Table 1.
Gene targets, forward and reverse RT-PCR primer sequences, and product lengths.
Fig 1.
Butyrate promotes the differentiation of CD138high PCs in isolated murine B cells.
(A) Flow cytometric gating strategy and representative histogram plots of CD138 expression after treatment of isolated murine splenic B cells with increasing concentrations of PA and/or BA after 4 days of cell culture. (B) Frequencies of CD138high PCs after treatment of isolated murine B cells with the indicated concentrations of PA and/or BA after 4 days of cell culture (n = 5 per group). (C) Gene expression analysis of PC transcription factors in isolated murine splenic B cells after one day of cell culture in the presence of 0.5 mM BA (n = 3). * p < 0.050, ** p < 0.010, *** p < 0.001.
Fig 2.
Butyrate promotes the expression of IL-10 in isolated murine B cells.
(A, B) Gene expression of anti-inflammatory cytokines after 4 days of B cell culture with increasing concentrations of (A) PA (n = 2–5) or (B) BA (n = 3–14). (C, D) Representative histogram plot and frequencies of IL-10 GFP protein expressing cells among all induced CD138high PCs after 4 days of B cell culture with increasing concentrations of PA and/ or BA (n = 5). (E, F) Representative contour plots and frequencies of IL-10+CD138high PCs among all murine B cells after 4 days of B cell culture with increasing concentrations of PA and/or BA (n = 5). (G) Representative histogram plot and frequencies of IgM + cells among IL-10+ and IL-10- CD138high PCs. * p < 0.050, ** p < 0.010, *** p < 0.001.
Fig 3.
Butyrate promotes the differentiation of CD138high PCs and IL-10+ CD138high PCs in Ova-immunized mice.
Wildtype or IL-10 reporter mice (IL-10 GFP or Vert-X) were treated for seven days with BA via drinking water or i.p. injection before i.p. immunization with Ova/CFA. 12 days after Ova/CFA treatment splenic cells were harvested and analyzed by flow cytometry. (A) Flow cytometric gating strategy and representative contour plots for CD138high PCs in control and BA-treated mice. (B+C) CD138high PC frequencies and cell counts for mice treated with either (B) drinking water (ctrl.) or BA (150 mM) (n = 9) or (C) daily i.p. injection of either PBS vehicle control or BA (100 mg/kg) (n = 5). (D) Flow cytometric gating strategy and representative histogram plots for IL-10+ CD138high PCs in BA-treated IL-10 reporter mice. (E, F) Frequencies and cell counts of IL-10+ cells among CD138high PCs for mice treated with either (E) drinking water (ctrl.) or BA (150 mM) (n = 4) or (F) daily i.p. injection of either PBS vehicle control or BA (100 mg/kg) (n = 5). * p < 0.050, ** p < 0.010, *** p < 0.001.
Fig 4.
BA induces IL-10+CD138high PCs that preferentially express IgM in vivo.
Wildtype or IL-10 reporter mice (IL-10 GFP) were treated for seven days with BA via drinking water or i.p. injection, before i.p. immunization with Ova/CFA. 12 days after Ova/CFA application splenic cells were harvested and analyzed by flow cytometry. (A) Flow cytometric gating strategy for gating IL-10 GFP+ or IgM+ PCs in B-I. (B) Representative histogram plot of IgM-expression in IL-10 GFP+ and IL-10 GFP- CD138high PCs after treatment with 150 mM BA in DW. (C+D) IgM+ PC frequencies and IgM MFI of IL-10 GFP+ and IL-10 GFP- CD138high PCs after treatment with (C) 150 mM BA in DW or (D) 100 mg/kg BA i.p. (E) Representative histogram plot of IL-10 GFP-expression in IgM+ and IgM- CD138high PCs after treatment with 150 mM BA in DW. (F+G) IL-10 GFP+ PC frequencies and IL-10 GFP MFI of IgM- and IgM+ CD138high PCs after treatment with (F) 150 mM BA in DW or (G) 100 mg/kg BA i.p. (H+I) Cell counts of IL-10+IgM+ CD138high PCs, IL-10 GFP MFI of IgM+ CD138high PCs, and total serum IgM levels after treatment with (H) 150 mM BA in DW or DW control or (I) 100 mg/kg BA i.p. in PBS or PBS control. OD = optical density. * p < 0.050, ** p < 0.010, *** p < 0.001.
Fig 5.
