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, 11 (1), 60

B Cell-Intrinsic Epigenetic Modulation of Antibody Responses by Dietary Fiber-Derived Short-Chain Fatty Acids

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B Cell-Intrinsic Epigenetic Modulation of Antibody Responses by Dietary Fiber-Derived Short-Chain Fatty Acids

Helia N Sanchez et al. Nat Commun.

Abstract

Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells, macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR), while decreasing at higher doses over a broad physiological range, AID and Blimp1 expression, CSR, somatic hypermutation and plasma cell differentiation. In human and mouse B cells, butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3'UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors, not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes, these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Dietary fibers and SCFAs dampen CSR, plasma cell differentiation and class-switched antibody responses.
After weaning, C57BL/6 mice were fed a fiber diet (regular chow) or no-fiber diet. Two weeks later, these mice were started on SCFAs water (SCFAs) or plain water (Nil). All mice were then administered OVA together with CT via intragastric gavage once a week for 4 weeks starting at 8 weeks of age, and killed a week after the last OVA and CT administration. a Total and OVA-binding IgM, IgG1, IgA, and IgE concentrations in serum of fiber or no-fiber-fed mice on SCFAs water or plain water, as measured by ELISA. Each symbol represents an individual mouse (n = 5 per group, pooled from two experiments). The bars represent the mean ± SD. b OVA-binding IgM, IgG1, IgA, and IgE concentrations in the feces of fiber-fed mice on SCFAs water or plain water, as measured by ELISA. Each symbol represents an individual mouse (n = 5 per group, pooled from two experiments). c IgM, IgA, and IgD-positive cells in the lamina propria, PPs, and MLNs of fiber or no-fiber-fed mice on SCFAs water or plain water as visualized by fluorescence microscopy. d ELISPOT analysis of IgM, IgG1, and IgA AFCs in MLNs, PPs, spleen, and bone marrow of fiber-fed mice on SCFAs water or plain water. e Proportion of surface CD19+IgG1+ and CD19+IgA+ B cells, germinal center B cells (CD19+GL-7+), and plasmablasts/plasma cells (CD138+) in MLNs, PPs, and the spleen of fiber-fed mice given SCFAs water or plain water, as analyzed by flow cytometry. Data in ce are representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ns: not significant (unpaired t test). Scale bar = 100 μm. The source data are provided in Source Data file.
Fig. 2
Fig. 2. Dietary fibers and SCFAs alter AID and Blimp1 expression, bacteria-bound/binding antibody responses, and gut bacteria composition.
C57BL/6 mice fed a fiber or no-fiber diet were on SCFAs water (SCFAs) or plain water (nil) and administered OVA together with CT (same as in Fig. 1). a, b AID and Blimp1 expression in spleen B cells of these mice, AID expression in spleen B cells of Aicda-/- mice, and Blimp1 expression in naive B cells of C57BL/6 mice, as analyzed by intracellular staining followed by fluorescence microscopy (a) and flow cytometry (b). c Quantification of AID+ and Blimp1+ cells in a and b. n = 3 per group. d Bacteria-bound IgM, IgD, IgG1, and IgA in the feces of fiber diet-fed C57BL/6 mice on SCFAs water or plain water, collected 1 week after the last OVA and CT administration, as well as bacteria-bound IgA in the feces of unimmunized Aicda–/– mice fed fiber diet and plain water, as analyzed by flow cytometry. e Bacteria-bound IgA in feces of fiber and no-fiber diet-fed mice on SCFAs water or plain water as analyzed by flow cytometry. f Concentration of bacteria-binding IgM and IgA in the serum as measured by ELISA; each symbol represents an individual mouse (n = 5 per group, pooled from two experiments). The bars represent the mean ± SD. g Relative abundance of gut bacteria classes (> 1% in any sample group; fiber, no-fiber, and no-fiber + SCFAs) in the mice (n = 4 per group, pooled from two experiments) as determined by high-throughput 16s rRNA gene miSeq sequencing. h Principal component analysis of gut bacterial composition in these three groups of mice on different diets. Data in a, b, d, and e are representative of three independent experiments. *p< 0.05, ***p< 0.001 values, ns: not significant (unpaired t test). Scale bar = 100 μm. The source data are provided in Source Data file.
