Itaconate ameliorates autoimmunity by modulating T cell imbalance via metabolic and epigenetic reprogramming

Abstract

Dysregulation of Th17 and Treg cells contributes to the pathophysiology of many autoimmune diseases. Herein, we show that itaconate, an immunomodulatory metabolite, inhibits Th17 cell differentiation and promotes Treg cell differentiation by orchestrating metabolic and epigenetic reprogramming. Mechanistically, itaconate suppresses glycolysis and oxidative phosphorylation in Th17- and Treg-polarizing T cells. Following treatment with itaconate, the S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and 2-hydroxyglutarate levels are decreased by inhibiting the synthetic enzyme activities in Th17 and Treg cells, respectively. Consequently, these metabolic changes are associated with altered chromatin accessibility of essential transcription factors and key gene expression in Th17 and Treg cell differentiation, including decreased RORγt binding at the Il17a promoter. The adoptive transfer of itaconate-treated Th17-polarizing T cells ameliorates experimental autoimmune encephalomyelitis. These results indicate that itaconate is a crucial metabolic regulator for Th17/Treg cell balance and could be a potential therapeutic agent for autoimmune diseases.

Fig. 1. Itaconate inhibits Th17 differentiation and enhances Treg differentiation.

Representative flow plots (a) and cumulative data (b) of the differentiation of murine naive CD4⁺ T cells from wild-type B6 mice activated under Th1, Th2, Th17, and Treg cell conditions in the presence or absence of itaconate (ITA; 0, 3, and 6 mM) after 3 days culture (Th1 and Th2, n = 5; Th17, n = 6; Treg, n = 4). c Expression of Th17- and Treg-related genes in the presence or absence of ITA (0 and 6 mM) (n = 4, each condition). d Mean fluorescence intensity (MFI) of RORγt expression under Th17-polarizing condition in the presence or absence of ITA (6 mM) (n = 5). P values are calculated using one-way ANOVA with Bonferroni post hoc test for (b) and two-tailed unpaired Student’s t-test for (c, d). Data are representative of mean ± s.e.m. Source data are provided as a Source Data file.

Fig. 2. Itaconate ameliorates experimental autoimmune encephalomyelitis.

a, b C57BL/6J mice were immunized with myelin oligodendrocyte glycoprotein (MOG)_35-55 and complete Freund’s adjuvant (CFA). Mice were intraperitoneally injected with 50 mg kg⁻¹ itaconate (ITA) every other day from day 0 to day 14. Clinical scores (a) and body weight (b) in EAE mice treated with PBS (Ctrl, n = 11) or ITA (n = 11). c–j For adoptive transfer EAE (tEAE), pathogenic Th17-polarizing CD4⁺ T cells from 2D2 mice were cultured with or without ITA ex vivo for 3 days. Then, the harvested cells were transferred to recipient Rag1-deficient mice intravenously. Clinical scores (c) and body weight (d) of control (Ctrl, n = 10) or ITA (n = 13) recipient mice in tEAE models. e Representative histology of spinal cord stained with hematoxylin and eosin (H&E) and luxol fast blue (LFB). Scale bar, 500 μm. f Inflammation scores of spinal cords are shown (Ctrl, n = 6; ITA, n = 8, biologically independent samples). Absolute numbers (left) and frequency (right) of IL-17A⁺ (g) and IL-17A⁺GM-CSF⁺ (h) CD4⁺ T cells in the spinal cord of recipient mice, as assessed using flow cytometry (n = 4 in each condition, biologically independent samples). i Absolute number of macrophages, Ly6C^hi monocytes, and neutrophils in the spinal cord of Rag1-deficient mice on day 14 after induction of EAE (Ctrl, n = 11; ITA, n = 10, biologically independent samples). j Absolute number of IL-1β-producing cells in three cell types as described in (i) (Ctrl, n = 11; ITA, n = 10, biologically independent samples). Data of e are representative of four independent experiments with similar results. P values are calculated using two-way ANOVA for (a–d) and two-tailed unpaired Student’s t-test for (f–j). Data are representative of mean ± s.e.m. Source data are provided as a Source Data file.

