Limits...
Characterization of the anti-inflammatory Lactobacillus reuteri BM36301 and its probiotic benefits on aged mice.

Lee J, Yang W, Hostetler A, Schultz N, Suckow MA, Stewart KL, Kim DD, Kim HS - BMC Microbiol. (2016)

Bottom Line: On the other hand, treatment of THP-1 directly with live bacterial cells identified a group of pro-inflammatory LAB, which stimulated significant production of TNF-α.Furthermore, while males treated with pro-inflammatory BM36304 developed higher serum levels of TNF-α and insulin, in contrast females did not experience such effects from this bacteria strain.It helped mice maintain healthy conditions as they aged.

View Article: PubMed Central - PubMed

Affiliation: Research and Development, Benebios LLC, 10527 Garden Grove Blvd, Garden Grove, CA, 92843, USA. joon.lee@benebios.com.

ABSTRACT

Background: The gut microbiota is playing more important roles in host immune regulation than was initially expected. Since many benefits of microbes are highly strain-specific and their mechanistic details remain largely elusive, further identification of new probiotic bacteria with immunoregulatory potentials is of great interest.

Results: We have screened our collection of probiotic lactic acid bacteria (LAB) for their efficacy in modulating host immune response. Some LAB are characterized by suppression of TNF-α induction when LAB culture supernatants are added to THP-1 cells, demonstrating the LAB's anti-inflammatory potential. These suppressive materials were not inactivated by heat or trypsin. On the other hand, treatment of THP-1 directly with live bacterial cells identified a group of pro-inflammatory LAB, which stimulated significant production of TNF-α. Among those, we chose the Lactobacillus reuteri BM36301 as an anti-inflammatory strain and the L. reuteri BM36304 as a pro-inflammatory strain, and further studied their in vivo effects. We supplied C57BL/6 mice with these bacteria in drinking water while feeding them a standard diet for 20 weeks. Interestingly, these L. reuteri strains evoked different consequences depending on the gender of the mice. That is, males treated with anti-inflammatory BM36301 experienced less weight gain and higher testosterone level; females treated with BM36301 maintained lower serum TNF-α as well as healthy skin with active folliculogenesis and hair growth. Furthermore, while males treated with pro-inflammatory BM36304 developed higher serum levels of TNF-α and insulin, in contrast females did not experience such effects from this bacteria strain.

Conclusion: The L. reuteri BM36301 was selected as an anti-inflammatory strain in vitro. It helped mice maintain healthy conditions as they aged. These findings propose the L. reuteri BM36301 as a potential probiotic strain to improve various aspects of aging issues.

No MeSH data available.


Related in: MedlinePlus

Immunomodulation by lactic acid bacteria in vitro. a Screening of lactic acid bacteria (LAB) with conditioned medium (CM) for anti-inflammatory activities against LPS-induced TNF-α production in THP-1. Without LPS, a non-detectable amount of TNF-α was observed (lane 1). With 150 ng/ml of LPS treatment, THP-1 cells produced significant amounts of TNF-α (lane 2). Each CM was treated to 5 % of the THP-1 culture volume to assess its inhibitory effects (lane 3, L. reuteri BM36301; lane 4, L. reuteri BM36304; lane 5, L. gasseri BM33601; and lane 6, B. animalis subsp. lactis BM10307). CM from control medium (MRS) was prepared and added in lanes 1 and 2. * indicates p < 0.03 from the t-test between the control (lane 2) and either BM36301 CM-treated (lane 3) or BM10307 CM-treated (lane 6). Bars show averages with standard deviation (SD) from 5 independent assays. b Screening of LAB with live cells for pro-inflammatory activities to induce TNF-α production in THP-1 cells. About 1.5 × 108 bacterial cells from exponentially growing cultures were applied to 6 × 105 THP-1 cells for 6 h. The cell-free supernatants were collected and assessed for the secreted TNF-α by ELISA method. The results show averages with SD from 4 independent experiments. c Summary of in vitro immunomodulation by various lactic acid bacteria
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4837529&req=5

Fig1: Immunomodulation by lactic acid bacteria in vitro. a Screening of lactic acid bacteria (LAB) with conditioned medium (CM) for anti-inflammatory activities against LPS-induced TNF-α production in THP-1. Without LPS, a non-detectable amount of TNF-α was observed (lane 1). With 150 ng/ml of LPS treatment, THP-1 cells produced significant amounts of TNF-α (lane 2). Each CM was treated to 5 % of the THP-1 culture volume to assess its inhibitory effects (lane 3, L. reuteri BM36301; lane 4, L. reuteri BM36304; lane 5, L. gasseri BM33601; and lane 6, B. animalis subsp. lactis BM10307). CM from control medium (MRS) was prepared and added in lanes 1 and 2. * indicates p < 0.03 from the t-test between the control (lane 2) and either BM36301 CM-treated (lane 3) or BM10307 CM-treated (lane 6). Bars show averages with standard deviation (SD) from 5 independent assays. b Screening of LAB with live cells for pro-inflammatory activities to induce TNF-α production in THP-1 cells. About 1.5 × 108 bacterial cells from exponentially growing cultures were applied to 6 × 105 THP-1 cells for 6 h. The cell-free supernatants were collected and assessed for the secreted TNF-α by ELISA method. The results show averages with SD from 4 independent experiments. c Summary of in vitro immunomodulation by various lactic acid bacteria

