6 research outputs found
Role of <i>Lactobacillus pentosus</i> Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement
<div><p>Lactic acid bacteria are well known to possess immune-modulating effects, but the mechanisms underlying their modulation of the gut immune system are not fully understood. Here, we examined the localization of heat-killed <i>Lactobacillus pentosus</i> strain b240 (b240) in intestinal tissues and the effect of b240 on adaptive immune cascades in the gut. Histological analysis showed that b240 co-localized with dendritic cells (DCs) in the subepithelial dome region of Peyer's patches (PPs). In a PP cell culture system, b240 promoted the production of immunoglobulin A (IgA), interleukin (IL)-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor, but not IL-4, IL-5, B-cell activating factors, IFN-α, IFN-β, and transforming growth factor-β1. The enhanced IgA production by b240 was attenuated by neutralizing IL-6, a potent IgA-enhancing cytokine. b240 stimulated DCs to produce an elevated amount of IL-6 in a Toll-like receptor (TLR) 2-, but not TLR4- or TLR9-dependent manner. Finally, we demonstrated that TLR2-mediated IL-6 production from PP DCs in response to b240 activated B cells to produce a large amount of IgA in a DC-B cell co-culture system. Our findings open up the possibility that the heat-killed form of <i>Lactobacillus pentosus</i> strain b240 can be used as a TLR2-mediated DC-activating biologic for enhancing IgA production in the intestine.</p></div
Localization of orally administered b240 in the small intestine.
<p>Histological analysis was performed to examine the localization of b240 in (A) the PPs and (B) the small intestine after mice were provided with autoclaved drinking water supplemented with 1 mg/ml of FITC-labeled b240 <i>ad libitum</i> for 3 days. Tissue sections were prepared and then stained with 4,6-diamidino-2-phenylindole (blue). b240 (green) internalized in PP are indicated by arrowheads. The bar indicates 50 μm. (C) The number of b240 inside PPs and in the luminal areas of the tissue sections was counted. Then, the proportion of b240 inside the PPs was calculated. Data are representative of 2 independent experiments producing similar results.</p
Involvement of TLR signals in the IL-6-inducing ability of b240.
<p>(A) PP cells (5.8×10<sup>5</sup> cells) from WT (white), TLR2<sup>−/−</sup> (black), TLR4<sup>−/−</sup> (gray), or TLR9<sup>−/−</sup> (dotted) mice were cultured with or without heat-killed b240 (4.7×10<sup>6</sup> counts) for 4 days. IL-6 in the culture supernatants was determined by CBA. <sup>*</sup><i>P</i><0.05 versus WT in b240 group by Dunnett's test. The saline group was not tested. (B) PP cells (5.8×10<sup>5</sup> cells) from WT (white) or TLR2<sup>−/−</sup> (black) mice were cultured with or without heat-killed b240 (4.7×10<sup>6</sup> counts), the intact cell wall (ICW), or the KOH-treated ICW for 4 days. The ICW and the KOH-treated ICW were applied in the same amount as heat-killed b240. IL-6 in the culture supernatants was determined by CBA. <sup>*</sup><i>P</i><0.05 versus b240 in the WT group by Dunnett's test, and the TLR2<sup>−/−</sup> group was not tested. <sup>#</sup><i>P</i><0.05 versus WT in the ICW group by Student's <i>t</i>-test. Data are expressed as mean ± SEM (n = 3–6). Data are (A) combined or (B) representative of 2 independent experiments.</p
Role of PP DCs in the IgA-enhancing ability of b240.