BA in drinking water increases Ova-specific PCs and serum levels of anti-Ova IgM, but blocks class switching to anti-Ova IgG2b.
Mice were treated for seven days with BA via drinking water or i.p. injection, before i.p. immunization with Ova/CFA. 12 days after Ova/CFA application splenic cells were harvested and analyzed by flow cytometry. (A) Flow cytometric gating strategy and representative histogram plots for staining of Ova-specific PCs in BA-treated mice (drinking water). (B, C) Ova-specific PC frequencies and cell counts of mice treated with either (B) water or BA (150 mM) or (C) PBS vehicle control or BA (100 mg/kg) by i.p. injection. (D-M) Serum levels of anti-Ova IgA (D, I), IgM (E, J), IgG1 (F, K), IgG2b (G, L), and (H, M) IgG2c antibodies for mice treated with either (D-H) water or BA (150 mM) or (I-M) PBS vehicle control or BA (100 mg/kg) by i.p. injection. OD = optical density. * p < 0.050, ** p < 0.010.
Fig 6.
Specific HDAC3 inhibition by RGFP966 promotes the differentiation of CD138high PCs, IL-10+ B cells, and IL-10+ CD138high PCs in isolated murine B cells.
(A) Flow cytometric gating strategy and representative histogram plots of H3K27 acetylation in isolated murine B cells after 3 days of cell culture. (B) Frequencies of H3K27ac+ cells in isolated murine B cells after 3 days of incubation with increasing concentrations of PA and BA (n = 3). Treatment with RGFP966 (10 μM) as a positive control. (C) Frequencies of CD138high PCs after 4 days of incubation of isolated murine B cells with HDAC inhibitor TSA or HDAC3-specific inhibitor RGFP966. (D) Gene expression of anti-inflammatory cytokines after 4 days of cell culture with 10 μM RGFP966 (n = 6). (E) Representative overlay histogram plot and IL-10 GFP protein expression among all CD138high PCs after 4 days of B cell culture with 10 μM RGFP966 (n = 6). (F) Representative contour plots and frequencies of IL-10+ CD138high PCs among all B cells after 4 days of B cell culture with 10 μM RGFP966. TSA = Trichostatin A. * p < 0.050, ** p < 0.010, *** p < 0.001.
Fig 7.
Seahorse analysis of BA effects on mitochondrial metabolism in isolated murine B cells.
(A) OCR measurements of isolated murine B cells in the Mito-Stress assay after one day of incubation with 0.5 mM BA. The following parameters were calculated (n = 3) from the initial measurements of the oxygen consumption rates at baseline and after injection of oligomycin (O), FCCP, and antimycin A/rotenone (AA/Rot): (B) Basal respiration, (C) ATP production, (D) maximal respiration, (E) spare respiratory capacity in %, (F) coupling efficiency in %, and (G) proton leak. * p < 0.050.
Fig 8.
BA decreases mitochondrial membrane potential and superoxide production after one day of cell culture.
(A) Representative contour plots of MT-Green/TMRE-stained B cells after incubation with 0.5 mM BA for 1 day. (B) P1 frequencies after incubation with 0.5 mM BA for 1 day indicating MT-Greenhigh TMREhigh B cells (n = 4). (C) P2 frequencies after incubation with 0.5 mM BA for 1 day indicating MT-Greenlow TMRElow B cells (n = 4). (D) Ratio of TMRE MFI and Mito-Green MFI after incubation with 0.5 mM BA for 1 day indicating MT-Greenlow TMRElow B cells (n = 4). (E) Incubation with 0.5 mM BA for 1 day decreased the levels of mitochondrial superoxide levels indicated by MitoSOX staining (n = 5). (F) Incubation with 10 μM RGFP966 for 1 day decreased the levels of mitochondrial superoxide levels indicated by MitoSOX staining (n = 3). (G) Representative contour plots of CD138 and IL-10 GFP expression in isolated splenic B cells from IL-10 reporter mice after treatment with 0.5 mM BA and/or 100ng/ml PMA. (H) Frequencies of CD138high PCs after treatment of isolated murine B cells with 0.5 mM BA and/or 100ng/ml PMA after 4 days of cell culture (n = 6). (I) Frequencies of IL-10+ B cells after treatment of isolated murine B cells with 0.5 mM BA and/or 100ng/ml PMA after 4 days of cell culture (n = 5). PMA = Phorbol-12-myristate-13-acetate. * p < 0.050, ** p < 0.010, *** p < 0.001.