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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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.
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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
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.
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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.
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.
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 b–d, data is representative of three experiments.
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.
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. i–k, 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.
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.
Supplementary information
Supplementary Figures
This file contains Supplementary Figure 1, the western blot source data with size marker indications and Supplementary Figure 2, flow cytometry gating.
Supplementary Table 1
A list of primers used in the study.
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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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DOI: https://doi.org/10.1038/s41586-018-0052-z
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