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Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis

Abstract

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite2. Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines2, including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17–IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI–IκBζ regulatory axis could be an important new strategy for the treatment of IL-17–IκBζ-mediated autoimmune diseases.

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Fig. 1: DI and itaconate induce electrophilic stress in macrophages.
Fig. 2: DI inhibits LPS-mediated IκBζ induction.
Fig. 3: DI induces an Nrf2-independent response and inhibits the IL-6–IκBζ axis via ATF3.
Fig. 4: DI inhibits IL-17-mediated IκBζ induction in keratinocytes and ameliorates psoriatic pathology.

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References

  1. Jha, A. K. et al. Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization. Immunity 42, 419–430 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. 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 

  3. 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 

  4. Gorrini, C., Harris, I. S. & Mak, T. W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Kobayashi, E. H. et al. Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription. Nat. Commun. 7, 11624 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sullivan, L. B. et al. The proto-oncometabolite fumarate binds glutathione to amplify ROS-dependent signaling. Mol. Cell 51, 236–248 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zheng, L. et al. Fumarate induces redox-dependent senescence by modifying glutathione metabolism. Nat. Commun. 6, 6001 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Medzhitov, R. & Horng, T. Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692–703 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Yamamoto, M. et al. Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ. Nature 430, 218–222 (2004).

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Dhamija, S. et al. IL-1-induced post-transcriptional mechanisms target overlapping translational silencing and destabilizing elements in IκBζ mRNA. J. Biol. Chem. 285, 29165–29178 (2010); erratum 291, 24801 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Harding, H. P. et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11, 619–633 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Komatsu, M. et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of KEAP1. Nat. Cell Biol. 12, 213–223 (2010).

    CAS  PubMed  Google Scholar 

  13. Otterbein, L. E., Soares, M. P., Yamashita, K. & Bach, F. H. Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol. 24, 449–455 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Gilchrist, M. et al. Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 441, 173–178 (2006).

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Labzin, L. I. et al. ATF3 is a key regulator of macrophage IFN responses. J. Immunol. 195, 4446–4455 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Okuma, A. et al. Enhanced apoptosis by disruption of the STAT3-IκB-ζ signaling pathway in epithelial cells induces Sjögren’s syndrome-like autoimmune disease. Immunity 38, 450–460 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Muromoto, R. et al. IL-17A plays a central role in the expression of psoriasis signature genes through the induction of IκB-ζ in keratinocytes. Int. Immunol. 28, 443–452 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Johansen, C. et al. IκBζ is a key driver in the development of psoriasis. Proc. Natl Acad. Sci. USA 112, E5825–E5833 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tsoi, L. C. et al. Enhanced meta-analysis and replication studies identify five new psoriasis susceptibility loci. Nat. Commun. 6, 7001 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Okada, K. et al. The α-glucosidase inhibitor acarbose prevents obesity and simple steatosis in sequestosome 1/A170/p62 deficient mice. Hepatol. Res. 39, 490–500 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Parada, E. et al. The microglial α7-acetylcholine nicotinic receptor is a key element in promoting neuroprotection by inducing heme oxygenase-1 via nuclear factor erythroid-2-related factor 2. Antioxid. Redox Signal. 19, 1135–1148 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hartman, M. G. et al. Role for activating transcription factor 3 in stress-induced beta-cell apoptosis. Mol. Cell. Biol. 24, 5721–5732 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vincent, E. E. et al. Mitochondrial phosphoenolpyruvate carboxykinase regulates metabolic adaptation and enables glucose-independent tumor growth. Mol. Cell 60, 195–207 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Sergushichev, A. An algorithm for fast preranked gene set enrichment analysis using cumulative statistic calculation. Preprint at https://www.biorxiv.org/content/early/2016/06/20/060012 (2016).

  25. 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 

  26. McGuire, V. A. et al. Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63 ubiquitin chain formation. Sci. Rep. 6, 31159 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lu, W. et al. Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone Orbitrap mass spectrometer. Anal. Chem. 82, 3212–3221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sahil et al. ElucidataInc/ElMaven: El-MAVEN v0.2.2. (2017).

  31. Harder, B.-J., Bettenbrock, K. & Klamt, S. Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli. Metab. Eng. 38, 29–37 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Cheng, Z. et al. Pervasive, coordinated protein-level changes driven by transcript isoform switching during meiosis. Cell 172, 910–923.e16 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Keshishian, H. et al. Multiplexed, quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury. Mol. Cell. Proteomics 14, 2375–2393 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Hildebrand, D. G. et al. IκBζ is a transcriptional key regulator of CCL2/MCP-1. J. Immunol. 190, 4812–4820 (2013).

    CAS  PubMed  Google Scholar 

  37. Stewart, S. A. et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9, 493–501 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. de Guzman Strong, C. et al. A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis. Hum. Mol. Genet. 19, 1453–1460 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  39. van der Fits, L. et al. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J. Immunol. 182, 5836–5845 (2009).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank H. Virgin for providing p62-deficient mice; I. Schukina, J. Middleton and L. Arthur for technical support; and R. Dolle for assistance. This work was supported by RO1-A1125618 to M.N.A. and MES of Russia (project 2.3300.2017/4.6) to A.Se.

