MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation

Abstract

Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1^f/fMb1^Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.

Fig. 1. Mct1 deficiency in B cells dampens the CSR.

a The schematic plot of assays in vivo. This image was adapted from Biorender. The titers of IgM (b), IgG (c) from Mb1^Cre and Mct1^f/fMb1^Cre mice upon immunization (Intraperitoneal injection, i.p.) with NP₃₃-KLH at week 1 and 2, n = 4 biological replicates, p = 0.0067 (c). d The titers of Anti-NP3/Anti-NP30 from Mb1^Cre (n = 4 biological replicates, p = 0.0004) and Mct1^f/fMb1^Cre (n = 3 biological replicates) mice upon immunization (i.p.) with NP₃₃-KLH at week 2. GCBC in Mb1^Cre and Mct1^f/fMb1^Cre mice upon i.p. with SRBC at day 7 were analyzed by flow cytometry (e) and the percentage of GCBC is illustrated (f), Mb1^Cre (n = 4 biological replicates) and Mct1^f/fMb1^Cre (n = 3 biological replicates), p = 0.006. g The representative immunohistochemical staining of spleens with PNA (GCBC marker) and Ki67 (proliferative cells marker) from Mb1^Cre and Mct1^f/fMb1^Cre mice at day 7 with SRBC immunization (i.p.). Black arrows indicated the positive cells (brown points). Scale Bar, 250 μm and 50 μm. The percentage of IgM (h) and IgG1 (i)-BCR expressing B cells in GCBC upon immunization (i.p.) with SRBC at day 7 was analyzed by flow cytometry, n = 4 biological replicates, p = 0.0005. j The schematic diagram shows the experimental procedure for the analysis of IgG1 B cells after stimulation for 3 days in vitro. Flow cytometry analysis of IgM, IgG1 expressing B cells stimulated with LPS and IL-4 at day 3 from Mb1^Cre and Mct1^f/fMb1^Cre mice (k) and the percentage of IgG1 B cells is presented (l), n = 4 biological replicates, p < 0.0001. m–o Naive B cells isolated from B6 mice were stimulated with LPS and IL-4 for 3 days with MCT1 inhibitor α-Cyano-4-hydroxycinnamic acid (CHC) or AZD3965. Cumulative data of CHC is shown in (m), n = 4 biological replicates, p < 0.001. Representative plots by flow cytometry and respective statistical analysis of AZD3965 are shown in (n, o), n = 4 biological replicates, p < 0.01. Data are presented as mean ± SEM of 4 mice or mean ± SD, two-way ANOVA followed by Sidak’s multiple-comparisons test (b, c, m, o) or unpaired two-tailed Student’s t-test (d, f, h, i, l), **p < 0.01, ***p < 0.001, ****p < 0.0001 n.s, not significant.

Fig. 2. Mct1 deficiency inhibits the glycolysis metabolism of B cells.

a The naive B cells isolated from the Mb1^Cre and Mct1^f/fMb1^Cre cells were stimulated with LPS and IL-4 for 3 days. The representative glucose consumption is shown, n = 3 biological replicates, p = 0.5546. b Naive B cells isolated from the Mb1^Cre and Mct1^f/fMb1^Cre mice were cultured with different levels of glucose and stimulated with LPS and IL-4 for 3 days. The expression of enzymes of glucose metabolism was presented by immunoblot (left) and its quantification (right), n = 3 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001. c Hexokinase activity of B cells isolated from the Mb1^Cre and Mct1^f/fMb1^Cre mice treated with LPS and IL-4 for 3 days was measured using a hexokinase activity assay kit, n = 3 biological replicates, **p < 0.01, ***p < 0.001. d The schematic diagram shows the experimental procedure for metabolite analysis from cells or supernatants after stimulation for 3 days in vitro. This image was adapted from Biorender. e–g Naive B cells isolated from Mb1^Cre and Mct1^f/fMb1^Cre mice were stimulated with LPS and IL-4 for 3 days, n = 3 biological replicates, **p < 0.01, ***p < 0.001. The concentration of lactate in intracellular (left) and supernatant (right) was determined by lactate assay kit (e). The relative level of lactate in intracellular (left) and supernatant (right) was determined by metabonomics (g). The concentration of pyruvate in intracellular (left) and supernatant (right) was determined by pyruvate assay kit (f). The relative level of pyruvate in intracellular (left) and supernatant (right) was determined by metabonomics (g). Data are presented as mean ± SD, two-way ANOVA followed by Sidak’s multiple-comparisons test (c) or unpaired two-tailed Student’s t-test (a, b, e–g), *p < 0.05, **p < 0.01, ***p < 0.001, n.s, not significant. Source data are provided as a Source Data file.

