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Itaconate inhibits TET DNA dioxygenases to dampen inflammatory responses

Nature Cell Biology volume 24, pages 353–363 (2022)Cite this article

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Abstract

As one of the most induced genes in activated macrophages, immune-responsive gene 1 (IRG1) encodes a mitochondrial metabolic enzyme catalysing the production of itaconic acid (ITA). Although ITA has an anti-inflammatory property, the underlying mechanisms are not fully understood. Here we show that ITA is a potent inhibitor of the TET-family DNA dioxygenases. ITA binds to the same site on TET2 as the co-substrate α-ketoglutarate, inhibiting TET2 catalytic activity. Lipopolysaccharide treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals that TET2 is a major target of ITA in suppressing lipopolysaccharide-induced genes, including those regulated by the NF-κB and STAT signalling pathways. In vivo, ITA decreases the levels of 5-hydroxymethylcytosine, reduces lipopolysaccharide-induced acute pulmonary oedema as well as lung and liver injury, and protects mice against lethal endotoxaemia, depending on the catalytic activity of Tet2. Our study thus identifies ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the inflammatory responses.

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Fig. 1: Irg1 expression reduces Tet2-catalysed 5hmC production in cells.
Fig. 2: Itaconate binds directly to TET2 in a manner similar to α-KG.
Fig. 3: Irg1 produces itaconate to inhibit Tet activity in vivo.
Fig. 4: Tet2 is a major target of itaconate to suppress LPS-induced genes in macrophages.
Fig. 5: RelA interacts with and recruits Tet2 to the Nfkbiz promoter in LPS-activated macrophages.
Fig. 6: Itaconate reduces LPS-induced mouse mortality in a Tet2-dependent manner.

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Data availability

The RNA-seq data that support the findings of this study have been deposited in the GEO under the following accession codes: GSE148143, GSE148145 and GSE148147. The hMeDIP–seq datasets have been deposited in the GEO under the accession number GSE158580. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Boyer, S. Parnham and the UNC Biomolecular NMR Spectroscopy Core Facility for the NMR analysis; M. Lee of Molecular Cornerstones for molecular modelling; Y. Xu (Fudan University) for providing human TET2CD expression vector; J. Wong (East China Normal University) for providing human TET3 expression vector; N. Brown (UNC-Chapel Hill) for providing advice on TET2 purification; and J. Chen and S. Gao (School of Life Sciences and Technology, Tongji University, Shanghai) for providing Tet2HxD-KI mutant mice. We also thank the staff members working at IBS, Fudan University for their help with the mass spectrometry analyses. This work was supported by the National Key R&D Program of China (grant nos 2020YFA0803202 and 2016YFA0501800 to D. Ye., and 2016YFC1305102 to Y. Zhou.), NSFC grants (grant nos 31871431 and 31821002 to D. Ye., 82103366 to L. Chen., and 81671561 and 81974248 to Y. Zhou.), Innovative research team of high-level local universities in Shanghai (grant no. SSMU-ZLCX20180501 to D. Ye.) and Program for Outstanding Medical Academic Leader (grant no. 2019LJ19 to Y. Zhou.). This study was also supported by a Samuel Waxman Research Foundation Investigator Award and NIH grant no. CA163834 (A. Baldwin. and Y. Xiong.).

Author information

Author notes

  1. Yue Xiong

    Present address: Cullgen Inc., San Diego, CA, USA

  2. These authors contributed equally: Lei-Lei Chen, Carmen Morcelle, Zhou-Li Cheng.

Authors and Affiliations

  1. Huashan Hospital, Fudan University, and Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), and Molecular and Cell Biology Laboratory, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, China

    Lei-Lei Chen, Carmen Morcelle, Zhou-Li Cheng, Xiufei Chen, Yajing Gao, Junbin Song, Chenxi He, Kwei‐Yan Liu, Lei Zhang, Cheng Zhang, Jinye Zhang, Yiping Sun, Jiayong Yin, Fei Lan, Maoxiang Qian, Yufeng Zhou & Dan Ye

