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Mechanisms involved in alleviation of intestinal inflammation by bifidobacterium breve soluble factors.

Heuvelin E, Lebreton C, Grangette C, Pot B, Cerf-Bensussan N, Heyman M - PLoS ONE (2009)

Bottom Line: Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive.Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production.The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.

View Article: PubMed Central - PubMed

Affiliation: INSERM U793, Paris, France.

ABSTRACT

Objectives: Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive. To decipher the mechanisms accounting for the cross-talk between bacteria/soluble factors and intestinal epithelium, we measured the capacity of the bacteria, its conditioned medium (Bb-CM) and other Gram(+) commensal bacteria to dampen inflammatory chemokine secretion.

Methods: TNFalpha-induced chemokine (CXCL8) secretion and alteration of NF-kappaB and AP-1 signalling pathways by Bb were studied by EMSA, confocal microscopy and western blotting. Anti-inflammatory capacity was also tested in vivo in a model of TNBS-induced colitis in mice.

Results: Bb and Bb-CM, but not other commensal bacteria, induced a time and dose-dependent inhibition of CXCL8 secretion by epithelial cells driven by both AP-1 and NF-kappaB transcription pathways and implying decreased phosphorylation of p38-MAPK and IkappaB-alpha molecules. In TNBS-induced colitis in mice, Bb-CM decreased the colitis score and inflammatory cytokine expression, an effect reproduced by dendritic cell conditioning with Bb-CM.

Conclusions: Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production. The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.

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Related in: MedlinePlus

Bb preserves the integrity of HT29-19A epithelial cells.Filter-grown HT29-19A monolayers were treated with 10 ng/ml TNFα with or without live Bb placed in the apical compartment for 4 h. As index of epithelial viability, we measured markers of apoptosis (M30) and epithelial tight junction integrity (ZO-1). After 4 hour-treatment with Bb, HT29-19A monolayers (Costar® clear 3460) were labelled with the monoclonal antibody M30 CytoDEATH recognizing caspase-cleaved cytokeratin 18 (1∶10, Roche Diagnostics) and rabbit polyclonal anti-ZO-1 (24 µg/ml, Zymed). Cells were observed with the laser scanning confocal microscope LSM 510 Carl Zeiss. Treatment with H2O2 (100 µM), used as a positive control, induced apoptosis (blue M30 labeling) and alteration of ZO-1 distribution (green). In contrast, in basal condition or after treatment with Bb at MOI 100, no M30 labeling was observed and ZO-1 distribution was preserved. Results are representative of three independent experiments.
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pone-0005184-g002: Bb preserves the integrity of HT29-19A epithelial cells.Filter-grown HT29-19A monolayers were treated with 10 ng/ml TNFα with or without live Bb placed in the apical compartment for 4 h. As index of epithelial viability, we measured markers of apoptosis (M30) and epithelial tight junction integrity (ZO-1). After 4 hour-treatment with Bb, HT29-19A monolayers (Costar® clear 3460) were labelled with the monoclonal antibody M30 CytoDEATH recognizing caspase-cleaved cytokeratin 18 (1∶10, Roche Diagnostics) and rabbit polyclonal anti-ZO-1 (24 µg/ml, Zymed). Cells were observed with the laser scanning confocal microscope LSM 510 Carl Zeiss. Treatment with H2O2 (100 µM), used as a positive control, induced apoptosis (blue M30 labeling) and alteration of ZO-1 distribution (green). In contrast, in basal condition or after treatment with Bb at MOI 100, no M30 labeling was observed and ZO-1 distribution was preserved. Results are representative of three independent experiments.

Mentions: The inhibitory effects of Bb on chemokine secretion were not due to the alteration of HT29-19A epithelial integrity as attested by stable electrical resistance of epithelial monolayers in the presence of bacteria at high concentration (MOI 100, R = 98±11 ohms.cm2) as compared to control epithelial cells (R = 116±9 Ω.cm2). In addition, integrity of tight junctions was also attested by a normal distribution of ZO-1 protein and by the lack of bacteria-induced apoptosis of epithelial cells (Fig. 2).