Reviewer information

Nature thanks N. Chandel, T. Horng and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations

Authors

Contributions

M.B. and M.N.A. conceived and designed the study and wrote the manuscript. M.B. performed western blot, cytokine, cytometry and glutathione analyses, metabolic labelling of protein synthesis and succinate dehydrogenase activity assays. L.G., D.K., M.B. and E.K. designed and performed human blood monocyte experiments. V.L., L.-H.H. and G.J.R. designed and performed in vivo psoriasis model experiments. A.Se. performed RNA-seq data analysis. E.L. prepared RNA-seq libraries and helped with PCR experiments. M.E.M. and C.d.G.S. designed and performed the isolation of mouse and human primary keratinocytes. H.K., K.J., H.B., T.P.R., S.A.B. and K.M.S. designed and performed mass spectrometry metabolomic measurements, and analysis and synthesis of Ita-GSH and DI-GSH conjugates. D.D. and K.A. synthesized 13C5-labelled DI. A.K. and M.J. designed and performed proteomic analysis. A.Sa., R.S.A., T.H., M.P.S., T.S. and S.A. provided animals and bones for the study. R.S. helped with the analysis of the metabolic data. B.J. and G.K.A. designed and performed the initial Nrf2 experiments.

Corresponding author

Correspondence to Maxim N. Artyomov.

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Competing interests

M.B., V.L. and M.N.A. are listed as inventors on provisional patent applications regarding the anti-inflammatory properties of itaconate derivatives.

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Extended data figures and tables

Extended Data Fig. 1 Detection of DI-GSH and Ita-GSH and electrophilic stress response.

a, Transcriptional comparison of KpCKO and wild-type BMDMs and enrichment of the DI gene signature. b, The reaction of DI with a thiol group in a Michael reaction. c, DI levels in media of BMDMs treated with DI for the indicated time, as determined by GC–MS. Mean of n = 2 cultures. d, Levels of the DI-GSH conjugate in the media of BMDMs treated with DI for the indicated time, as detected by LC–MS. Mean of n = 2 cultures. Data from Fig. 1e are overlaid with data for cell-free media. e, Levels of DI-GSH conjugate in BMDMs (left) and in their media (right) after treatment with 13C5-labelled DI for the indicated time, as detected by LC–MS. Mean of n = 2 cultures. f, g, Representative extracted ion chromatograms of DI-GSH detected in the media of BMDMs treated with DI for 6 h compared to the synthesized DI-GSH standard (f), and Ita-GSH detected in BMDMs stimulated with LPS for 24 h compared to the synthesized Ita-GSH standard (g). n = 10 technical replicates. h, Detection of reactive oxygen species in BV2 cells treated with DI for the indicated time, as determined by flow cytometry. Mean of n = 2 experiments. i, Cytokine production in BMDMs treated with DI in the presence of EtGSH and stimulated with LPS for 4 h, mean ± s.e.m., n = 3 experiments. j, Western blot of HO-1 expression in BMDMs treated with DMF. Representative of three experiments. For gel source data, see Supplementary Fig. 1. Statistical tests used were two-tailed t-tests.

Source Data

Extended Data Fig. 2 DI downregulates secondary transcriptional response to TLR stimulation.

a, Western blot of IκBζ expression in wild-type or Nfkbiz−/− BMDMs stimulated with LPS. b, Cytokine production in wild-type and Nfkbiz−/− BMDMs stimulated with LPS for 4 h, mean ± s.e.m., n = 3 experiments. c, RNA-seq analysis of BMDMs treated with DI and stimulated with LPS and IFNγ. d, mRNA expression show the induction of the indicated target genes in wild-type and Nfkbiz−/− BMDMs treated with DI and stimulated with LPS for 4 h, mean ± s.e.m., n = 3 experiments. e, Western blot of IκBζ expression in DI-treated BMDMs stimulated with LPS for 1 h. f, mRNA expression in human blood monocytes treated with DI and stimulated with LPS. g, Western blot of IκBζ expression in human blood monocytes treated with DI and stimulated with LPS. h, i, Western blot of IκBα (h) and IRAK1 expression and IKK phosphorylation (i) in BMDMs treated with DI and stimulated with LPS. j, p65 localization in DI-treated, LPS-stimulated BMDMs. Nuclei are stained with DAPI. Scale bars, 25 µm. Representative of two cultures. k, Western blot of IκBζ expression in BMDMs treated with DI in the presence of EtGSH and stimulated with LPS for 1 h. l, Western blot of IκBζ expression in human blood monocytes treated with DI in the presence of EtGSH and stimulated with LPS for 1 h. m, Cytokine production in wild-type or Nfkbiz−/− BMDMs treated with DI in the presence of NAC, stimulated with LPS for 4 h. Mean of n = 2 cultures. Representative data from two experiments (a), three experiments (e, h, i, k), three donors (f, g) and two donors (l). For gel source data, see Supplementary Fig. 1. Statistical tests used were two-tailed t-tests.