Fig. 3
Fig. 3. SCFAs reduce class-switched and hypermutated T-dependent and T-independent antibody responses.
ad C57BL/6 mice fed fiber diet on SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks, and i.p. injected with NP16-CGG (in Alum) or NP-LPS (in PBS) at the age of 8 weeks. The mice were boost-injected 3 weeks later with NP16-CGG or NP-LPS, respectively, and killed a week after the boost injection. a, b Titers of total and NP3-binding serum IgM, IgG1, and IgA in NP16-CGG injected C57BL/6 mice (a), as well as total and NP3-binding serum IgM, IgG3, and IgG2b titers in NP-LPS injected C57BL/6 mice (b), as measured by ELISA (n = 4 per group, pooled from two experiments). Bars represent the mean ± SD. c, d Somatic point mutations in the V186.2 regions from V186.2DJH-Cγ1 transcripts of NP16-CGG injected C57BL/6 mice (c), or from V186.2DJH-Cγ3 transcripts of the NP-LPS injected C57BL/6 mice (d). Sequence data were pooled from three mice in each group. Pie charts depict the proportions of sequences that carry different numbers of point mutations; listed below the pie charts is the overall mutation frequency (change/base). Donut charts depict the spectrum of point mutations. eh Tcrβ–/–Tcrδ–/– mice on SCFAs water or plain water were injected with NP-LPS. Total and NP3-specific serum IgM, IgG3, and IgG2b titers were measured by ELISA (n= 5 per group) (e). Somatic point mutations in the V186.2 region of V186.2DJH-Cγ3 and V186.2DJH-Cγ2b transcripts. Sequence data were pooled from three mice in each group (f). AID and Blimp1 expression in spleen B cells of these mice, AID expression in spleen B cells of Aicda-/- mice, and Blimp1 expression in naive B cells of C57BL/6 mice, as analyzed by intracellular staining followed by fluorescence microscopy (g), and flow cytometry (h). Data in g and h are representative of three independent experiments. i Quantification of AID+ and Blimp1+ cells in h and g. n = 3 per group. *p< 0.05, **p< 0.01, ***p< 0.001 (unpaired t test). Scale bar = 10 μm. The source data are provided in Source Data file.
Fig. 4
Fig. 4. Dose-dependent and B-cell-intrinsic modulation of Aicda and Prdm1 expression, CSR, SHM and plasma cell differentiation by SCFAs in vivo.
ac C57BL/6 mice on plain water (Nil), SCFAs “lower dose” water (pH = 7.4, 20 mM sodium butyrate, and 30 mM sodium propionate) or SCFAs “higher dose” water (pH = 7.4, 20 mg ml−1 tributyrin emulsion, 140 mM sodium butyrate and 150 mM sodium propionate, SCFAs higher dose. The SCFAs water is referred here as SCFAs “higher dose” as opposed to the “lower dose” used in these experiments for the sake of clarify) were i.p. injected with NP-LPS. a Total and NP3-binding IgM, IgG2b, and IgG3 induced by low dose of SCFAs, as measured by ELISA. Each symbol represents an individual mouse (n = 4–5 per group, pooled from two experiments). Bars represent mean ± SD. b AID and Blimp1 expression in spleen B cells from the same mice, as analyzed by intracellular staining followed flow cytometry. c IgG3+, IgG2b+ B cells, and CD19lowCD138+ plasmablasts/plasma cells in the spleen, as analyzed by flow cytometry. Data in b and c are representative of three independent experiments. di NSG/B mice on plain water or SCFAs water (pH = 7.4, 20 mg ml−1 tributyrin emulsion, 140 mM sodium butyrate, and 150 mM sodium propionate) were injected with NP-LPS. d Titers of total and NP3-binding serum IgM, IgG3, IgG2b, and IgA, as analyzed by ELISA. Each symbol represents an individual mouse (n = 5 per group from one experiments). e NP3-binding IgM, IgG3, IgG2b, and IgA AFCs in spleen as analyzed by ELISPOT. f Viable (7-AAD) spleen CD19+ B cells as analyzed by flow cytometry. g Aicda, Prdm1, germline IH-CH, and post-recombination Iμ-CH transcripts, as analyzed by qRT-PCR and normalized to Gapdh. Data are ratios to B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Proportion of IgG3+ and IgG2b+ B cells, and CD138+ plasmablasts/plasma cells in the spleen, as analyzed by flow cytometry. i Somatic point mutations in the VH region of VHDJH-Cγ2b transcripts. Mutations are in pooled sequences from two out of five mice per group. Pie charts depict the proportions of sequences that carry different numbers of point mutations; listed below the pie charts is the overall mutation frequency. Donut charts depict the spectrum of point mutations. Histograms depict frequencies of silent and replacement mutations in FR and CDR regions. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (unpaired t test). The source data are provided in Source Data file.
