATA FIG. 6 PRODUCTION OF ITACONATE UPON TLR STIMULATION LEADS TO INCREASED PEROXIDE ACCUMULATION. A Representative immunoblot of

Irg1 expression in WT or Irg1-/- BMDMs non-stimulated or stimulated with R848 (1 μg/ml, 24 h). GAPDH serves as loading control (n = 3). B Intracellular levels of itaconate in BMDMs

stimulated as in (A). To rescue itaconate levels in Irg1-/- cells were treated with itaconic acid (IA) (1 mM, at 4 h post stimulation) (n = 3 biological replicates). C-D mtPY1 levels and

representative histograms in BMDMs non-stimulated or stimulated as with LPS (0.1 ug/ml) or R848 (1 μg/ml) or poly(I:C) (20 μg/ml) for 24 h (n = 6 (C), n = 5 (D) independent cultures). E

Intracellular itaconate levels in WT and Irg1-/- BMDMs treated as in (C) (n = 3 biological replicates). F Nitrite+Nitrate levels in supernatant of WT or Irg1-/- BMDMs stimulated as in (C) (n

 = 4 biological replicates). G Nitrite levels in supernatants of BMDMs stimulated as in (C) (n = 4 independent cultures) H Randomized design assignment of TMT10 reagents used for

multiplexing of enriched samples. Data represent mean ±SEM. Statistical analysis: C-D One sample t-test. Source data EXTENDED DATA FIG. 7 METHYL SUCCINIC ACID PHENOCOPIES ITACONIC ACID

EFFECTS IN ACTIVATED MACROPHAGES. A Sensogram for the interaction between Prdx5 and 2-methylsuccinate from Fig. 7h before bulk shift correction. B Survival of BMDMs pretreated with

methylsuccinic acid (MetSA, 5 mM, 16 h), or media measured by LDH activity in the supernatant (left) or ATP levels (right) (n = 3-4 independent cultures) C Intracellular levels of indicated

metabolites in BMDMs pretreated with itaconic acid or MetSA (5 mM, 16 h) (n = 3). D Hallmark gene set enrichment analysis in samples RNAseq data from BMDMs pretreated with metabolites as in

c. E Survival of human Monocyte-derived macrophages (hMoDMs) pretreated with IA or MetSA as in c (n = 2 individual donors). F IFNβ release from hMoDMs stimulated with poly(I:C) (30 ug/ml),

LPS (100 ng/ml) or STING agonis diABZI (1 μM) for 24 h (n = 4 individual donors). G IFNβ release from hMoDM pretreated with IA or MetSA as in e and stimulated with poly(I:C) as in f (n = 3

individual donors). Data represent mean ±SEM. Statistical analysis: G Brown-Forsythe and Welsh ANOVA with Dunnet’s T3 multiple comparison test. Source data EXTENDED DATA FIG. 8 GATING

STRATEGY TO IDENTIFY LIVE SINGLET CELLS. A First debris and dublets were removed gating on FSC-Amid FSC-Wlow. Subsequently, live cells were gated as Live/Dead NIRlow. Fluorescent intensity

of select probe was determined in the Live cells population. SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY TABLE 1. Metabolomics dataset of WT and Irg1-/- BMDMs non-stimulated or

stimulated with LPS, poly(I:C) or R848 for 24 h. Alternatively macrophages were pretreated with media, itaconic acid, or 2-methylsuccinic acid (5 mM, 16 h). Sample list, ion_matrix,

annotation, ions. 2. List of identified peptides in WT BMDMs treated with media for 16 h. List of peptides observed in WT BMDMs treated with 5 mM itaconic acid for 16 h. List of PSMs

itaconated on cysteine. 3. List of oxidized cysteines in WT and Irg1-/- BMDMs non-activated or stimulated with LPS for 24 h. 4. List of reagents and used software and algorithms. SOURCE DATA

SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 1 Unprocessed western blots. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 2 Unprocessed western blots. SOURCE

DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 3 Unprocessed western blots. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA FIG. 6

Statistical source data. SOURCE DATA FIG. 6 Unprocessed western blots. SOURCE DATA FIG. 7 Statistical source data. SOURCE DATA FIG. 7 Unprocessed western blots. SOURCE DATA EXTENDED DATA

FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG.2 Statistical source data. SOURCE DATA EXTENDED DATA FIG.2 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 3

Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 6 Statistical

source data. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 7 Statistical source data. RIGHTS AND PERMISSIONS Springer Nature or its licensor

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T., Echalar, B., Potuckova, L. _et al._ Itaconate modulates immune responses via inhibition of peroxiredoxin 5. _Nat Metab_ (2025). https://doi.org/10.1038/s42255-025-01275-0 Download

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