Figure 1.
Oral treatment of B. longum JCM 1222T (B.l) alleviates DSS-induced acute colitis.
Mice were monitored daily for weight loss (a) and DAI (b). On day 5, the entire colon was removed (c), and the length was measured (d). The data are representative of four experiments. Colonic tissue sections were stained with hematoxylin-eosin for histological examination (e). The data are representative of two experiments. Scale bars represent 100 μm. The data are Results are expressed as means ± standard error (n = 4). **p<0.01 versus control mice (Control); #p<0.05 and ##p<0.01 versus DSS-treated mice (DSS).
Figure 2.
Oral treatment of B. longum JCM 1222T (B.l) suppresses Th17-specific cytokines and transcription factors.
(a) Cytokine production from colonic tissue culture was measured by ELISA. (b) mRNA expression of transcription factors in LPL was analyzed by quantitative PCR. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to control mice. (c) LPL was analyzed for cytokine-expression profiles by intracellular cytokine staining. The frequency of CD4+ T cells expressing the indicated cytokines is shown (n = 3-6). The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). *p<0.05 and **p<0.01 versus control mice (Control); #p<0.05 versus DSS-treated mice (DSS).
Figure 3.
IECs from DSS mice, but not B. longum JCM 1222T (B.l) -treated mice, induces IL-17A response.
IECs were co-cultured with pan T cells (a), CD4+ T cells (b), and CD8+ T cells (c) from control mice in the presence of anti-CD3 antibody. The cytokine concentration in the supernatant was measured by ELISA. The data are representative of four experiments. Results are expressed as means ± standard error (n = 4). **p<0.01 versus co-culture of IECs from control mice and T cells (Control-IEC/T); ##p<0.01 versus co-culture of IECs from DSS-treated mice and T cells (DSS-IEC/T). (d) IECs from DSS-treated mice were co-cultured with CD4+ T cells in a 96-well plate or Transwell plate. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). **p<0.01 versus CD4+ T cells alone (CD4+T); ##p<0.01 versus co-culture of IECs from DSS-treated mice and CD4+ T cells in a 96-well plate (DSS-IEC/CD4+T).
Figure 4.
Oral treatment of B. longum JCM 1222T (B.l) suppresses the expression of costimulatory molecules in IEC.
(a) mRNA expression of costimulatory molecules in IECs was analyzed by quantitative PCR. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to control mice. The data are representative of three experiments. Results are expressed as means ± standard error (n = 6 (Control) or 7 (DSS, and DSS+B.l)). (b) IECs were stained for cytokeratin and CD80 or CD40 and analyzed by flow cytometry. Debris was gated out by forward and side scatter. Representative plots (upper panel) and the mean and standard error values of the percentage of cytokeratin/CD80 or CD40 positive cells (lower panel) are shown (n = 4). The data are representative of two experiments. (c) Cryosections of colonic tissue were labeled for CD40 (green) and E-cadherin (red). The data are representative of two experiments. Scale bars represent 50 μm. **p<0.01 versus control mice (Control); #p<0.05 and ##p<0.01 versus DSS-treated mice (DSS).
Figure 5.
IECs from DSS mice induce IL-17A response via CD80/CD86 and CD40 dependent costimulation.
(a) IECs from DSS mice were co-cultured with CD4+ T cells from control mice in the presence of anti-CD3 antibody. In the indicated groups, IECs were pretreated with CD80 or CD86 blocking antibodies, and CD4+ T cells were pretreated with CD40L blocking antibody before co-culturing. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). **p<0.01 versus CD4+ T cells alone (CD4+T); ##p<0.01 versus co-culture of CD4+ T cells and IECs from DSS-treated mice (DSS-IEC). (b) IECs were cultured in the absence (gray bars) and presence (black bars) of CD40 agonist antibody for 24 h. IL-6 concentration in the supernatant was measured by ELISA. The data are representative of three experiments. (c) CD4+ T cells were cultured for 3 days in fresh medium or supernatant from IECs (IECs sup) cultured as described in (b) in the presence of anti-CD3/CD28 antibodies. The IL-17A concentration in the supernatant was measured by ELISA. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). **p<0.01 versus the supernatant from IECs stimulated with CD40 agonist antibody (CD40 agonist Ab). (d) CD4+ T cells were cultured with fresh medium or supernatant from CD40-stumulated IEC of DSS mice (DSS-IEC sup) as described in (c) in the presence of IL-6-neutralizing antibody (anti-IL-6) or IgG control antibody (isotype). The IL-17A concentration in the supernatant was measured by ELISA. Results are expressed as means ± standard error (n = 6). **p<0.01 versus fresh medium; #p<0.05 versus the supernatant from CD40-stumulated IEC of DSS mice (DSS-IEC sup).
Figure 6.
B. longum JCM 1222T (B.l) suppresses the expression of costimulatory molecules in Colon-26 cells.
(a) mRNA expression of CD80 and CD40 was analyzed by quantitative real-time PCR in Colon-26 cells after stimulation with IFN-γ. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to before stimulation (0 h). Results are expressed as means ± standard error (n = 4). *p<0.05 and **p<0.01 versus before stimulation (0 h). (b) Colon-26 cells were pre-incubated with B. longum JCM 1222T, and then treated with penicillin and streptomycin and incubated with IFN-γ. Messenger RNA expression of CD80 and CD40 was analyzed after stimulation with IFN-γ for 6 and 72 h, respectively. The data are representative of three experiments. (c) Colon-26 cells were incubated with B. longum JCM 1222T and IFN-γ. Cell surface protein expression of CD80 and CD40 was analyzed by flow cytometry after stimulation with IFN-γ. Debris was gated out by forward and side scatter. Histograms show a representative experiment (specific antibody stains are shown in filled histograms, and isotype control antibody stains are shown in open histograms) and bar figures are representative of two experiments. **p<0.01 versus non-treated cells (Control); #p<0.05 and ##p<0.01 versus IFN-γ-stimulated cells.