Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism

Abstract

Aerobic glycolysis (the Warburg effect) is a metabolic hallmark of activated T cells and has been implicated in augmenting effector T cell responses, including expression of the proinflammatory cytokine interferon-γ (IFN-γ), via 3' untranslated region (3'UTR)-mediated mechanisms. Here, we show that lactate dehydrogenase A (LDHA) is induced in activated T cells to support aerobic glycolysis but promotes IFN-γ expression independently of its 3'UTR. Instead, LDHA maintains high concentrations of acetyl-coenzyme A to enhance histone acetylation and transcription of Ifng Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells. These findings reveal an epigenetic mechanism by which aerobic glycolysis promotes effector T cell differentiation and suggest that LDHA may be targeted therapeutically in autoinflammatory diseases.

Fig. 1. LDHA dictates aerobic glycolysis in activated CD4⁺ T cells

(A and B) Naïve CD4⁺ T cells isolated from wild-type (WT) or CD4^CreLdha^fl/fl (KO) mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 for 2 days. Cells were replenished with fresh medium, which was harvested 24 h later. Lactate production (A) and glucose consumption (B) were determined with triplicates. (C – I) Naïve WT or KO CD4⁺ T cells were stimulated as in (A). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured with a glycolysis stress test kit (C, E and F) or a Mito stress test kit (D, G – I). Baseline ECAR (E) and stressed ECAR (F) of activated CD4⁺ T cells were calculated according to (C). Baseline OCR (G), stressed OCR (H) and baseline OCR/ECAR (I) were calculated according to (D). Statistics (E – I) were from one of two independent experiments each with 8 biological replicates (n = 8), data represent mean ± SD, two-tailed unpaired t-test, ***P ≤ 0.001.

Fig. 2. LDHA regulates IFN-γ expression independent of its 3′UTR

(A and B) Naïve wild-type (WT) or CD4^CreLdha^fl/fl (KO) CD4⁺ T cells were differentiated under Th1 conditions for 3 days, and restimulated with phorbol myristate acetate (PMA) and ionomycin for 4 h. IFN-γ expression was determined by intracellular staining. Representative plots (A) and mean fluorescence intensity (MFI) (B) are shown. (C – E) Naïve WT or KO CD4⁺ T cells were activated for 2 days and transduced with green fluorescent protein (GFP) constructs fused with the 3′ untranslated region (3′UTR) of the Ifng or Gapdh gene. GFP expression was measured 48 h after transduction. Representative plots (C) and MFI of GFP in GFP⁺ cells are shown (D). (E) MFI of GFP from IFN-γ 3′UTR was normalized to that of GAPDH 3′UTR control. (F) A diagram of IFN-γ mRNA expressed from the WT Ifng or Yeti allele. (G and H) Naïve CD4⁺ T cells from Yeti/Yeti or KOYeti/Yeti mice were differentiated as in (A). The expression of IFN-γ and eYFP were determined by flow cytometry. Representative plots and respective statistic analysis are shown in (G) and (H). Statistics (B, E and H) were from one of two independent experiments each with 3 biological replicates (n = 3), data represent mean ± SD, two-tailed unpaired t-test, *P ≤ 0.05; ***P ≤ 0.001; ns, not significant.

Fig. 3. LDHA promotes IFN-γ expression through an epigenetic mechanism

(A) Representative H3K9 acetylation (H3K9Ac) peaks at the Cd3e or Ifng promoter and enhancer (CNS22) regions from one of two ChIP-Seq experiments are shown. (B) H3K9Ac at the Ifng promoter and CNS22 enhancer regions in WT or KO Th1 cells were assessed by ChIP-qPCR. Enrichment was normalized to H3K9Ac at the Cd3e promoter region. (C) Naïve WT or KO CD4⁺ T cells were differentiated under Th1 conditions, and acetyl-CoA levels were measured by an acetyl-CoA assay kit. (D and E) Naïve WT or KO CD4⁺ T cells were differentiated under Th1 conditions for 3 days, and either left untreated or supplemented with 20 mM sodium acetate for another 24 h. Cells were subsequently restimulated with phorbol myristate acetate (PMA) and ionomycin for 4 h. The expression of IFN-γ protein (D) and mRNA (E) were determined by flow cytometry and qPCR, respectively. mRNA level of IFN-γ was normalized to that of β-Actin. MFIs of IFN-γ are shown in (D). (F) T cells were cultured as in (D), and H3K9Ac at the Ifng promoter and CNS22 enhancer regions were assessed by ChIP-qPCR. Enrichment was normalized to H3K9Ac at the Cd3e promoter region. Statistics (B, C, E and F) were from one of three independent experiments each with 3 biological replicates (n = 3), data represent mean ± SD, two-tailed unpaired t-test, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.

Fig. 4. LDHA deficiency in T cells protects Yeti/Yeti mice from a lethal autoinflammatory disease

(A) The survival curve of Yeti/Yeti (n = 5) and CD4^CreLdha^fl/flYeti/Yeti (KO Yeti/Yeti) mice (n = 6). (B) Representative haematoxylin and eosin staining of liver sections from wild-type (WT), CD4^CreLdha^fl/fl (KO), Yeti/Yeti and KO Yeti/Yeti mice. Arrows indicate the necrotic regions. (C and D) Splenocytes from mice of the indicated genotypes were stimulated with phorbol myristate acetate (PMA) and ionomycin for 4 h. IFN-γ and YFP expression in CD4⁺ T cells and NK1.1⁺TCR⁻ NK cells were determined by flow cytometry. Representative plots (C) and statistics (D) are shown, data represent mean ± SD, n = 4 – 5 mice per genotype, two-tailed paired t-test, ***P ≤ 0.001; ns, not significant.