  2. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Lei-Lei Chen, Yanping Xu, Zhijun Li, Matthew D. Smith, Meng Cheng, Zhi-Zhong Ren, Xian-Ming Tan, Albert S. Baldwin & Yue Xiong

  3. Institute of Pediatrics, Children’s Hospital of Fudan University, Shanghai, China

    Yajing Gao, Kwei‐Yan Liu & Yufeng Zhou

  4. Life Sciences Institute and Stem Cell Institute, Zhejiang University, Hangzhou, China

    Miao Shi, Yezhang Zhu & Li Shen

  5. Key Laboratory of Pediatric Hematology and Oncology Ministry of Health and Pediatric Translational Medicine Institute, Shanghai Children’s Medical Center, Shanghai Jiao Tong University of Medicine, Shanghai, China

    Neng Zhou & Caiwen Duan

  6. Department of Critical Care Medicine, Children’s Hospital of Fudan University, Shanghai, China

    Guoping Lu

  7. Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Tuan Qi & Xing Chang

  8. Department of Neurosurgery, Huashan Hospital, Fudan Universit, Shanghai, China

    Yingying Lyu & Hui Yang

  9. Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China

    Ying Liu

  10. Department of Hematology and Oncology, Children’s Hospital of Fudan University, Shanghai, China

    Maoxiang Qian

  11. Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, USA

    Kun-Liang Guan

  12. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, USA

    Yue Xiong

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  1. Lei-Lei Chen
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Contributions

Y. Xiong., K. Guan. and D. Ye. conceived and supervised the project. L. Chen. designed the experiments and performed most of the experiments with Z. Cheng. L. Chen. wrote the draft with Y. Xiong. and D. Ye. C. Morcelle. participated in the initiation of the project. A. Joshua. and Q. Zhang. performed the NMR studies using the TET2 protein provided by Z. Ren. C. Morcelle., C. Zhang., J. Zhang. and K. Liu. measured the intracellular levels of ITA and other metabolites. X. Chen and C. Morcelle. performed immunofluorescence staining for 5hmC in HEK293T cells and generated constructs overexpressing Irg1 and the catalytically defective mutant. J. Song. generated Irg1-KO RAW264.7 cells. M. Shi., Y. Zhu and C. Morcelle. performed the 5hmC hMeDIP–seq under the supervision of L. Shen. J. Yin. performed the 5hmC hMeDIP–seq analyses under the supervision of M. Qian. H. Yang. and Y. Liu. performed the 5hmC staining. Z. Cheng. and L. Zhang. quantified genomic 5hmC and 5mC in cells by dot blot and LC–MS/MS, respectively. C. He. performed the in vitro assay for KDM activity under the supervision of F. Lan. (data not shown). Y. Gao., K. Liu., C. Morcelle., G. Lu. and T. Qi. conducted LPS administration of mice under the supervision of Y. Zhou and X. Chang. N. Zhou. performed the mouse bone-marrow transplantations under the supervision of C. Duan. Y. Xu made the initial finding of the NF-κB and TET2 interaction. A. Baldwin. participated in the design of NF-κB-related experiments. X. Tan. performed the statistical analyses.

Corresponding author

Correspondence to Dan Ye.

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The authors declare no competing interests.

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Nature Cell Biology thanks Tiffany Horng, Francois Fuks and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Itaconate inhibits the catalytic activity of Tet2 in vitro.

a, Computational modelling of TET2-metabolites binding. Current TET2 crystal reveals the binding of indicated metabolites with TET2. The high resolution 2.03 Å structure of TET2 in complex with N-oxalylglycine (NOG) in PDB entry 4NM6 was used as the basis for modelling α-KG and the related oncometabolites. While keeping the Fe(II), protein and solvent heavy atoms fixed, polar hydrogens in the binding site and the ligands were optimized using the MMFF94 energy potential with a reaction field solvation model as implemented in MOE.2018. b, IC50 of indicated metabolites toward purified mTET2CD measured by using a commercial kit (EpiGentek). Enzyme activity was analysed using GraphPad software and IC50 values were calculated using the equation of log (inhibitor) vs. response (three parameters). Data represent three independent experiments with similar results. c, R1896M or S1898F mutation reduces TET2 enzyme activity. As shown, both R1896 and S1898 are α-KG binding sites in the catalytic domain (CD) of human TET2 (upper panel). In vitro TET2 activity was determined by 5hmC dot-blot assays using human TET2CD purified from bacteria, as well as methylated dsDNA oligonucleotides as substrates (lower panel). Data represent two independent experiments with similar results. d, R1896M mutation, but not S1898F mutation, weakens the binding of TET2 with α-KG. STD NMR spectroscopy was used to determine the binding of α-KG with recombinant proteins of TET2CD mutant. Data represent two independent experiments with similar results.