Fig. 3. Mct1 deficiency promotes the glucose flux into the mitochondrial pyruvate oxidation.

a The schematic diagram shows the experimental procedure for metabolite analysis from cells or supernatants after stimulation with LPS and IL-4 for 2 days, replacing with U-¹³C₆ glucose tracing 30 min, 60 min and 180 min to do LC-MS/MS in vitro. This image was adapted from Biorender. b The schematic plot of ¹³C₆ glucose tracing. c–e Naive B cells isolated from the Mb1^Cre and Mct1^f/fMb1^Cre mice were stimulated with LPS and IL-4 for 2 days. Cells were replenished with U-¹³C₆ glucose medium, which was harvested for glucose flux after 30, 60 and 180 min. The glucose (m + 6), pyruvate (m + 3) and lactate (m + 3) levels were checked in the supernatant (c) and intracellular (d), n = 4 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001. The intracellular levels of citrate (m + 2), succinate (m + 2) and malate (m + 2) (e), n = 4 biological replicates, **p < 0.01, ***p < 0.001. f Naive B cells isolated from the Mb1^Cre and Mct1^f/fMb1^Cre mice were stimulated with LPS and IL-4 for 2 days. Oxygen consumption rate (OCR) was determined by Seahorse with six biological replicates (n = 6 biological replicates, p = 0.0076). Baseline OCR (right) was calculated according to OCR (left). Oligo: Oligomycin, 2 μM; FCCP, 1 μM; A: Antimycin A, 1 μM; R: rotenone, 1 μM. g B cells from Mb1^Cre and Mct1^f/fMb1^Cre mice with LPS and IL-4-treated for 3 days were harvested for ATP detection by a ATP assay kit with triplicate (n = 3 biological replicates). h EM analysis of mitochondrial morphology in B cells with LPS/IL-4 treatment for 2 days. Scale Bar, 2 μm (upper) and 200 nm (lower). i RT-PCR analysis of Mfn1 and Mfn2 in B cells after LPS/IL-4 activation for 2 days, n = 3 biological replicates, p = 0.0004(left), p = 0.007(right). j Schematic depicting rewiring of the metabolism in Mct1-deficient B cells. Data are presented as mean ± SD, two-way ANOVA followed by Sidak’s multiple-comparisons test (c–e) or unpaired two-tailed t-test (f, g, i), *p < 0.05, **p < 0.01, ***p < 0.001, n.s not significant.

Fig. 4. Pyruvate supports the CSR via securing the expression of AID.

a Scatterplot of gene expression in B cells from Mb1^Cre and Mct1^f/fMb1^Cre mice stimulated with LPS and IL-4 for 3 days, as determined by RNA-seq. Yellow: upregulated genes. Blue: downregulated genes. b Naive B cells isolated from Mb1^Cre and Mct1^f/fMb1^Cre mice were stimulated with LPS and IL-4 for 3 days. mRNA expression of Mct1 and Aicda was analyzed by qPCR, n = 3 biological replicates, **p < 0.01, ***p < 0.001. c As above conditions, analysis protein level of MCT1 and AID by immunoblot, n = 4 biological replicates, p < 0.001. d, e The B cells from WT control Mb1^Cre mice were treated with LPS and IL-4 for 3 days and cultured in the presence or absence of the Pyr (Sodium pyruvate, 1 mM, 2 mM). The proportion of IgG1 B cells caused by Pyr was analyzed by flow cytometry (d n = 7 biological replicates, p < 0.05). The expression of AID caused by Pyr was assessed by immunoblot (e n = 3 biological replicates, p < 0.001). f–h Mct1-deficient B cells were pretreated with Pyr (f n = 7 biological replicates) or methyl-pyruvate (MePyr, g, n = 7 biological replicates, p < 0.001) co-stimulated by LPS and IL-4 for 3 days. Protein abundance of AID was analyzed by western blotting (h, n = 3 biological replicates, p < 0.01). i Western blotting bands and their quantification analysis of MCT1 and AID expression of control and sgMct1 A20 cells, n = 4 biological replicates, p = 0.000346(left), p = 0.002247(right). j, k. The control A20 cells were cultured with various concentrations of Pyr (j) for 24 h, n = 3 biological replicates, **p < 0.01, ***p < 0.001. The sgMct1 A20 cells were cultured with various concentrations of Pyr or MePyr (k) for 24 h, n = 3 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± SD, two-way ANOVA followed by Sidak’s multiple-comparisons test (b) or two-tailed unpaired t-test (c–k), *p < 0.05, **p < 0.01, ***p < 0.001, n.s, not significant. Source data are provided as a Source Data file.

