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In vitro evaluation of immunological properties of extracellular polysaccharides produced by Lactobacillus delbrueckii strains.

Kishimoto M, Nomoto R, Osawa R - Biosci Microbiota Food Health (2014)

Bottom Line: After incubation, the amounts of TNF-α and several cytokines that had been released by either RAW264.7 or Caco-2 cells were then quantified by cytotoxic activity on L929 cells or the RT-PCR method.It was found that the EPS-stimulated RAW264.7 cells express different profiles of cytokine production via Caco-2 cells but that the profile difference could not be related to the above TLC grouping.The evidence suggests that the EPSs of L. delbrueckii strains are diverse not only in their biochemical structure but also in their immunological properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresource Science, Organization of Advanced Science and Technology, Kobe University, 1-1 Rokko-dai, Nada-ku, Kobe 657-8501, Japan.

ABSTRACT
We investigated the variation in immunological properties of the extracellular polysaccharides (EPSs) produced by different Lactobacillus delbrueckii strains as well as that of their monosaccharide composition. The monosaccharide composition of each EPS produced by L. delbrueckii strains, as determined by thin layer chromatography (TLC), showed an appreciable variation in a strain-dependent manner, which could be broadly assigned to 4 TLC groups. Meanwhile, the immunological properties of the EPSs produced by 10 L. delbrueckii strains were evaluated in a semi-intestinal model using a Transwell co-culture system, which employed human intestinal epithelial Caco-2 cells on the apical side and murine macrophage RAW264.7 cells on the basolateral side. Each EPS was added to the apical side to allow direct contact with Caco-2 cells and incubated for 6 hr. After incubation, the amounts of TNF-α and several cytokines that had been released by either RAW264.7 or Caco-2 cells were then quantified by cytotoxic activity on L929 cells or the RT-PCR method. It was found that the EPS-stimulated RAW264.7 cells express different profiles of cytokine production via Caco-2 cells but that the profile difference could not be related to the above TLC grouping. The evidence suggests that the EPSs of L. delbrueckii strains are diverse not only in their biochemical structure but also in their immunological properties.

No MeSH data available.


Direct and indirect effects of EPSs purified by several strains on the production of TNF-α in RAW264.7 cells.(a) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, a 100 µg/ml of EPS sample and 5 ng/ml of LPS were applied to the cells and incubated for 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of the TNF-α content. Values represent the means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group. (b) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, the Transwell inserts on which the Caco-2 cells had been cultured were placed into the 24-well plates preloaded with RAW264.7 cells. Next, 100 µg/ml of EPS was applied to the apical side of the wells and incubated for 3 hr. Then, LPS was added into the basolateral side at the final concentration of 5ng/ml only as a positive control group, followed by incubation for an additional 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of TNF-α content. Values represent means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group.
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fig_002: Direct and indirect effects of EPSs purified by several strains on the production of TNF-α in RAW264.7 cells.(a) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, a 100 µg/ml of EPS sample and 5 ng/ml of LPS were applied to the cells and incubated for 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of the TNF-α content. Values represent the means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group. (b) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, the Transwell inserts on which the Caco-2 cells had been cultured were placed into the 24-well plates preloaded with RAW264.7 cells. Next, 100 µg/ml of EPS was applied to the apical side of the wells and incubated for 3 hr. Then, LPS was added into the basolateral side at the final concentration of 5ng/ml only as a positive control group, followed by incubation for an additional 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of TNF-α content. Values represent means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group.

Mentions: In order to determine whether an EPS itself had any immunological effect on RAW264.7 cells, the production of TNF-α was measured in the supernatants of RAW264.7 cells cultured in direct contact with EPSs produced by L. delbrueckii strains. Treatment with the EPS of L. delbrueckii TU-1 showed the highest production, followed by the EPS of L. delbrueckii KM-1, compared with LPS. In addition, the treatments of RAW264.7 cells with the EPSs of L. delbrueckii JCM1002T, L. delbrueckii JCM1012T, L. delbrueckii JCM1248T, L. delbrueckii JCM15610T, L. delbrueckii KM-4 and L. delbrueckii KM-5 showed higher production than the control. Moreover, treatment with the whey sample also showed higher production than the control. On the other hand, treatments with the EPSs produced byL. delbrueckii KM-2 and L. delbrueckii KM-3 showed the same level as the control (Fig. 2aFig. 2.


In vitro evaluation of immunological properties of extracellular polysaccharides produced by Lactobacillus delbrueckii strains.