Source Data

Extended Data Fig. 3 DI regulates IκBζ at the post-transcriptional level.

a, Comparison of the effects of DI on IL-6, TNF and IκBζ on the protein and mRNA levels. Cytokine production is shown in BMDMs treated with DI (left) or DMF (middle) and stimulated with LPS for 4 h (DI), mean of n = 2 experiments, or 24 h (DMF), mean ± s.e.m., n = 3 experiments. Right, densitometric quantification of IκBζ protein and mRNA expression is shown for BMDMs treated with DI, stimulated with LPS for 1 h. Mean of n = 3 experiments, mRNA representative of two experiments. b, Western blot of IκBζ expression in BMDMs treated with DI and stimulated with LPS for 1 h. MG132 or bafilomycin A (BafA) were added 30 min before LPS stimulation. c, Nfkbiz 3′ UTR reporter expressing GFP in BV2 cells treated with DI (250 µM) for 12 h and stimulated with LPS for 1 h. EMPTY vector expressed GFP only; GFP expression determined by flow cytometry. d, Western blot of phosphorylated and total eIF2α in DI-treated BMDMs. e, Western blot of nascent protein synthesis detected using biotin–alkyne click chemistry in BMDMs treated with DI and stimulated with LPS for 1 h. The same membrane was reprobed for IκBζ. Representative of two experiments. f, Densitometric quantification of the biotin signal in the membrane in e. g, log fold change of proteomic signal in unstimulated and LPS-stimulated cells. h, log fold change of transcript and protein. For bd, data is representative of three experiments.

Source Data

Extended Data Fig. 4 BSO potentiates the inhibitory effect of DI.

a, Western blot of Nrf2 expression in BMDMs treated with BSO or DI. b, GSH levels in BMDMs treated with BSO and stimulated with LPS. Mean ± s.e.m., n = 3 cultures. c, Cytokine production in BMDMs treated with BSO and stimulated with LPS. Mean ± s.e.m., n = 3 experiments. d, Cytokine production in BMDMs treated with DI and BSO and stimulated with LPS for 4 h. Mean ± s.e.m., n = 3 experiments. e, Cytokine production in BMDMs treated with 4EI (10 mM) and BSO and stimulated with LPS for 4 h. Mean ± s.e.m., n = 3 experiments. f, Western blot of IκBζ expression in BMDMs tolerized with LPS in the presence of BSO for 18 h and restimulated for 1 h (see Fig. 2l), asterisk shows the different exposures. Western blot data are representative of three experiments. For gel source data, see Supplementary Fig. 1. Statistical tests used were two-tailed t-tests.

Source Data

Extended Data Fig. 5 Nrf2-independent action of DI.

a, Western blot of IκBζ expression in wild-type or Nrf2−/− BMDMs treated with DI and stimulated with LPS for 1 h. b, Western blot of p62 and HO-1 in wild-type or Nrf2−/− BMDMs treated with DI and stimulated with LPS. c, Western blot of IκBζ expression in wild-type and p62-deficient BMDMs treated with DI and stimulated with LPS. d, Western blot of IκBζ expression in wild-type and Hmox1-deficient BMDMs treated with DI and stimulated with LPS. e, Transcriptional comparison of Nrf2−/− and wild-type BMDMs treated with DI and GSEA statistics for unfolded protein response (UPR) and IFNα pathways. f, Pathways regulated by DI in an Nrf2-independent manner. Gene ranks, normalized enrichment score (NES), P and adjusted P (padj) are shown. g, h, Western blot of Nrf2 expression (g) or phosphorylated and total eIF2α (h) in DI-treated wild-type or Atf3−/− BMDMs. ik, Western blot of ATF3 in BMDMs (i, j) and human blood monocytes (k) treated with DI in combination with NAC or EtGSH and stimulated with LPS. Data are representative of three experiments (a, g, i, j), two experiments (b, c, h), one experiment (d) and from two donors (k). For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6 Viability of keratinocytes after DI treatment.

Mouse and human primary keratinocytes were treated with DI for 12 h and viability was determined by propidium iodide staining and flow cytometry. Percentage of propidium iodide-negative cells is shown. Representative of two mice or donors.

Source Data

Extended Data Fig. 7 DI shows a lack of in vivo toxicity.

a, Schematic of DI administration for the analysis of succinate dehydrogenase (SDH) activity in the heart and the liver. b, SDH activity in the heart and the liver of mice treated as in a. Mean of n = 2 technical replicates. Representative data from two mice. c, Western blot of SDH and GAPDH in mitochondrial and cytoplasmic fractions from the heart and the liver of mice treated as in a. Representative of two mice. For gel source data, see Supplementary Fig. 1.

Source Data

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Bambouskova, M., Gorvel, L., Lampropoulou, V. et al. Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis. Nature 556, 501–504 (2018). https://doi.org/10.1038/s41586-018-0052-z

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