Source data

Extended Data Fig. 2 Octyl-ITA (OI) can enter the cells and be hydrolyzed to itaconate.

a, OI can enter and accumulate in the cell. RAW264.7 cells were treated with different concentrations of OI for different lengths of time, followed by LC–MS/MS analysis of intracellular ITA. Data shown represent average values with S.D. of three independent experiments. b, Unmodified ITA can enter and accumulate in the cell. RAW264.7 cells were treated with increased concentrations of ITA as indicated, and the intracellular concentration of ITA and succinate was measured by LC–MS/MS. Data shown represent average values with S.D. of four independent experiments. c, Intracellular accumulation of cell-permeable metabolites. HEK293T cells were treated with cell-permeable octyl-ITA and octyl-L-2-HG for 12 hours as indicated, and the intracellular levels of L-2-HG and ITA were quantified by LC–MS/MS. Shown are average values of 2 independent experiments. The intracellular concentrations of indicated metabolites are shown.

Source data

Extended Data Fig. 3 Itaconate inhibits Tet activity in cells.

a, HEK293T cells were transiently overexpressed with HA-tagged mTET2CD or catalytic inactive mutant (referred to as mTET2CM), and then treated with indicated cell-permeable metabolites. Global 5hmC was detected by immunofluorescence. Scale bar, 20 μM. b, TET2-overexpressing HEK293T cells in (a) were treated with indicated OI either alone or together with increasing amount of Dimethyl-α-KG. Global 5hmC was detected by immunofluorescence. Scale bar, 20 μM. Data in (a-b) represent two independent experiments with similar results. c, HEK293T cells were transiently overexpressed with Flag-tagged human full-length TET3. The expression of TET3 was determined by western blot with indicated antibodies. A representative blot of two independent experiments is shown. d, TET3-overexpressing HEK293T cells in (c) were treated with indicated concentrations of OI or ITA. The intracellular levels of ITA and succinate were measured by LC–MS/MS. Data shown are average values of 2 independent experiments. e, f, TET3-overexpressing HEK293T cells in (c) were treated with indicated concentrations of OI or unmodified ITA, and then global 5hmC was detected by dot-blot (e) and LC–MS/MS (f). Data shown in (f) are average values with S.D. of 3 independent experiments. g, TET2-overexpressing HEK293T cells in (a) were treated with different amounts of cell-permeable ITA or L-2-HG as indicated, and histone methylation markers were determined by western blot with indicated antibodies. A representative blot of two independent experiments is shown. h, Tet2+/+ and Tet2-/- BMDMs were treated with 0.5 mM OI or increased concentrations of unmodified ITA for 8 hours, followed by LC–MS/MS analysis to determine intracellular ITA. Data shown are average values with S.D. of 3 independent experiments. i, Wild-type BMDMs were treated with DMSO or 0.5 mM OI for 8 hours, followed by 5hmC mapping by hMeDIP–seq. The normalized density profile for 5hmC across gene body + 5kb flanking regions is shown. The experiment was performed once. P values are calculated using one-way ANOVA for multiple comparisons (f). ****denotes p < 0.0001 for the indicated comparison.