Mechanisms involved in alleviation of intestinal inflammation by bifidobacterium breve soluble factors.

Heuvelin E, Lebreton C, Grangette C, Pot B, Cerf-Bensussan N, Heyman M - PLoS ONE (2009)

Bb preserves the integrity of HT29-19A epithelial cells.Filter-grown HT29-19A monolayers were treated with 10 ng/ml TNFα with or without live Bb placed in the apical compartment for 4 h. As index of epithelial viability, we measured markers of apoptosis (M30) and epithelial tight junction integrity (ZO-1). After 4 hour-treatment with Bb, HT29-19A monolayers (Costar® clear 3460) were labelled with the monoclonal antibody M30 CytoDEATH recognizing caspase-cleaved cytokeratin 18 (1∶10, Roche Diagnostics) and rabbit polyclonal anti-ZO-1 (24 µg/ml, Zymed). Cells were observed with the laser scanning confocal microscope LSM 510 Carl Zeiss. Treatment with H2O2 (100 µM), used as a positive control, induced apoptosis (blue M30 labeling) and alteration of ZO-1 distribution (green). In contrast, in basal condition or after treatment with Bb at MOI 100, no M30 labeling was observed and ZO-1 distribution was preserved. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667252&req=5

pone-0005184-g002: Bb preserves the integrity of HT29-19A epithelial cells.Filter-grown HT29-19A monolayers were treated with 10 ng/ml TNFα with or without live Bb placed in the apical compartment for 4 h. As index of epithelial viability, we measured markers of apoptosis (M30) and epithelial tight junction integrity (ZO-1). After 4 hour-treatment with Bb, HT29-19A monolayers (Costar® clear 3460) were labelled with the monoclonal antibody M30 CytoDEATH recognizing caspase-cleaved cytokeratin 18 (1∶10, Roche Diagnostics) and rabbit polyclonal anti-ZO-1 (24 µg/ml, Zymed). Cells were observed with the laser scanning confocal microscope LSM 510 Carl Zeiss. Treatment with H2O2 (100 µM), used as a positive control, induced apoptosis (blue M30 labeling) and alteration of ZO-1 distribution (green). In contrast, in basal condition or after treatment with Bb at MOI 100, no M30 labeling was observed and ZO-1 distribution was preserved. Results are representative of three independent experiments.
Mentions: The inhibitory effects of Bb on chemokine secretion were not due to the alteration of HT29-19A epithelial integrity as attested by stable electrical resistance of epithelial monolayers in the presence of bacteria at high concentration (MOI 100, R = 98±11 ohms.cm2) as compared to control epithelial cells (R = 116±9 Ω.cm2). In addition, integrity of tight junctions was also attested by a normal distribution of ZO-1 protein and by the lack of bacteria-induced apoptosis of epithelial cells (Fig. 2).

Bottom Line: Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive.Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production.The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.

View Article: PubMed Central - PubMed

Affiliation: INSERM U793, Paris, France.

ABSTRACT

Objectives: Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive. To decipher the mechanisms accounting for the cross-talk between bacteria/soluble factors and intestinal epithelium, we measured the capacity of the bacteria, its conditioned medium (Bb-CM) and other Gram(+) commensal bacteria to dampen inflammatory chemokine secretion.

Methods: TNFalpha-induced chemokine (CXCL8) secretion and alteration of NF-kappaB and AP-1 signalling pathways by Bb were studied by EMSA, confocal microscopy and western blotting. Anti-inflammatory capacity was also tested in vivo in a model of TNBS-induced colitis in mice.

Results: Bb and Bb-CM, but not other commensal bacteria, induced a time and dose-dependent inhibition of CXCL8 secretion by epithelial cells driven by both AP-1 and NF-kappaB transcription pathways and implying decreased phosphorylation of p38-MAPK and IkappaB-alpha molecules. In TNBS-induced colitis in mice, Bb-CM decreased the colitis score and inflammatory cytokine expression, an effect reproduced by dendritic cell conditioning with Bb-CM.

Conclusions: Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production. The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.

Show MeSH
Related in: MedlinePlus