Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1

Abstract

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood1,2,3. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

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Figure 1: Itaconate activates Nrf2.
Figure 2: Itaconate alkylates cysteines.
Figure 3: OI limits IL-1β in an Nrf2-dependent manner and protects against LPS lethality.
Figure 4: A feedback loop exists between itaconate and IFN-β.

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Acknowledgements

We thank M. McMahon and J. D. Hayes for plasmids, and Cancer Research UK (C20953/A18644) and the BBSRC (BB/L01923X/1) for financial support for ATDK. This work was supported by a Wellcome Trust Investigator award to R.C.H. (110158/Z/15/Z), a grant to M.P.M. from the Medical Research Council UK (MC_U105663142), a Wellcome Trust Investigator award to MPM (110159/Z/15/Z), and a grant to E.R.S.K. and M.S.K. from the Medical Research Council UK (MC_U105663139). B.M.K. and R.F. are supported by the Kennedy Trust Fund. We acknowledge Metabolon for their assistance with the metabolic work and analysis. The O’Neill laboratory acknowledges the following grant support: European Research Council (ECFP7-ERC-MICROINNATE), Science Foundation Ireland Investigator Award (SFI 12/IA/1531), GlaxoSmithKline Visiting Scientist Programme and The Wellcome Trust (oneill-wellcometrust-metabolic, grant number 205455). E.T.C. is supported by the Claudia Adams Barr Program.

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E.L.M. and D.G.R. designed and performed experiments and analysed the data. E.L.M. wrote the manuscript with assistance from all other authors. D.M., M.M.H., M.C.R. and A.F.M. performed in vitro experiments using OI. R.G.C., D.C.S., A.S.H.C. and C.F. assisted with the metabolomics analysis. Z.Z., P.G.F. and E.H. assisted with the in vivo mouse LPS trials. S.T.C. and R.C.H. were responsible for the design and synthesis of octyl esters. H.A.P., E.R.S.K., M.S.K. and L.M.B. assessed the effect of OI and itaconate on mitochondrial parameters and itaconate transport. D.D., M.H. and A.T.D.-K. performed the NQO1 assay and KEAP1 wild-type and Cys151Ser mutant experiments. J.F.M., R.F., B.M.K., E.T.C., M.P.J. and J.S. assisted with mass spectrometry experiments. L.K.M. and G.B. provided guidance and advice. E.V.K., P.J.M. and M.L.J.A. assisted with experiments in Nrf2-deficient mice. L.A.O’N. conceived ideas and oversaw the research programme. M.P.M. provided advice, reagents and oversaw a portion of the work.

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Correspondence to Luke A. O’Neill.

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Reviewer Information Nature thanks N. S. Chandel, R. Rossignol, S. Werner and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 The effect of itaconate on complex II activity.

a, Complex II and III activity in bovine heart mitochondrial membranes incubated with succinate plus malonate or itaconate (n = 3 independent experiments). b, Effect of malonate or itaconate on the oxygen consumption rate (OCR) of rat liver mitochondria in the presence of succinate (1 mM) and FCCP (1 μM; n = 3 independent experiments). c, d, Complex II and III activity in bovine heart mitochondrial membranes incubated with itaconate (IA; 1 mM unless indicated), with subsequent removal and addition of succinate (1 mM; n = 3 independent experiments) (see Methods for further details). Data are mean ± s.e.m. P values calculated using one or two-way ANOVA.

Extended Data Figure 2 DMI activates Nrf2 and limits cytokine production.

a, c, LPS (100 ng ml−1)-induced Nrf2 (a, 24 h) and HMOX1 (c, 6 h) protein expression with or without the itaconate derivative DMI. b, Nrf2-dependent mRNA expression after treatment with LPS (6 h) and DMI where indicated (n = 3). d, Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels after treatment with LPS and DMI (n = 5). e, f, LPS (24 h)-induced Il1b mRNA (e), IL-1β and HIF-1α protein (f) expression in mouse macrophages with or without DMI (n = 3). Data are mean ± s.e.m. P values calculated using one-way ANOVA. Blots are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 OI is the best tool to assess itaconate-dependent Nrf2 activity.

a, Reactivity of DMI, itaconate and OI with thiols. b, c, Itaconate ester reactivity with GSH and glutathione-S-transferase (GST) as detailed in the Methods (n = 3). d, Itaconate levels in mouse C2C12 cells plus itaconate esters (n = 3). MI, 4-methyl itaconate. e, i, Itaconate (e) or GSH (i) levels plus LPS (6 h) and OI as indicated (n = 5). f, NQO1 activity in mouse Hepa1c1c7 cells treated with DMI or OI (48 h) and GSH (n = 8). g, h, Metabolic intermediates in GSH synthesis (h, average of five biological replicates). i, GSH levels after treatment with LPS (6 h) and/or OI (n = 5). j, GSH/GSSG ratio after treatment with OI (2 h) and H2O2 (100 μM, 24 h; n = 3) as indicated. k, HMOX1 protein levels after treatment with OI and/or H2O2 (24 h). l, Nrf2, HMOX1 and IL-1β protein levels in BMDMs pre-treated with OI, 4-octyl 2-methylsuccinate (OMS) or octyl succinate (OS), all 125 μM for 3 h with or without LPS (6 h). m, LPS-induced Nrf2 (24 h) and HMOX1 (6 h) protein expression with or without dimethyl malonate (DMM). Data are mean ± s.e.m. P values calculated using one- or two-way ANOVA. Blots are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Itaconate is transported by the mitochondrial oxoglutarate, dicarboxylate and citrate carriers.

a, Itaconate uptake into vesicles of Lactococcus lactis membranes expressing the indicated carriers loaded with itaconate (1 mM), and transport initiated by the addition of [3H]itaconate (1 μM). b, Initial transport rates of each carrier with either canonical substrate (homo-exchange) or canonical substrate/itaconate (hetero-exchange). n = 4 independent experiments; data are mean ± s.d. P values calculated using two-tailed Student’s t-test.