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Extended Data Fig. 4 LPS induces Itaconate and metabolic reprogramming in macrophages.

a, b, Irg1 mRNA and protein expression in RAW264.7 cells after LPS treatment, as determined by qRT-PCR and western blot, respectively. Data shown in (a) are average values of 2 (time points: 1–12 hours) or 3 (time points: 0/0.5/24 hours) independent experiments. c, d, LPS-induced ITA accumulation in Irg1-WT but not Irg1-KO RAW264.7 cells. Irg1 protein was detected by western blot (c), and the intracellular concentration of ITA was measured by LC–MS/MS (d). Data shown in (d) are average values of 2 independent experiments. A representative blot of two independent experiments is shown in (b) and (c). e, Succinate but not fumarate and 2-HG is accumulated, while α-KG is decreased upon LPS treatment in RAW264.7 cells. The intracellular concentrations of indicated metabolites were measured by LC–MS/MS. Data shown are average values of 2 independent experiments (left) or average values with S.D. of 3 independent experiments (right).

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Extended Data Fig. 5 LPS-induced Itaconate inhibits Tet activity in macrophages.

a, In RAW264.7 and mouse BMDMs, relative mRNA expression of Tet1, Tet2 and Tet3 was determined by RNA-seq and plotted according to the fragments per kilobase of transcript per million mapped reads (FPKM) values. Fold changes in Tet gene expression for the indicated comparison are shown. Data shown are average values with S.D. of 3 independent experiments. b, c, LPS treatment reduces genome-wide 5hmC in Irg1-WT but not Irg1-KO RAW264.7 cells. 5hmC was determined by IF staining (b) and FACS (c) in cells after LPS treatment for indicated time. Scale bar, 50 μm. Data shown in (c, right) are average values of 2 independent experiments. d, LPS-triggered 5hmC decline is not due to succinate accumulation in macrophages. Irg1-KO RAW264.7 cells were treated with 3-NPA (1 mM), which specifically inhibits SDH and causes intracellular accumulation of succinate (left panel, data shown are average values with 2 independent experiments). Genomic levels of 5mC and 5hmC were determined by LC–MS/MS in these cells after LPS treatment for indicated time (middle and right panels, data shown are average values with S.D. of 3 independent experiments). e, α-KG restores LPS-induced 5hmC decrease in RAW264.7 cells. Macrophages were challenged with LPS for 12 hours and then treated dimethyl-α-KG (1 mM) for 12 hours. Genomic 5hmC was detected by IF staining. Scale bar, 25 μm. f, RAW264.7 cells were treated with LPS for indicated time, and then histone methylation markers were determined by western blot using indicated antibodies. g, h, Mouse BMDMs (g) and thioglycolate elicited peritoneal macrophages (h) were from Irg1+/+ or Irg1-/- mice (n=3 mice per group), and then were either unstimulated or treated with LPS for indicated time. The levels of Tet2 and Irg1 proteins were determined by western blot using indicated antibodies. P values are calculated using one-way ANOVA (g, h) for multiple comparisons. *denotes p < 0.05, **denotes p < 0.01, and ***denotes p < 0.001 for the indicated comparison. n.s. = not significant. Data represent two independent experiments with similar results in (b, e, f).

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Extended Data Fig. 6 Tet2 is the major target of Itaconate in macrophages during LPS response.

a, Principal component analysis (PCA) on the effect of OI treatment and Tet2 deletion on gene expression in LPS-treated RAW264.7 cells. 3D representation of different colour-coded RNA-seq datasets corresponding to different Tet2 genotype, LPS and OI treatments. RNA-seq was performed using three biological replicates. b, 1,846 genes which are downregulated by OI in Tet2-WT RAW264.7 cells are compared with 807 gene which are downregulated by OI in Tet2-KO cells (FC ≥ 2). As shown, only 493 (of 1,846) genes were commonly downregulated by OI in both Tet2-WT and Tet2-KO cells. c, Identification of LPS-induced genes that are inhibited by OI. The overlap of LPS-induced genes (FC ≥ 2) and OI-inhibited genes (FC ≥ 2) was displayed by Venn diagram (P-value < 10−10). 712 genes were identified and then used for KEGG pathway analysis by online DAVID analysis. Top 10 pathways are listed. d, 712 genes which are induced by LPS and downregulated by OI in Tet2-WT RAW264.7 cells are compared with 807 gene which are downregulated by OI in Tet2-KO cells (FC ≥ 2). As shown, only 135 (of 712) genes were commonly downregulated by OI in both Tet2-WT and Tet2-KO cells. e, f, Verification of the Tet2 H1795R KI mice. The KI mutation was confirmed by DNA sequencing (e). Catalytic inactivation of Tet2 was confirmed by 5hmC dot-blot using genomic DNA from hepatocytes (f). The experiments in e–f were performed once. g, Overlap between LPS-induced genes downregulated by OI and by H1795R catalytical inactivation in Tet2 in BMDMs is displayed by Venn diagram (p-value < 10−10). Top 10 pathways enriched among the overlapping genes were identified by KEGG pathway analysis.

