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. 2014 Oct;80(20):6506-16.
doi: 10.1128/AEM.02104-14. Epub 2014 Aug 8.

Distinct immunomodulation of bone marrow-derived dendritic cell responses to Lactobacillus plantarum WCFS1 by two different polysaccharides isolated from Lactobacillus rhamnosus LOCK 0900

Sabina Górska  1 , Martin Schwarzer  2 , Wojciech Jachymek  3 , Dagmar Srutkova  4 , Ewa Brzozowska  3 , Hana Kozakova  4 , Andrzej Gamian  3
Affiliations

Distinct immunomodulation of bone marrow-derived dendritic cell responses to Lactobacillus plantarum WCFS1 by two different polysaccharides isolated from Lactobacillus rhamnosus LOCK 0900

Sabina Górska et al. Appl Environ Microbiol. 2014 Oct.

Abstract

The structures of polysaccharides (PS) isolated from Lactobacillus rhamnosus LOCK 0900 and results from stimulation of mouse bone marrow-derived dendritic cells (BM-DC) and human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR) upon exposure to these antigens were studied. L. rhamnosus LOCK 0900 produces PS that differ greatly in their structure. The polymer L900/2, with a high average molecular mass of 830 kDa, is a branched heteropolysaccharide with a unique repeating unit consisting of seven sugar residues and pyruvic acid, whereas L900/3 has a low average molecular mass of 18 kDa and contains a pentasaccharide repeating unit and phosphorus. Furthermore, we found that both described PS neither induce cytokine production and maturation of mouse BM-DC nor induce signaling through TLR2/TLR4 receptors. However, they differ profoundly in their abilities to modulate the BM-DC immune response to the well-characterized human isolate Lactobacillus plantarum WCFS1. Exposure to L900/2 enhanced interleukin-10 (IL-10) production induced by L. plantarum WCFS1, while in contrast, L900/3 enhanced the production of IL-12p70. We conclude that PS, probably due to their chemical features, are able to modulate the immune responses to third-party antigens. The ability to induce regulatory IL-10 by L900/2 opens up the possibility to use this PS in therapy of inflammatory conditions, such as inflammatory bowel disease, whereas L900/3 might be useful in reverting the antigen-dependent Th2-skewed immune responses in allergies.

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Figures

FIG 1
FIG 1
Selected part of the 1H-13C HSQC NMR spectrum for the L900/2 polysaccharide.
FIG 2
FIG 2
The structure of the heptasaccharide repeating unit of the L. rhamnosus LOCK 0900 polysaccharide L900/2 (L900/4).
FIG 3
FIG 3
Selected part of the 1H-13C HSQC NMR spectrum for the L900/3 polysaccharide.
FIG 4
FIG 4
The structure of the pentasaccharide repeating unit of the L. rhamnosus LOCK 0900 polysaccharide L900/3.
FIG 5
FIG 5
Maturation of DC and cytokine production induced by L. rhamnosus LOCK 0900. BM-DC from naive BALB/c mice were cultured with medium alone (CTRL), ultrapure LPS from E. coli (1 μg/ml), PAM3 (1 μg/ml), or 107 CFU/ml formalin-inactivated L. rhamnosus LOCK 0900 for 20 h. Production of IL-10 (A) and IL-12p70 (B) in culture supernatant was determined by ELISA. Pooled results from three independent experiments are shown. Means ± SEM are shown. BM-DC were gated as MHC-II+ CD11c+ and analyzed by flow cytometry for CD40, CD80, and CD86 expression (C, D, and E). Numbers shown are mean fluorescence units. ISO, isotype controls. Pooled results from three independent experiments are shown.
FIG 6
FIG 6
Maturation of DC and cytokine production induced by PS L900/2, PS L900/3, L. plantarum WCFS1, and their mixtures. BM-DC from naive BALB/c mice were cultured with medium alone (CTRL), ultrapure LPS from E. coli (1 μg/ml), PAM3 (1 μg/ml), 10 μg/ml of polysaccharide L900/2 or L900/3, 107 CFU/ml of formalin-inactivated L. plantarum, or the mixture of L. plantarum and corresponding PS (L. plantarum plus L900/2 and L. plantarum plus L900/3) for 20 h. Production of IL-10 (A) and IL-12p70 (B) in culture supernatant was determined by ELISA. Pooled results from three independent experiments are shown. Data are means ± SEM. BM-DC were gated as MHC-II+ CD11c+ and analyzed by flow cytometry for CD40, CD80, and CD86 expression (C, D, and E). Numbers shown are mean fluorescence units. ISO, isotype controls. Pooled results from three independent experiments are shown. *, P < 0.05; **, P < 0.01; n.s., not significant.
FIG 7
FIG 7
Activation of TLR receptors by PS L900/2, PS L900/3, L. plantarum WCFS1, and their mixture. HEK293 cells stably transfected with an expression vector for human TLR2 (293-hTLR2/CD14) (A) or TLR4 (293-hTLR4/MD2/CD14) (B) were cultured for 20 h with 10 μg/ml of polysaccharide L900/2, L900/3, 107 CFU/ml of formalin-inactivated L. plantarum, or the mixture of L. plantarum and the corresponding PS (L900/2 or L900/3). PAM3 (1 μg/ml) and ultrapure LPS from E. coli (1 μg/ml) were used as positive controls for TLR2 and TLR4, respectively. Unstimulated cells (CTRL) were used as controls. Stimulation was evaluated by measurement of IL-8 production; results are expressed as means ± SEM. One representative experiment out of three is shown. n.s., not significant.
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