Probiotic Bifidobacterium breve induces IL-10-producing Tr1 cells in the colon

Abstract

Specific intestinal microbiota has been shown to induce Foxp3(+) regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103(+) dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103(+) DCs from Il10(-/-), Tlr2(-/-), and Myd88(-/-) mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103(+) DCs failed to induce IL-10 production from co-cultured Il27ra(-/-) T cells. B. breve treatment of Tlr2(-/-) mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103(+) DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4(+) T cells from wild-type mice, but not Il10(-/-) mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Figure 1. Induction of IL-10-producing CD4⁺ T cells by B. breve in the colonic lamina propria.

6-week-old C57BL/6 mice were fed L. casei or B. breve 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 L. casei (A) or B. breve (C) were shown. *P<0.05. (B, D) Representative FACS dot plots showing production of IL-10 and IFN-γ gated on colonic CD4⁺ T cells in the indicated mice.

Figure 2. Induction of Foxp3⁻ IL-10-producing CD4⁺ T cells by B. breve.

6-week-old BALB/c mice were administered with B. breve 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 (A), and CD4⁺ Foxp3⁺ T cells (B) are shown. *P<0.02. Representative FACS dot plots for IL-10⁺ and IL-17⁺ T cells (C), and histogram for Foxp3⁺ T cells (D) gated on CD4⁺ T cells are shown. (E) Foxp3 ^GFP mice were fed with B. breve for 4 weeks. CD4⁺ T cells in the colonic lamina propria were analyzed for expression of GFP and IL-10. (F) 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.

Figure 3. Intestinal DCs mediate B. breve-dependent Tr1 cell development.

CD11c⁺ DCs (5×10⁴) were isolated from the colonic lamina propria, and cultured with B. breve, L. casei, B. adolescentis, B. longum, or B. bifidum (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. (A) 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) 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. (C) T cells were collected, and then stained for CD4 and Foxp3. Foxp3 expression in CD4⁺ cells is shown. (D) 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 cMaf, Il21, and Ahr 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.

Figure 4. CD103⁺ DCs, but not CX3CR1⁺ DCs, induce B. breve-dependent Tr1 cell differentiation.

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 B. breve for 24 h. After washing, splenic naïve CD4⁺ T cells were co-cultured with B. breve-treated CD103⁺ DCs or CX₃CR1⁺ DCs in the presence of anti-CD3 mAb for 4 days. (A) T cells were then harvested and re-stimulated for 24 h to analyze IL-10 production by ELISA. *P<0.05. (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. (C) C57BL/6J mice (n = 5) were fed with B. breve 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.

Figure 5. IL-10/IL-27-dependent Tr1 cell development by B. breve-treated DCs.

(A) CD103⁺ DCs were isolated from the colonic lamina propria, and incubated with B. breve for 4 h. Total RNA was extracted and analyzed for mRNA expression of Il27p28, Ebi3, and Il10 by quantitative real-time RT-PCR. N.D, not detected. **P<0.01, ***P<0.001. (B) Naïve T cells were co-cultured with B. breve-treated CD103⁺ DC in the presence of the indicated neutralizing antibody for 4 days. T cells were then harvested and re-stimulated with anti-CD3 and CD28 mAbs for 24 h. IL-10 concentrations in the supernatants were measured by ELISA. *P<0.05, **P<0.01, ***P<0.001, N.S, not significant. (C) CD103⁺ DCs were isolated from the colonic lamina propria of wild-type and Il10 ^−/− mice (C57BL/6 background) and incubated with B. breve. Naïve CD4⁺ T cells from wild-type C57BL/6 mice were then co-cultured with B. breve-treated DCs. T cell production of IL-10 was analyzed by ELISA. N.D, not detected. (D) CD103⁺ DCs were isolated from the colonic lamina propria of wild-type BALB/c mice and incubated with B. breve. Naïve CD4⁺ T cells from wild-type and Il27ra ^−/− mice (BALB/c background) were then co-cultured with B. breve-treated DCs. T cell production of IL-10 was measured by ELISA. Data are representative of three independent experiments and show mean values ± S.D. of triplicate determinations. *P<0.05.

Figure 6. B. breve induces Tr1 cells in a TLR2/MyD88-dependent manner.

(A, C) CD103⁺ DCs were isolated from the colonic lamina propria of wild-type, Myd88 ^−/− (A) and Tlr2 ^−/− (C) mice, incubated with B. breve for 4 h, and then analyzed for mRNA expression of Il27p28, Ebi3, and Il10. *P<0.05, **P<0.01. (B, D) Wild-type, Myd88 ^−/− (B) and Tlr2 ^−/− (D) CD103⁺ DCs were incubated with B. breve 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. (E) 6-week-old wild-type and Tlr2 ^−/− mice (BALB/c background) were fed with B. breve 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. (F) Representative FACS plots of IL-10- and IFN-γ-producing CD4⁺ T cells were shown.

Figure 7. IL-10-dependent amelioration of intestinal inflammation by B. breve.

(A, C) 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 Il10 ^−/− mice (BALB/c background) (B). The mice were orally administered daily with B. breve 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. (C) Production of IL-10, IL-17 and IFN-γ from the colon of wild-type T cell-transferred SCID mice daily administered with B. breve or placebo (n = 5 per group). *P<0.0064, **P<0.0005. (D) Hematoxylin and eosin staining of colon sections at 4 weeks after the transfer. Original magnification, ×400. (E) 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.