Fig. 3. Itaconate decreases glycolysis-related genes under Th17- and Treg-polarizing conditions.

a–f Th17- and Treg-polarizing T cells from B6 mice in the presence or absence of itaconate (ITA) after 2 days of culture were subjected to RNA sequencing (n = 3 in each condition, independent experiments). Heatmap (a) shows the top 100 and bottom 100 modified genes from RNA-seq in Th17- (left) and Treg- (right) polarizing T cells in the presence or absence of ITA. Principle component analysis of global gene expression from RNA-seq in four T cell populations (b). Volcano plot of differential gene expression in ITA-treated Th17- (c) or Treg- (d) polarizing T cells compared to Control (Ctrl). Venn diagram (e) displays the overlapping DEGs between ITA-treated Th17- and Treg-polarizing T cells. The top gene ontology pathways in the overlapping group are shown. P value for (e) indicates gene enrichment analysis test implemented in Metascape without adjustment for multiple comparisons. Heatmap (f) shows relative expression (z-score) of glycolysis and HIF-1α-related genes according to RNA-seq. Source data are provided as a Source Data file.

Fig. 4. Itaconate inhibits glycolysis without suppressing HIF-1α.

Intracellular HIF-1α expression in Th17- and Treg-polarizing T cells with or without itaconate (ITA) (a), percentage of HIF-1α⁺ cells under each growth condition (b) (Th17, n = 3; Treg, n = 4). Extracellular acidification rate (ECAR) of Th17- (c) and Treg-polarizing T cells (d) with or without ITA measured using glycolytic rate assay (Th17, n = 4; Treg, n = 3). Basal glycolysis and compensatory glycolysis were calculated. Mitochobdrial oxygen consumption rate (OCR) using an extracellular flux analyzer in Th17- (e) and Treg-polarizing T cells (f) with or without ITA (n = 4, each condition). P values are calculated using two-tailed unpaired Student’s t-test for (b–f). Data are representative of mean ± s.e.m. Source data are provided as a Source Data file.

Fig. 5. Metabolic reprogramming and enzymatic inhibition in Itaconate-treated Th17- and Treg-polarizing T cells.

a Intracellular levels of metabolites implicated in TCA cycle, glutaminolysis, and methionine metabolism from Th17- and Treg-polarizing T cells in the presence or absence of itaconate (ITA) after 2 days of culture (n = 3 independent experiments). b Heatmap of glycolytic metabolites from Th17- and Treg-polarizing T cells in the presence or absence of ITA. c Methylation index (SAM/SAH ratio) of Th17- and Treg-polarizing T cells in the presence or absence of ITA (n = 3, each condition). Enzymatic activity of methionine adenosyltransferase (MAT) (d) (Th17, n = 3; Treg, n = 4) and isocitrate dehydrogenase (IDH)1 and 2 (e) in Th17- and Treg-polarizing T cells with or without ITA (n = 4, each condition). IDH1 (f) and 2 (g) activity were measured in the presence of a variable concentration of ITA. IDH activity was calculated as the ratio of measured data to the average control value. Cumulative data of the differentiation of murine naive CD4⁺ T cells from Nrf2-knockout mice activated under Th17- (h), and Treg cell conditions (i) in the presence or absence of ITA (n = 4, each condition). P values are calculated using two-tailed unpaired Student’s t-test for (a, c–e) and one-way ANOVA with Bonferroni post hoc test for (h, i). arb.units, arbitrary unit. Data are representative of mean ± s.e.m. Source data are provided as a Source Data file.

Fig. 6. Itaconate altered the chromatin accessibility of essential transcription factors in Th17 and Treg cell differentiation.

a Chromatin immunoprecipitation (ChIP) analysis of RORγt at the Il17a promoter regions in naive CD4⁺ and Th17-polarizing T cells in the presence or absence of itaconate (ITA) (Naive CD4⁺, n = 3; Th17, n = 3). b–f Assay for transposase-accessible chromatin sequencing (ATAC-seq) was performed using Th17- and Treg-polarizing T cells from B6 mice in the presence or absence of ITA after 2 days of culture. Two replicates (n = 2, each group) were used for ATAC-seq. Venn diagram displaying the overlap between DEGs between ITA-treated and control T cells under Th17 or Treg conditions and between genes that show differential chromatin accessibility (DA) (b). Heatmap displaying relative expression (z-score) of genes that show DA and DE between ITA-treated and control T cells under Th17 or Treg conditions (c). d Representative ATAC-seq tracks in Th17- and Treg-polarizing T cells. e Motif discovery of the peaks which significantly changed to be more closed in ITA-treated Th17 cells compared to Ctrl. f Motif discovery of the peaks which significantly changed to be more open in ITA-treated Treg cells compared to Ctrl. P values are calculated using one-way ANOVA with Bonferroni post hoc test for (a) and two-sided Wald test with Benjamini and Hochberg method for (e, f). Data are representative of mean ± s.e.m. Source data are provided as a Source Data file.