Fig. 5
Fig. 5. SCFAs inhibit Aicda and Prdm1 expression, and reduce CSR and plasma cell differentiation in mouse B cells.
af Mouse B cells were stimulated in the presence of Nil, butyrate (500 μM, But), propionate (2000 μM, Prop), or butyrate (500 μM) plus propionate (2000 μM). Proportions of surface CD19+ IgG1+ B cells and CD138+ plasmablasts/plasma cells (a, b), surface CD19+ IgA+ B cells (c, d), intracellular CD19+ IgE+ B cells (e), and surface CD19+ IgM IgD+ B cells (f) as analyzed by flow cytometry 96 h after stimulation. g, h CD19+ B cells were labeled with CFSE and stimulated for 96 h with LPS plus IL-4 in the presence of nil, butyrate (500 μM), or propionate (2000 μM). CFSE intensity and surface IgG1 expression as analyzed by flow cytometry. Progressive left shift of fluorescence intensity indicates CD19+ B-cell division. Data are representative of three independent experiments yielding comparable results (g). Proportion of surface IgG1+ B cells at each cell division. Data are mean and SE from three independent experiments (h). i Titers of IgG1 and IgA in culture fluids as analyzed by ELISA. Data are from three independent experiments (mean and SE). j Aicda, Prdm1, Irf4, Xbp1, Sdc1, post-recombination Iμ-Cγ1, Iμ-Cε, and Iμ-Cα transcripts as well as germline Iγ1-Cγ1, Iε-Cε, and Iα-Cα transcripts in B cells stimulated with LPS plus IL-4, or CD154 plus IL-4, IL-5, TGF-β, anti-δ mAb-dextran in the presence of butyrate, propionate, or butyrate plus propionate for 60 h, as analyzed by qRT-PCR and normalized to Gapdh transcripts. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). k, l B cells were stimulated with LPS plus IL-4 in the presence of nil, butyrate, propionate, or butyrate plus propionate for 72 h. The B-cell expression of AID and Blimp1 as determined by intracellular staining and flow cytometry (k). AID, Blimp1, and β-Actin proteins, as detected by immunoblotting (l). Data are one representative of three independent experiments yielding comparable results. *p< 0.05, **p< 0.01, ***p< 0.001 (unpaired t test). The source data are provided in Source Data file.
Fig. 6
Fig. 6. Butyrate and propionate inhibit AICDA and PRDM1 expression, and reduce CSR and plasma cell differentiation in human B cells.
a Human B cells purified from PBMCs of healthy subjects were stimulated with CD154 plus IL-4 and IL-21, in the presence of nil, butyrate (250, 500, or 1000 μM), propionate (2000 or 4000 μM), or butyrate (500 μM) plus propionate (2000 μM). The proportions of CD19+ IgA+ and CD19+ IgG+ as well as viable (7-AAD) CD19+ B cells were analyzed 120 h post stimulation by flow cytometry. Data are representative of three independent experiments yielding comparable results. bd Human B cells were stimulated with CD154 plus IL-4 and IL-21, in the presence of nil, butyrate (500 μM), propionate (2000 μM), or butyrate (500 μM) plus propionate (2000 μM). CD19loCD138+ plasma cells were analyzed 120 h post stimulation by flow cytometry (b). IgG and IgA titers in culture fluids of these B cells were analyzed 120 h post stimulation by ELISA. Data are from three independent experiments (mean and SE) (c). Expression of AICDA and PRDM1, germline Iγ1-Cγ1, Iα-Cα, and Iε-Cε, as well as mature VHDJH-Cγ1, VHDJH-Cε, and VHDJH-Cα transcripts were analyzed 72 h post stimulation by qRT-PCR and normalized to the expression of HRPT. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments) (d). e Human B cells were labeled with CFSE and stimulated with CD154 plus IL-4 and IL-21 in the presence of nil, butyrate (500 μM), or propionate (2000 μM) for 96 h. CFSE intensity were analyzed by flow cytometry. Progressive left shift of fluorescence intensity indicates CD19+ B-cell division. Data are representative of three independent experiments yielding comparable results. **p< 0.01, ***p< 0.001 (unpaired t test). The source data are provided in Source Data file.