Extended Data Figure 5 KEAP1 is alkylated by OI on major redox sensing cysteine residues.

a, Modification of cysteine by fumarate or itaconate. Tandem mass spectrometry spectrum of KEAP1 Cys257 (b), Cys257 (c) and Cys288 (d) peptides, indicating alkylation of these sites after OI treatment (left) but not in the corresponding carbamidomethylated (CAM) peptides (right). e, f, LDHA Cys84 alkylation after treatment with LPS (e, 24 h) or OI (f, 250 μM, 4 h) (n = 4). Detected N- and C-terminal fragment ions of both peptides are assigned in the spectrum and depicted as follows: b: N-terminal fragment ion; y: C-terminal fragment ion; asterisk: fragment ion minus NH3; 0 or asterisk: fragment ion minus H2O; and 2+: doubly charged fragment ion. Representative of one independent experiment.

Extended Data Figure 6 Identification of an itaconate-cysteine adduct.

ae, 13C6-glucose (ac) or 13C5-glutamine (d, e) labelling experiment tracking itaconate-cysteine adduct formation in BMDMs treated with LPS (n = 5; 24 h). Data in b and e are expressed as the percentage isotopologue of the total pool. Data in c and f represent changes in the total pool after LPS treatment. Data are mean ± s.e.m., for five replicates. P values calculated using two-way ANOVA.

Extended Data Figure 7 OI decreases LPS-induced cytokine production, extracellular acidification rate, ROS and nitric oxide.

a, Percentage cytotoxicity (LDH release) in BMDMs after treatment with LPS and OI as indicated (n = 3). b, LPS-induced extracellular acidification rate (ECAR) after treatment with OI and/or LPS as indicated, analysed on the Seahorse XF-24 in BMDMs (trace representative of three independent experiments). c, d, LPS-induced Il10 mRNA (c, 4 h) and protein (d, 24 h) and TNF protein (f; n = 7) after OI treatment as indicated (n = 3). e, Phosphorylated p65 (pp65) protein levels (a measure of NF-κΒ activity) after treatment with LPS and OI as indicated. h, Representative gating strategy for FACS analysis of ROS production in cells as treated in d (image representative of three independent experiments). i, LPS-induced NOS2 expression (n = 6), with or without OI treatment. j, LPS-induced TNF (n = 4) and IL-1β (n = 3) protein levels after OI treatment in PBMCs. k, Nrf2 and HMOX1 protein levels or Nrf2-dependent gene expression (n = 5) in peritoneal macrophages from mice (m) injected intraperitoneally with OI (50 mg kg−1, 6 h) or vehicle control. l, Serum IL-10 from mice injected intraperitoneally with vehicle control or OI (50 mg kg−1, 2 h) and LPS (2.5 mg kg−1, 2 h, n = 3 vehicle, OI; n = 15 LPS, OI plus LPS). Data are mean ± s.e.m. P values calculated using one-way ANOVA. Blots are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Figure 8 The effects of OI on cytokine production are Nrf2-dependent.

ae, Nrf2, HMOX1 and IL-1β protein levels (a, c, d) and Il1b mRNA expression (b, e) in mouse BMDMs transfected with two different Nrf2 siRNAs (50 nM) compared with a non-silencing scrambled control siRNA plus LPS (6 h; ac, e; 24 h; d) and/or OI (n = 6). NT, non-transfected. f, Il1b mRNA expression in wild-type and Nrf2-knockout BMDMs treated with LPS (24 h; WT n = 2, Nrf2 KO n = 4) and/or OI. gk, Il1b (g) and Nos2 (j) mRNA, and IL-1β (h), IL-10 (i), TNF and nitrite (k) production with or without LPS (24 h), diethyl maleate (DEM; 100 μM) or 15-deoxy-Δ12,14-prostaglandin J2 (J2; 5 μM) pre-treatment for 3 h (n = 3). Data are mean ± s.e.m. P values calculated using one-way ANOVA. Blots are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9 An Nrf2-dependent feedback loop exists between itaconate and IFN-β.

a, Metabolite levels after treatment with IFN-β (1,000 U ml−1; 27 h; n = 5). b, c, Isg20 and Irf5 mRNA expression in BMDMs treated with LPS (b) or poly(I:C) (c, 40 μg ml−1; 24 h) and/or OI (n = 6). d, Il10 mRNA (n = 3) and IL-10 protein (n = 5) expression after treatment with LPS for 4 h (left) or 24 h (right) and/or IFN-β treatment (1,000 U ml−1) for 3 h. e, Isg20 expression in BMDMs transfected with two different Nrf2 siRNAs (50 nM) compared with non-silencing control plus LPS (6 h) and/or OI (n = 6). f, Isg20 mRNA expression in wild-type (n = 2) and Nrf2-knockout (n = 4) BMDMs plus LPS (6 h) and/or OI. g, Isg20 mRNA expression after pre-treatment with LPS (24 h) and/or diethyl maleate (100 μM) or 15-deoxy-Δ12,14-prostaglandin J2 (5 μM) for 3 h (n = 3). Data are mean ± s.e.m. P values calculated using one-way ANOVA.

Extended Data Table 1 Mass spectrometry analysis of itaconate-induced cysteine alkylation

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Mills, E., Ryan, D., Prag, H. et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature 556, 113–117 (2018). https://doi.org/10.1038/nature25986

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