Kishimoto M, Nomoto R, Osawa R - Biosci Microbiota Food Health (2014)

Direct and indirect effects of EPSs purified by several strains on the production of TNF-α in RAW264.7 cells.(a) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, a 100 µg/ml of EPS sample and 5 ng/ml of LPS were applied to the cells and incubated for 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of the TNF-α content. Values represent the means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group. (b) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, the Transwell inserts on which the Caco-2 cells had been cultured were placed into the 24-well plates preloaded with RAW264.7 cells. Next, 100 µg/ml of EPS was applied to the apical side of the wells and incubated for 3 hr. Then, LPS was added into the basolateral side at the final concentration of 5ng/ml only as a positive control group, followed by incubation for an additional 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of TNF-α content. Values represent means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group.
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Related In: Results  -  Collection

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fig_002: Direct and indirect effects of EPSs purified by several strains on the production of TNF-α in RAW264.7 cells.(a) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, a 100 µg/ml of EPS sample and 5 ng/ml of LPS were applied to the cells and incubated for 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of the TNF-α content. Values represent the means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group. (b) RAW264.7 cells were cultured overnight in a 24-wells plate in 5% CO2 to allow them to adhere to the bottom of the plate. Then, the Transwell inserts on which the Caco-2 cells had been cultured were placed into the 24-well plates preloaded with RAW264.7 cells. Next, 100 µg/ml of EPS was applied to the apical side of the wells and incubated for 3 hr. Then, LPS was added into the basolateral side at the final concentration of 5ng/ml only as a positive control group, followed by incubation for an additional 3 hr. LPS-stimulated cultures were used for the positive control. The culture media were then collected for measurement of TNF-α content. Values represent means ± SE (n=3). **p<0.01, *p<0.05; there was a significant difference between the EPS-treated group and the control group.
Mentions: In order to determine whether an EPS itself had any immunological effect on RAW264.7 cells, the production of TNF-α was measured in the supernatants of RAW264.7 cells cultured in direct contact with EPSs produced by L. delbrueckii strains. Treatment with the EPS of L. delbrueckii TU-1 showed the highest production, followed by the EPS of L. delbrueckii KM-1, compared with LPS. In addition, the treatments of RAW264.7 cells with the EPSs of L. delbrueckii JCM1002T, L. delbrueckii JCM1012T, L. delbrueckii JCM1248T, L. delbrueckii JCM15610T, L. delbrueckii KM-4 and L. delbrueckii KM-5 showed higher production than the control. Moreover, treatment with the whey sample also showed higher production than the control. On the other hand, treatments with the EPSs produced byL. delbrueckii KM-2 and L. delbrueckii KM-3 showed the same level as the control (Fig. 2aFig. 2.

Bottom Line: After incubation, the amounts of TNF-α and several cytokines that had been released by either RAW264.7 or Caco-2 cells were then quantified by cytotoxic activity on L929 cells or the RT-PCR method.It was found that the EPS-stimulated RAW264.7 cells express different profiles of cytokine production via Caco-2 cells but that the profile difference could not be related to the above TLC grouping.The evidence suggests that the EPSs of L. delbrueckii strains are diverse not only in their biochemical structure but also in their immunological properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresource Science, Organization of Advanced Science and Technology, Kobe University, 1-1 Rokko-dai, Nada-ku, Kobe 657-8501, Japan.

ABSTRACT
We investigated the variation in immunological properties of the extracellular polysaccharides (EPSs) produced by different Lactobacillus delbrueckii strains as well as that of their monosaccharide composition. The monosaccharide composition of each EPS produced by L. delbrueckii strains, as determined by thin layer chromatography (TLC), showed an appreciable variation in a strain-dependent manner, which could be broadly assigned to 4 TLC groups. Meanwhile, the immunological properties of the EPSs produced by 10 L. delbrueckii strains were evaluated in a semi-intestinal model using a Transwell co-culture system, which employed human intestinal epithelial Caco-2 cells on the apical side and murine macrophage RAW264.7 cells on the basolateral side. Each EPS was added to the apical side to allow direct contact with Caco-2 cells and incubated for 6 hr. After incubation, the amounts of TNF-α and several cytokines that had been released by either RAW264.7 or Caco-2 cells were then quantified by cytotoxic activity on L929 cells or the RT-PCR method. It was found that the EPS-stimulated RAW264.7 cells express different profiles of cytokine production via Caco-2 cells but that the profile difference could not be related to the above TLC grouping. The evidence suggests that the EPSs of L. delbrueckii strains are diverse not only in their biochemical structure but also in their immunological properties.

No MeSH data available.