Lactobacillus gasseri SBT2055 induces TGF-β expression in dendritic cells and activates TLR2 signal to produce IgA in the small intestine.

Probiotic bacteria provide benefits in enhancing host immune responses and protecting against infection. Induction of IgA production by oral administration of probiotic bacteria in the intestine has been considered to be one reason for this beneficial effect, but the mechanisms of the effect are poorly understood. Lactobacillus gasseri SBT2055 (LG2055) is a probiotic bacterium with properties such as bile tolerance, ability to improve the intestinal environment, and it has preventive effects related to abdominal adiposity. In this study, we have found that oral administration of LG2055 induced IgA production and increased the rate of IgA(+) cell population in Peyer's patch and in the lamina propria of the mouse small intestine. The LG2055 markedly increased the amount of IgA in a co-culture of B cells and bone marrow derived dendritic cells (BMDC), and TLR2 signal is critical for it. In addition, it is demonstrated that LG2055 stimulates BMDC to promote the production of TGF-β, BAFF, IL-6, and IL-10, all critical for IgA production from B cells. Combined stimulation of B cells with BAFF and LG2055 enhanced the induction of IgA production. Further, TGF-β signal was shown to be critical for LG2055-induced IgA production in the B cell and BMDC co-culture system, but TGF-β did not induce IgA production in a culture of only B cells stimulated with LG2055. Furthermore, TGF-β was critical for the production of BAFF, IL-6, IL-10, and TGF-β itself from LG2055-stimulated BMDC. These results demonstrate that TGF-β was produced by BMDC stimulated with LG2055 and it has an autocrine/paracrine function essential for BMDC to induce the production of BAFF, IL-6, and IL-10

<i>Lactobacillus gasseri</i> SBT2055 Induces TGF-β Expression in Dendritic Cells and Activates TLR2 Signal to Produce IgA in the Small Intestine

<div><p>Probiotic bacteria provide benefits in enhancing host immune responses and protecting against infection. Induction of IgA production by oral administration of probiotic bacteria in the intestine has been considered to be one reason for this beneficial effect, but the mechanisms of the effect are poorly understood. <i>Lactobacillus gasseri</i> SBT2055 (LG2055) is a probiotic bacterium with properties such as bile tolerance, ability to improve the intestinal environment, and it has preventive effects related to abdominal adiposity. In this study, we have found that oral administration of LG2055 induced IgA production and increased the rate of IgA⁺ cell population in Peyer's patch and in the lamina propria of the mouse small intestine. The LG2055 markedly increased the amount of IgA in a co-culture of B cells and bone marrow derived dendritic cells (BMDC), and TLR2 signal is critical for it. In addition, it is demonstrated that LG2055 stimulates BMDC to promote the production of TGF-β, BAFF, IL-6, and IL-10, all critical for IgA production from B cells. Combined stimulation of B cells with BAFF and LG2055 enhanced the induction of IgA production. Further, TGF-β signal was shown to be critical for LG2055-induced IgA production in the B cell and BMDC co-culture system, but TGF-β did not induce IgA production in a culture of only B cells stimulated with LG2055. Furthermore, TGF-β was critical for the production of BAFF, IL-6, IL-10, and TGF-β itself from LG2055-stimulated BMDC. These results demonstrate that TGF-β was produced by BMDC stimulated with LG2055 and it has an autocrine/paracrine function essential for BMDC to induce the production of BAFF, IL-6, and IL-10.</p></div

Significance of dendritic cell for the induction of IgA production by LG2055.

<p>(A) Whole PP cells or CD11c⁺ cell-depleted PP cells were cultured with or without heat treated LG2055 (10 µg/ml) for 7 days. (B) B cells from the spleen were co-cultured with or without CD11c⁺ cells derived from PP cells (left) or BMDC (right) in the presence or absence of the LG2055 for 7 days. (C) B cells were co-cultured with or without BMDC in the presence or absence of LG2055 in Transwell system for 7 days. The amounts of IgA in culture supernatants were determined by ELISA. Representative data from three for PP cells or four for BMDC independent experiments are shown. Each experiment was done with triplicate cultures; data are shown as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 was shown by <i>t</i>-test.</p

Critical factors for induction of IgA production by LG2055.

<p>(A) B cells and BMDC were co-cultured with or without LG2055 in the presence or absence of the TGF-β type I receptor inhibitor SB505124 or RAR antagonist LE135 for 7 days. (B) B cells were cultured with or without LG2055 or LPS (10 µg/ml) in the presence or absence of TGF-β for 7 days. B cells were cultured with or without LG2055 (C) or LA2062 (D), in the presence or absence of BAFF (500 ng/ml) for 7 days. IgA amounts in the supernatants were determined by ELISA. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are shown as the mean ± SD. ** P<0.01, was shown by <i>t</i>-test.</p

Significance of TLR2 in the induction of IgA production by LG2055.

<p>B cells or both B cells and BMDC were cultured with or without LG2055 in the presence or absence of the anti TLR2 antibody for 7 days. (B) Pam3CSK4 (TLR1/2 ligand) or FSL-1 (TLR2/6 ligand) was added to the B cell and BMDC co-culture system, and cultured for 7 days. The amounts of IgA in culture supernatants were determined by ELISA. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are shown as the mean ± SD. ** P<0.01 was shown by <i>t</i>-test.</p

Augmentation of IgA production by LG2055 <i>in vitro</i> and <i>in vivo</i>.

<p>Whole PP cells were cultured with or without each of the four <i>Lactobacillus</i> strains (LG2055, <i>L. gasseri</i> JCM1131^t (LG1131T), <i>L. helveticus</i> SBT2171 (LH2171), <i>L. acidophilus</i> SBT2062 (LA2062), 10 µg/ml) for 7 days (A). Whole PP cells were cultured with 0, 0.1, 1.0, and 10 µg/ml of LG2055 for 7 days (B). Whole PP cells were cultured with or without LG2055 (10 µg/ml) for 3, 5, and 7 days (C) The amounts of IgA in culture supernatants were determined by ELISA. Each experiment was done with tripricate cultures; data are shown as the mean ± SD. The values for cells cultured with lactic acid bacteria are compared with that of without the bacteria by one-way ANOVA, Dunnett's post test (A and B) and the <i>t</i>-test (C). Significant differences are indicated by * P<0.05, ** P<0.01, *** P<0.001. LG2055 was orally administrated to BALB/c mice for 5 weeks. Amounts of total IgA in intestinal tissue extracts (D) were determined by ELISA. The population of IgA⁺ B220⁺ cells in PP cells (E) and IgA⁺ B220^- cells in LP cells (F) was analyzed by FACS. Representative data from two independent experiments are shown. Data are shown as the mean ± SD (number of mice n = 10). Significant difference from control group at *<i>P</i><0.05, **<i>P</i><0.01 was shown by the <i>t</i>-test (D, E, and F).</p

Gene expression and cytokine production of BMDC stimulated by LG2055.

<p>BMDC was cultured with or without LG2055 for 48 hours. Gene expression of APRIL (<i>tnfsp13</i>), BAFF (<i>tnfsp13b</i>), RALDH2 (<i>aldh1a2</i>) in BMDC was determined by quantitative PCR. Amounts of TGF-β, IL-5, IL-6, and IL-10 in the culture supernatants were determined by ELISA. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are shown as the mean ± SD. ** P<0.01, *** P<0.001 was shown by <i>t</i>-test.</p