Mentions: We aimed to screen LAB for their potential to suppress intestinal inflammation. To this end, we adapted an in vitro tissue culture system where the human myeloid cells (THP-1) secrete TNF-α upon activation of their Toll-like receptors (TLR) by bacterial lipopolysaccharide (LPS) [28, 29]. First, we verified that THP-1 cells of 5 × 104 in 1 ml culture treated with LPS at 150 ng/ml dosage for 3.5 h usually resulted in a production of 200–300 pg/ml TNF-α (Fig. 1a, lanes 1 and 2). Next, we collected the supernatants of bacterial cultures grown for 24 h, vacuum-dried them, and reconstituted the conditioned medium (CM) (Methods). This CM contains complex activities to both suppress and induce TNF-α, depending on the LAB and culture conditions [23, 29]. Indeed, we observed slight production of TNF-α by the CMs from BM36304 and BM36301 without LPS, but this amount was less than 20 % of LPS-stimulated induction (data not shown). Finally, we added each CM up to 5 % of THP-1 culture (v/v) in the presence of LPS (Fig. 1a, lanes 3–6). The TNF-α production with LPS (208 ± 49 pg/ml, lane 2) was suppressed to 40 % with the CM from L. reuteri BM36301 (90.90 ± 49.3 pg/ml, lane 3), and to 52 % with the CM from B. animalis subsp. lactis BM10307 (118.8 ± 19.0 pg/ml, lane 6). For our screening purposes, we considered the suppression of TNF-α close to or less than 50 % of expression by LPS treatment as anti-inflammatory. However, the CMs from L. reuteri BM36304 and L. gasseri BM33601 were not able to suppress the TNF-α production to that point. We verified this inert activity by preparing these CMs under various culture conditions (data not shown).Fig. 1


Characterization of the anti-inflammatory Lactobacillus reuteri BM36301 and its probiotic benefits on aged mice.

Lee J, Yang W, Hostetler A, Schultz N, Suckow MA, Stewart KL, Kim DD, Kim HS - BMC Microbiol. (2016)

Immunomodulation by lactic acid bacteria in vitro. a Screening of lactic acid bacteria (LAB) with conditioned medium (CM) for anti-inflammatory activities against LPS-induced TNF-α production in THP-1. Without LPS, a non-detectable amount of TNF-α was observed (lane 1). With 150 ng/ml of LPS treatment, THP-1 cells produced significant amounts of TNF-α (lane 2). Each CM was treated to 5 % of the THP-1 culture volume to assess its inhibitory effects (lane 3, L. reuteri BM36301; lane 4, L. reuteri BM36304; lane 5, L. gasseri BM33601; and lane 6, B. animalis subsp. lactis BM10307). CM from control medium (MRS) was prepared and added in lanes 1 and 2. * indicates p < 0.03 from the t-test between the control (lane 2) and either BM36301 CM-treated (lane 3) or BM10307 CM-treated (lane 6). Bars show averages with standard deviation (SD) from 5 independent assays. b Screening of LAB with live cells for pro-inflammatory activities to induce TNF-α production in THP-1 cells. About 1.5 × 108 bacterial cells from exponentially growing cultures were applied to 6 × 105 THP-1 cells for 6 h. The cell-free supernatants were collected and assessed for the secreted TNF-α by ELISA method. The results show averages with SD from 4 independent experiments. c Summary of in vitro immunomodulation by various lactic acid bacteria
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837529&req=5