Fig. 7
Fig. 7. SCFAs inhibit AICDA/Aicda and PRDM1/Prdm1 expression, CSR, and plasma cell differentiation through their HDAC inhibitory activity.
a, b Human and mouse B cells were stimulated with CD154 plus IL-4 and IL-21, or CD154 plus IL-4, respectively, in the presence of nil, butyrate (500 μM), propionate (2000 μM), butyrate (500 μM) plus propionate (2000 μM) alone, or butyrate (2000 μM) plus propionate (2000 μM) together with increasing doses of GPR43 inhibitor GLPG0974. Human B cell AICDA and PRDM1 transcripts as well as mouse B cell Aicda and Prdm1 transcripts were analyzed 72 h post stimulation by qRT-PCR and normalized to HRPT and Gapdh expression, respectively. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments) (a). Proportion of CD19+IgG+ B cells were analyzed 120 h post stimulation by flow cytometry. Data are representative of three independent experiments yielding comparable results (b). cf Mouse B cells were stimulated with LPS plus IL-4, in the presence of increasing doses of butyrate, propionate, palmitate, and SAHA. Expression of Aicda and Prdm1 transcripts were analyzed 72 h post stimulation by qRT-PCR and normalized to Gapdh expression. Data are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments) (c). The proportion of CD19+ IgG1+ B cells and CD138+ plasmablasts/plasma cells were analyzed 120 h post stimulation by flow cytometry. Data are representative of three independent experiments yielding comparable results (df). *p< 0.05, **p< 0.01, ***p< 0.001 (unpaired t test). The source data are provided in Source Data file.
Fig. 8
Fig. 8. SCFAs increase expression of Aicda- and Prdm1-targeting miRNAs by enhancing histone acetylation of related miRNA host genes.
a Primary mouse B cells, CH12F3 B cells, and human B cells were stimulated with indicated stimuli for 60 h in the presence of nil, butyrate (500 μM), or propionate (2000 μM). Ex vivo B cells were isolated from mice injected with NP-LPS and on plain water or SCFA water for 21 days. Acetylated-histone H3 (H3K9ac), histone H3, and β-actin proteins as detected by immunoblotting. Data are one representative of three independent experiments. b Relative densities of the Ac-histone H3 bands normalized to histone H3 and β-actin levels. c, d B cells stimulated with LPS plus IL-4 in the presence of nil or butyrate for 60 h. Expression of the Aicda- and Prdm1-targeting miRNAs, and irrelevant miRNAs as analyzed by qRT-PCR (c). Relative abundance of H3K9ac/K14ac in miRNA host genes (HGs) as analyzed by ChIP-qPCR (d). e Schematic diagram of the luciferase reporter constructs-containing 3′UTRs of Aicda and Prdm1 mRNAs and their mutant (mut) counterparts. f Surface CD19 and IgA on CH12F3 B cells stimulated for 96 h in the presence of nil, butyrate (500 μM), propionate (2000 μM), or VPA (500 μM) as analyzed by flow cytometry. Data are one representative of 3 independent experiments yielding comparable results. g, miRNA expression in CH12F3 cells stimulated for 24 h in the presence of nil or butyrate, as analyzed by qRT-PCR. Data in c, d, and g are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Luciferase activity in CH12F3 cells transfected with luciferase reporter vectors-containing wild-type or mutated Aicda or Prdm1 3′UTRs after 24 h treatment with nil or butyrate. Luciferase activity was measured 6 h after transfection, and normalized relative to luciferase activity in B cells cultured with nil. Data in h are ratios to transfected B cells cultured with nil (set as 100%; means ± SEM of three independent experiments). *p< 0.05, **p< 0.01, ns: not significant (unpaired t test). The source data are provided in Source Data file.
Fig. 9
Fig. 9. Butyrate downregulates AICDA and PRDM1 expression while upregulating AICDA- and PRDM1-targeting miRNAs in human B cells.
Purified human B cells were stimulated with CD154 plus IL-4 and IL-21 in the presence of nil or butyrate (500 μM) for 60 h before preparation of RNA for mRNA-Seq and miRNA-Seq. a Profile of RNA-Seq reads at the AICDA and PRDM1 loci. b Scatter plots of miRNA expression as defined by miRNA-Seq (RPKs) in B cells treated with butyrate versus that in B cells treated with nil. c Bar graphs depict the fold changes in the average miRNA expression (RPMs) comparing B cells treated with nil or butyrate. d B-cell expression of AICDA- or PRDM1-targeting miRNAs, as analyzed by miRNA-Seq.
Fig. 10
Fig. 10. SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice.
Female MRL/Faslpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, anti-histone, and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse (n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/Faslp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c, d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy (c) and flow cytometry (d). e Spleen surface CD138+ plasmablasts/plasma cells, and intracellular CD138+ IgG1+ and CD138+ IgG2a+ plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H&E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ns: not significant (unpaired t test). Scale bar = 50 μm (b), 20 μm (c), and 100 μm (f). The source data are provided in Source Data file.

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