<p>(A, C) In the presence (black) or absence (white) of heat-killed b240 (1.6×10<sup>6</sup> counts), (A) purified WT PP IgD<sup>+</sup> cells (2×10<sup>5</sup> cells/well) were cultured with or without purified WT PP CD11c<sup>+</sup>B220<sup>−</sup> DCs (1×10<sup>5</sup> cells/well), (B) purified WT PP CD11c<sup>+</sup>B220<sup>−</sup> DCs and purified WT PP IgD<sup>+</sup> B cells were co-cultured with (black) or without (white) anti-IL-6 mAb (10 μg/ml) in the presence or absence of heat-killed b240 (1.6×10<sup>6</sup> counts), and (C) purified WT or TLR2<sup>−/−</sup> PP CD11c<sup>+</sup>B220<sup>−</sup> DCs and purified WT or TLR2<sup>−/−</sup> PP IgD<sup>+</sup> B cells were co-cultured for 7 days. IgA and IL-6 in the culture supernatants were determined by ELISA and CBA, respectively. Data are expressed as mean ± SEM (n = 1–3). ND, not detected. <sup>*</sup><i>P</i><0.05 versus (A) saline group by Welch's <i>t</i>-test, (B) isotype Ig group by Student's t-<i>t</i>est, (C) WT DCs + WT B cells + b240 co-culture by Dunnett's test. Data are representative of 2 independent experiments producing similar results.</p
Important factor for the enhancement IgA production from PP cells by b240.
<p>(A) PP cells (1.5×10<sup>6</sup> cells) were cultured with saline (open circles), 1.2×10<sup>6</sup> counts of heat-killed b240 (closed squares), or 1.2 × 10<sup>7</sup> counts of heat-killed b240 (closed circles) for 1, 3, 5, and 7 days. (B, C) In the presence or absence of heat-killed b240 (4.7×10<sup>6</sup> counts), PP cells (5.8×10<sup>5</sup> cells) were cultured with (B) anti-IL-6 mAb (10 μg/ml), anti-IFN-γ mAb (10 μg/ml), anti-TNF mAb (10 μg/ml), rat IgG1 k isotype control (10 μg/ml), (C) LE540 (1 μM), BCMA-Ig+ TACI-Ig (5 μg/ml each), dimethyl sulfoxide, or human IgG1 Fc antibody (10 μg/ml) for 4 days. The stimulation index of each sample was calculated (for example, (b240-treatment and anti-IL-6 Ab treatment)/(saline-treatment and anti-IL-6 Ab treatment) is the stimulation index for anti-IL-6 Ab treatment). (D) PP cells (5.8×10<sup>5</sup> cells) were cultured with a low dose (light gray), medium dose (dark gray), and high dose (black) of rIL-6 (0.4, 2, or 10 ng/ml), rIFN-γ (0.6, 3, or 15 ng/ml), rTNF (0.08, 0.4, or 2 ng/ml), or heat-killed b240 (4.7×10<sup>6</sup> counts) for 4 days. IgA or cytokine in the culture supernatants was determined by ELISA or CBA. Data are expressed as mean ± SEM (n = 3). (A, B) <sup>*</sup><i>P</i><0.05 versus control group by Dunnett's test. (C) Student's <i>t</i>-test was conducted. (D) Statistical analysis was not conducted. Data are representative of 2 independent experiments producing similar results.</p
IL-6 producing cells in the PPs in response to b240.
<p>(A) Purified CD11c<sup>+</sup>B220<sup>−</sup> cells as DCs, CD4<sup>+</sup> cells as T cells, CD19<sup>+</sup> cells as B cells from the PPs, or PP cells (1×10<sup>5</sup> cells) were cultured with (black) or without (white) heat-killed b240 (1.6×10<sup>6</sup> counts) for 3 days. IL-6 in the culture supernatants was determined by CBA. Data are expressed as mean ± SEM (n = 3). ND, not detected. The detection limit was 20 pg/ml. <sup>*</sup><i>P</i><0.05 by Student's <i>t</i>-test. Data are representative of 2 independent experiments producing similar results. (B) After the ligated intestinal loop assay with 1 mg/ml of FITC-labeled b240, histological analysis was performed to examine the co-localization of b240 (green) with DCs in PP. Tissue sections were prepared and stained with anti-CD11c antibody (red) and 4,6-diamidino-2-phenylindole (blue). Magnification of the area in the first image (white square) shows the contact between b240 and CD11c<sup>+</sup> cells (arrowhead). White and yellow bars indicate 100 and 25 μm, respectively. (C) After PP CD11c<sup>+</sup>B220<sup>−</sup> DCs were cultured with FITC-labeled b240 on a Cell Desk, the interaction between PP CD11c<sup>+</sup>B220<sup>−</sup> DCs and b240 was analyzed using a fluorescence microscope. The bar indicates 10 μm.</p