Fig1: Immunomodulation by lactic acid bacteria in vitro. a Screening of lactic acid bacteria (LAB) with conditioned medium (CM) for anti-inflammatory activities against LPS-induced TNF-α production in THP-1. Without LPS, a non-detectable amount of TNF-α was observed (lane 1). With 150 ng/ml of LPS treatment, THP-1 cells produced significant amounts of TNF-α (lane 2). Each CM was treated to 5 % of the THP-1 culture volume to assess its inhibitory effects (lane 3, L. reuteri BM36301; lane 4, L. reuteri BM36304; lane 5, L. gasseri BM33601; and lane 6, B. animalis subsp. lactis BM10307). CM from control medium (MRS) was prepared and added in lanes 1 and 2. * indicates p < 0.03 from the t-test between the control (lane 2) and either BM36301 CM-treated (lane 3) or BM10307 CM-treated (lane 6). Bars show averages with standard deviation (SD) from 5 independent assays. b Screening of LAB with live cells for pro-inflammatory activities to induce TNF-α production in THP-1 cells. About 1.5 × 108 bacterial cells from exponentially growing cultures were applied to 6 × 105 THP-1 cells for 6 h. The cell-free supernatants were collected and assessed for the secreted TNF-α by ELISA method. The results show averages with SD from 4 independent experiments. c Summary of in vitro immunomodulation by various lactic acid bacteria
Mentions: We aimed to screen LAB for their potential to suppress intestinal inflammation. To this end, we adapted an in vitro tissue culture system where the human myeloid cells (THP-1) secrete TNF-α upon activation of their Toll-like receptors (TLR) by bacterial lipopolysaccharide (LPS) [28, 29]. First, we verified that THP-1 cells of 5 × 104 in 1 ml culture treated with LPS at 150 ng/ml dosage for 3.5 h usually resulted in a production of 200–300 pg/ml TNF-α (Fig. 1a, lanes 1 and 2). Next, we collected the supernatants of bacterial cultures grown for 24 h, vacuum-dried them, and reconstituted the conditioned medium (CM) (Methods). This CM contains complex activities to both suppress and induce TNF-α, depending on the LAB and culture conditions [23, 29]. Indeed, we observed slight production of TNF-α by the CMs from BM36304 and BM36301 without LPS, but this amount was less than 20 % of LPS-stimulated induction (data not shown). Finally, we added each CM up to 5 % of THP-1 culture (v/v) in the presence of LPS (Fig. 1a, lanes 3–6). The TNF-α production with LPS (208 ± 49 pg/ml, lane 2) was suppressed to 40 % with the CM from L. reuteri BM36301 (90.90 ± 49.3 pg/ml, lane 3), and to 52 % with the CM from B. animalis subsp. lactis BM10307 (118.8 ± 19.0 pg/ml, lane 6). For our screening purposes, we considered the suppression of TNF-α close to or less than 50 % of expression by LPS treatment as anti-inflammatory. However, the CMs from L. reuteri BM36304 and L. gasseri BM33601 were not able to suppress the TNF-α production to that point. We verified this inert activity by preparing these CMs under various culture conditions (data not shown).Fig. 1

Bottom Line: On the other hand, treatment of THP-1 directly with live bacterial cells identified a group of pro-inflammatory LAB, which stimulated significant production of TNF-α.Furthermore, while males treated with pro-inflammatory BM36304 developed higher serum levels of TNF-α and insulin, in contrast females did not experience such effects from this bacteria strain.It helped mice maintain healthy conditions as they aged.

View Article: PubMed Central - PubMed

Affiliation: Research and Development, Benebios LLC, 10527 Garden Grove Blvd, Garden Grove, CA, 92843, USA. joon.lee@benebios.com.

ABSTRACT

Background: The gut microbiota is playing more important roles in host immune regulation than was initially expected. Since many benefits of microbes are highly strain-specific and their mechanistic details remain largely elusive, further identification of new probiotic bacteria with immunoregulatory potentials is of great interest.

Results: We have screened our collection of probiotic lactic acid bacteria (LAB) for their efficacy in modulating host immune response. Some LAB are characterized by suppression of TNF-α induction when LAB culture supernatants are added to THP-1 cells, demonstrating the LAB's anti-inflammatory potential. These suppressive materials were not inactivated by heat or trypsin. On the other hand, treatment of THP-1 directly with live bacterial cells identified a group of pro-inflammatory LAB, which stimulated significant production of TNF-α. Among those, we chose the Lactobacillus reuteri BM36301 as an anti-inflammatory strain and the L. reuteri BM36304 as a pro-inflammatory strain, and further studied their in vivo effects. We supplied C57BL/6 mice with these bacteria in drinking water while feeding them a standard diet for 20 weeks. Interestingly, these L. reuteri strains evoked different consequences depending on the gender of the mice. That is, males treated with anti-inflammatory BM36301 experienced less weight gain and higher testosterone level; females treated with BM36301 maintained lower serum TNF-α as well as healthy skin with active folliculogenesis and hair growth. Furthermore, while males treated with pro-inflammatory BM36304 developed higher serum levels of TNF-α and insulin, in contrast females did not experience such effects from this bacteria strain.

Conclusion: The L. reuteri BM36301 was selected as an anti-inflammatory strain in vitro. It helped mice maintain healthy conditions as they aged. These findings propose the L. reuteri BM36301 as a potential probiotic strain to improve various aspects of aging issues.

No MeSH data available.


Related in: MedlinePlus