Induction of IL-10-producing CD4⁺ T cells by <i>B. breve</i> in the colonic lamina propria.

<p>6-week-old C57BL/6 mice were fed <i>L. casei</i> or <i>B. breve</i> or placebo daily (each, 1×10⁹) by oral gavage for 3 months (n = 8). Intestinal lamina propria lymphocytes were analyzed for cytokine production by flow cytometry. Percentages of IL-10-, IL-17-, and IFN-γ-producing CD4⁺ T cells of mice administered with <i>L. casei</i> (<b>A</b>) or <i>B. breve</i> (<b>C</b>) were shown. *P<0.05. (<b>B</b>, <b>D</b>) Representative FACS dot plots showing production of IL-10 and IFN-γ gated on colonic CD4⁺ T cells in the indicated mice.</p

CD103⁺ DCs, but not CX3CR1⁺ DCs, induce <i>B. breve</i>-dependent Tr1 cell differentiation.

<p>CD103⁺ CX₃CR1⁻ CD11b⁻ CD11c⁺ DCs (CD103⁺ DCs) and CX₃CR1⁺ CD11b⁺ CD11c⁺ DCs (CX₃CR1⁺ DCs) were isolated from the colonic lamina propria, and treated with the same numbers of <i>B. breve</i> for 24 h. After washing, splenic naïve CD4⁺ T cells were co-cultured with <i>B. breve</i>-treated CD103⁺ DCs or CX₃CR1⁺ DCs in the presence of anti-CD3 mAb for 4 days. (<b>A</b>) T cells were then harvested and re-stimulated for 24 h to analyze IL-10 production by ELISA. *P<0.05. (<b>B</b>) T cells were collected, and re-stimulated with PMA and ionophore for 8 h. Intracellular expression of Foxp3 and IL-10 was then analyzed by flow cytometry. (<b>C</b>) C57BL/6J mice (n = 5) were fed with <i>B. breve</i> for 3 weeks. Then, CD103⁺ DCs were isolated from MLN and the colonic lamina propria, and co-cultured with splenic naïve CD4⁺ T cells. The co-cultured T cells were re-stimulated and IL-10 concentration in the supernatants was analyzed by ELISA. Data are representative of four independent experiments and show mean values ± S.D. of triplicate determinations. *P<0.05, **P<0.01.</p

Intestinal DCs mediate <i>B. breve</i>-dependent Tr1 cell development.

<p>CD11c⁺ DCs (5×10⁴) were isolated from the colonic lamina propria, and cultured with <i>B. breve</i>, <i>L. casei, B. adolescentis, B. longum</i>, or <i>B. bifidum</i> (5×10⁴) for 24 h. After washing, DCs were co-cultured with splenic naïve CD4⁺ T cells (5×10⁴) in the presence of soluble anti-CD3 mAb for 4 days. (<b>A</b>) T cells were harvested and re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for 24 h. IL-10 concentrations in the culture supernatants were analyzed by ELISA. *P<0.001. (<b>B</b>) T cells were harvested and re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for 24 h. IL-10 concentrations in the culture supernatants were analyzed by ELISA. *P<0.001. (<b>C</b>) T cells were collected, and then stained for CD4 and Foxp3. Foxp3 expression in CD4⁺ cells is shown. (<b>D</b>) T cells were harvested, and stimulated with anti-CD3 and anti-CD28 mAbs for 4 h. Total RNA was then extracted to analyze expression of <i>cMaf</i>, <i>Il21</i>, and <i>Ahr</i> by quantitative real-time RT-PCR. Data are representative of five independent experiments and show mean values ± S.D. of triplicate determinations. *P<0.05, **P<0.01.</p

Induction of Foxp3⁻ IL-10-producing CD4⁺ T cells by <i>B. breve</i>.

<p>6-week-old BALB/c mice were administered with <i>B. breve</i> or placebo orally for 1–4 weeks (n = 5). At the indicated time point, mice were sacrificed and CD4⁺ T cells in the colonic lamina propria were analyzed by flow cytometry. Percentages of CD4⁺ IL-10⁺ T cells (<b>A</b>), and CD4⁺ Foxp3⁺ T cells (<b>B</b>) are shown. *P<0.02. Representative FACS dot plots for IL-10⁺ and IL-17⁺ T cells (<b>C</b>), and histogram for Foxp3⁺ T cells (<b>D</b>) gated on CD4⁺ T cells are shown. (<b>E</b>) <i>Foxp3</i>^GFP mice were fed with <i>B. breve</i> for 4 weeks. CD4⁺ T cells in the colonic lamina propria were analyzed for expression of GFP and IL-10. (<b>F</b>) Percentages of IL-10⁺ T cells in Foxp3⁺ or Foxp3⁻ CD4⁺ T cells (n = 5). Data are representative of two independent experiments; means ± S.D. *P<0.02.</p

IL-10-dependent amelioration of intestinal inflammation by <i>B. breve</i>.

<p>(<b>A, C</b>) 6 week-old SCID mice (n = 8 per group) were intraperitoneally injected with PBS or 3×10⁵ of naïve CD4⁺ T cells from wild-type BALB/c mice (A) or <i>Il10</i>^−/− mice (BALB/c background) (<b>B</b>). The mice were orally administered daily with <i>B. breve</i> from 1 week before the T cell transfer to the end of experiment. Changes in body weight were monitored daily and presented relative to initial body weight. *P<0.05, Error bars, S.E.M. (<b>C</b>) Production of IL-10, IL-17 and IFN-γ from the colon of wild-type T cell-transferred SCID mice daily administered with <i>B. breve</i> or placebo (n = 5 per group). *P<0.0064, **P<0.0005. (<b>D</b>) Hematoxylin and eosin staining of colon sections at 4 weeks after the transfer. Original magnification, ×400. (<b>E</b>) Clinical scores for colitis were shown in the indicated group. Data are representative of two independent experiments. *P<0.05, **P<0.01. N.S, not significant.</p

<i>B. breve</i> induces Tr1 cells in a TLR2/MyD88-dependent manner.

<p>(<b>A, C</b>) CD103⁺ DCs were isolated from the colonic lamina propria of wild-type, <i>Myd88</i>^−/− (A) and <i>Tlr2</i>^−/− (C) mice, incubated with <i>B. breve</i> for 4 h, and then analyzed for mRNA expression of <i>Il27p28</i>, <i>Ebi3</i>, and <i>Il10</i>. *P<0.05, **P<0.01. (<b>B, D</b>) Wild-type, <i>Myd88</i>^−/− (B) and <i>Tlr2</i>^−/− (D) CD103⁺ DCs were incubated with <i>B. breve</i> for 24 h, and then co-cultured with naïve CD4⁺ T cells from wild-type mice for 4 days. T cells were harvested and re-stimulated for 24 h. IL-10 production in the supernatants was analyzed by ELISA. Data are representative of three independent experiments and show mean values ± S.D. of triplicate determinations. *P<0.05. N.D, not detected. (<b>E</b>) 6-week-old wild-type and <i>Tlr2</i>^−/− mice (BALB/c background) were fed with <i>B. breve</i> or placebo for 3 weeks (n = 5). Then, the mice were sacrificed and colonic lamina propria lymphocytes were analyzed for IL-10 production by flow cytometry. The percentage of IL-10⁺ cells gated on CD4⁺ T cells is shown in the indicated mice. Data are representative of three independent experiments and show mean values ± S.D. of triplicate determinations. *P<0.05. (<b>F</b>) Representative FACS plots of IL-10- and IFN-γ-producing CD4⁺ T cells were shown.</p