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Extended Data Fig. 7 Itaconate inhibits Tet2-mediated LPS induction of Nfkbiz and other inflammatory genes.

a, Unmodified ITA inhibits LPS-induced genes in a Tet2-dependent manner. Tet2-WT and Tet2-KO RAW264.7 cells were pre-treated with unmodified ITA (3 mM) and then stimulated with LPS for 4 hours, following detection of the indicated gene mRNA expression by qRT-PCR. b, Addition of OI in a limited time window inhibits LPS-induced genes, mimicking the effect of Tet2 deletion. RAW264.7 cells were stimulated with LPS for 2 hours before endogenous ITA starts to accumulate, and then treated with OI for another 2 hours, following determination of indicated gene mRNA expression by qRT-PCR. Shown are average values with S.D. of three independent experiments. P values are calculated using two-way ANOVA for multiple comparisons. *denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001, and ****denotes p < 0.0001 for the indicated comparison. n.s. = not significant.

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Extended Data Fig. 8 Itaconate inhibits Tet2-mediated LPS induction of IκBζ.

a, b, Gene expression of all detectable NfкB family members in RAW164.7 cells (a) and BMDMs (b) with indicated treatments was determined by RNA-seq and represented by heatmaps. c, Tet2 deletion inhibits LPS induction of Nfkbiz gene expression in RAW264.7 cells. Tet2-WT and Tet2-KO macrophages were treated with LPS for indicated time. Nfkbiz mRNA was determined by qRT-PCR. Data shown are average values with S.D. of 3 independent experiments. d, Both ITA and Tet2 deletion inhibits LPS induction of Nfkbiz. Tet2-WT and Tet2-KO RAW264.7 cells were treated with either LPS alone or with OI and the level of Nfkbiz mRNA was determined by qRT-PCR. Data shown are average values with S.D. of 3 independent experiments. e, α-KG activates Nfkbiz mRNA expression in RAW264.7 cells. Macrophages were treated with increased concentrations (0.5, 1, 2 mM) of Dimethyl-α-KG together with LPS as indicated. Nfkbiz mRNA was determined by qRT-PCR. Data shown are average values with S.D. of 3 independent experiments. f, α-KG elevates Nfkbiz mRNA level in Tet2-dependent manner. Tet2-WT and Tet2-KO RAW264.7 cells were treated with either LPS alone or with cell-permeable α-KG, and the level of Nfkbiz mRNA was determined by qRT-PCR. Data shown are average values with S.D. of 3 independent experiments. g, h, α-KG restores IκBζ protein blocked by ITA in a Tet2-dependnet manner. Tet2-WT (g) but not Tet2-KO (h) RAW264.7 cells were treated with LPS and cell-permeable ITA and α-KG as indicated. The levels of IκBζ protein were measured by western blot and normalized against Gapdh. A representative blot of two independent experiments is shown in (g, h). P values are calculated using two-tailed Student’s t-test for paired comparisons (c), one-way ANOVA (e, d) and two-way ANOVA (f) for multiple comparisons. **denotes p < 0.01, ***denotes p < 0.001, and ****denotes p < 0.0001 for the indicated comparison. n.s. = not significant.