Fig. 5. MCT1 supports CSR by glucose metabolism, which provides substrates for the histone acetylation.

a Western blotting bands and their quantification analysis of H3K27Ac, H3K9Ac, H3K14Ac, H3K18Ac and H3K27me3 proteins in B cells from Mb1^Cre and Mct1^f/fMb1^Cre mice stimulated with LPS and IL-4 for 3 days, n = 4 biological replicates, **p < 0.01, ***p < 0.001. b Western blotting bands and their quantification analysis of CBP, HDAC and SIRT in B cells treated with LPS and IL-4 for 3 days, n = 4 biological replicates, p < 0.001. c Analysis of H3K27 acetylation at the Aicda gene promoter with ChIP-seq and ATAC-seq in B cells from Mb1^Cre and Mct1^f/fMb1^Cre mice stimulated with LPS and IL-4 for 3 days, n = 3 biological replicates. d Chromatin accessibility at the Aicda promoter analyzed by the Chip-qPCR in B cells from Mb1^Cre and Mct1^f/fMb1^Cre mice stimulated with LPS and IL-4 for 3 days (n = 6 biological replicates). e, f Naive B cells isolated from Mb1^Cre and Mct1^f/fMb1^Cre mice were co-stimulated with LPS and IL-4 and added NAM (Nicotinamide, sirtuins inhibitor, 5 mM) for 3 days. The percentage of IgG1 B cells was analyzed by flow cytometry with four biological replicates (n = 4 biological replicates, p < 0.001) (e). The protein level of AID and H3K27Ac was quantified by western blotting (n = 3 biological replicates, * p < 0.05, ** p < 0.01, *** p < 0.001) (f). g Quantitative proteomic analysis of histone extracts from A20 cells cultured with U-¹³C₃-pyruvate for 0, 6, 12 and 24 h with triplicate (n = 3 biological replicates). h, i Naive B cells isolated from Mb1^Cre (h) and Mct1^f/fMb1^Cre (i) mice were cultured with Pyr or MePyr containing LPS and IL-4 for 3 days (n = 3 biological replicates, p < 0.001). The protein quantification analysis of H3K27Ac was determined by western blottingting. Data are presented as mean ± SD, unpaired two-tailed t-test (a–d, f, h, i), or two-way ANOVA followed by Sidak’s multiple-comparisons test (e), *p < 0.05, **p < 0.01, ***p < 0.001, n.s not significant. Source data are provided as a Source Data file.

Fig. 6. Mct1 deficiency in B cells suppresses IgG autoantibody production and GC formation in bm12-induced murine lupus model.

a MCT1 expression in a cohort of African-American females (9 subjects with SLE and 12 healthy controls, from GEO accession: GSE118254). Activated Naive B cells (aN), Switched Memory B cells (SM), Resting Naive B cells (rN), Double negative B cells (DN), Transitional 3 B cells (T3), Antigen Secreting Cell (ASC) (Pbpc). b MCT1 expression in B cells of 21 SLE patients and 18 healthy controls, from data of GSE10325 and GSE4588. c qPCR analysis of MCT1 expression in healthy control (n = 19, n indicates different donors) and SLE samples (n = 24, n indicates different donors), p < 0.0001. d–h The schematic diagram of bm12-induced SLE model, n = 6 biological replicates (d). The spleen size of Mb1^Cre and Mct1^f/fMb1^Cre mice was calculated in bm12-induced SLE model (e p = 0.0105). The percentage of GCBC (f p = 0.0067) and IgG1 B cells (g p = 0.0046) were analyzed in the bm12-induced SLE model. Anti-dsDNA IgG in Mb1^Cre and Mct1^f/fMb1^Cre mice serum was analyzed by ELISA (h), p = 0.0059(left), p = 0.0103(right). i–k H&E staining of kidney of Mb1^Cre and Mct1^f/fMb1^Cre mice. Black arrows indicated the inflammatory cell. Scale Bar, 25 μm (i). Immunofluorescence of IgG in the glomerulus of Mb1^Cre and Mct1^f/fMb1^Cre mice. White arrows indicated the glomerulus. Scale Bar, 100 μm (k). l The schematic diagram of CHC treatment procedure in SLE model. m The titer of anti-dsDNA IgG was determined by ELISA in WT or CHC-treated mice (n = 4 biological replicates, p = 0.0467). d and k were adapted from Biorender.Statistics (e–k, m) are representative one of three independent experiments. Data are presented as mean ± SEM, unpaired two-tailed t-test (a, c, e, f, g, h, m), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. not significant.