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 interferon

biological rationale for the predominantly immune specification of itaconate.

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.

No new code has been generated for this work. Sources for the code used are cited in the Methods.

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.

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

Kurt Schwabe Institute for Sensor Technologies, Waldheim, Germany

Pirogov Russian National Research Medical University, Moscow, Russia

Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, Moscow, Russia

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

Biological Sciences Division, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA

Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

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

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA

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.

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.

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

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.

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.

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.

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.

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).

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.

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.

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.

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.

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