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Extended Data Fig. 9 Irg1-deficient mice are more susceptible to LPS-induced acute lung injury and mortality.

a, Flowchart for establishing LPS-induced sepsis mouse model. Briefly, age and sex-matched Irg1-/- or Irg1+/+ mice were i.p. injected with PBS or LPS (20 mg/kg). At 4- or 24-hours post LPS injection, peritoneal leukocytes, serum, and lung were harvested for further analysis. b, LPS induces ITA accumulation in Irg1+/+ peritoneal leukocytes, but not in those from Irg1-/- mice. Peritoneal leukocytes were freshly isolated as described above in (a), and the intracellular concentration of ITA was measured by LC–MS/MS (n=2–3 mice per group). c, LPS-challenged Irg1-/- mice exhibit higher serum Il-6 levels than Irg1+/+ controls, as determined by ELISA (n=3-7 mice per group). d,e, LPS-challenged Irg1-/- mice exhibit more severe lung injury than Irg1+/+ controls. As described above in (a), mouse lungs were harvested and then subjected to histopathological analysis (n=3-6 mice per group). Representative photomicrographs of HE staining are shown (d). Scale bar, 200 μm (upper panels) & 100 μm (lower panels). f, Irg1-/- mice are more susceptible to LPS-induced mortality. Kaplan–Meier survival curves were determined as described in Methods (n=10 mice per group). Shown in b, c and e are average values with S.D. P values are calculated using one-way ANOVA for multiple comparisons (c, e). As for the percent survival, P values were determined using log-rank (Mantel–Cox) test comparing each 2 groups (f); *denotes p < 0.05, **denotes p < 0.01 and ****denotes p < 0.0001 for the indicated comparison.

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Extended Data Fig. 10 Itaconate reduces LPS-induced mouse mortality in a manner dependent on Tet2.

a, Mice were i.p. injected with ITA prior to LPS challenge (25 mg/kg, n=3 mice per group; left panel). At 2 hours post LPS, peritoneal leukocytes and lung were freshly isolated and then subjected to determination of genomic 5hmC and 5mC by LC–MS/MS (right panels). b,c,d As described above in (a), mice were i.p. injected with ITA prior to LPS challenge (n=6-10 mice per group). At 2 hours post LPS, lung tissues were harvested and then subjected to the wet/dry weight ratio calculation (b). Meanwhile, lung histopathological injury was assessed by HE staining and the injury score was determined as described in method (c, d). Representative photomicrographs are shown. Scale bar, 200 μm (upper panels) & 100 μm (lower panels). e,f, As described above in (a), mice were i.p. injected with ITA prior to LPS challenge (Tet2+/+, n=13 mice; Tet2+/+, n=14 mice; Tet2-/-, n=7 mice per group). The animal survival was carefully monitored and Kaplan–Meier survival curves were determined as described in Methods. g, Flowchart for developing chimaeric mice by bone marrow transplantation (BMT). h, i, The KI mutations were confirmed by DNA sequencing in bone marrow samples of transplanted animals (h). Catalytic inactivation of Tet2 was also confirmed by 5hmC LC–MS/MS using genomic DNA from bone marrow samples of transplanted animals. j, A schematic model for Itaconate inhibits TET2 to dampen inflammatory response. Shown are average values with S.D. (a, i) or SEM (b, d). P- values are calculated using two-tailed Student’s t-test for paired comparisons (i) or one-way ANOVA for multiple comparisons (a, b, d); As for the percent survival, P values were determined using log-rank (Mantel–Cox) test comparing each 2 groups; *denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001, and ****denotes p < 0.0001 for the indicated comparison. n.s. = not significant.

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Supplementary information

Supplementary Information

Supplementary Fig. 1. Flow cytometry gating strategies used in this study.

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Supplementary Table 1

Supplementary Table 1. Primer information. Supplementary Table 2. Detailed sequencing information of the hMeDIP–seq data.

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Chen, LL., Morcelle, C., Cheng, ZL. et al. Itaconate inhibits TET DNA dioxygenases to dampen inflammatory responses. Nat Cell Biol 24, 353–363 (2022). https://doi.org/10.1038/s41556-022-00853-8

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