Mechanisms Involved in Alleviation of Intestinal Inflammation by Bifidobacterium Breve Soluble Factors

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: TNFa-induced chemokine (CXCL8) secretion and alteration of NF-kB 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-kB transcription pathways and implying decreased phosphorylation of p38-MAPK and IkB-a 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 phosphorylation

Protective effect of <i>Bb</i> and <i>Bb</i>-CM in TNBS-induced colitis in mice.

<p>A. Wallace scores were used to semi-quantify colitis activity in BALB/c mice after colonic instillation of TNBS (control group) with or without intragastric pre-treatment with 10⁸ CFU <i>Bb</i> (<i>Bb</i> i.g.), intraperitoneal injection of <i>Bb</i>-CM (<i>Bb</i>-CM i.p.), intraperitoneal injection of non treated-DC (DC) or <i>Bb</i>-CM conditioned-DC (DC<i>_Bb</i>_-CM). The mild inhibition of colitis observed with <i>Bb</i> i.g. became significant with <i>Bb-CM</i> and DC<i>_Bb</i>_-CM. B. Percentage protection in mice of the different groups compared to control (TNBS only). C. Quantitative RT-PCR analysis of mRNA expression of pro-inflammatory cytokines/mediators in the colon, 48 hours after TNBS-induced colitis with or without treatment. A significant inhibition of pro-inflammatory molecules in colonic mucosa (IL-1β, CXCL-1, IL-23, IL-6, COX2) was observed in the presence of <i>Bb</i>-CM or <i>Bb</i>-CM pre-conditioned DC. Scatter plots are presented with medians. *p<0.05, †<i>p</i><0.01 <i>vs</i> TNBS; n = 7 to 8 mice per group.</p

<i>Bb</i> and its soluble factors inhibit TNFα-induced phosphorylation of p38-MAPK in epithelial cells.

<p>A–C. HT29-19A cells were incubated for 4 hours on the apical surface with live <i>Bb</i> or commensal bacteria <i>B.breve</i> ATCC 15698, <i>L.rhamnosus</i> 10863, <i>E.rectale</i> L15 at MOI 100, together with basolateral TNFα. Activation of AP-1 assessed by EMSA was inhibited by ∼35% in the presence of <i>Bb</i>-CM and by ∼40% in the presence of <i>Bb</i>, but not in the presence of other bacteria. n = 2 independent experiments. D. HT29-19A cells were treated with TNFα±<i>Bb</i>-CM. Total proteins were analysed by SDS-PAGE and revealed with antibodies to phospho-p38-MAPK, p38-MAPK and β-actin. <i>Bb</i>-CM inhibited TNFα-induced phosphorylation of p38-MAPK from 15 to 30 min of incubation. n = 3 independent experiments.</p

<i>Bb</i> and its soluble factors inhibit TNFα-induced NF-κB in epithelial cells.

<p>A. HT29-19A cells were incubated for 30 min with TNFα±<i>Bb</i>-CM and localization of the NF-κB p65 subunit was observed by confocal microscopy. Following exposure to TNFα, p65 (green) was translocated into the nuclei (blue) and co-localization was observed (white spots). <i>Bb</i>-CM inhibited TNFα-induced p65 subunit translocation. n = 3 independent experiments. B. HT29-19A cells were incubated for 4 hours with TNFα±<i>Bb</i>-CM. Electrophoretic migration shift assay (EMSA) was performed following extraction of nuclear proteins. TNFα condition was taken as 100% activation and <i>Bb</i>-CM induced a 40% inhibition of NF-κB activation (n = 2 independent experiments). C. Epithelial cells were incubated with TNFα±<i>Bb</i> at MOI 100. Activation of NF-κB was inhibited by ∼40% with <i>Bb</i> but not with commensal bacteria <i>B.breve</i> ATCC 15698, <i>L.rhamnosus</i> 10863, <i>E.rectale</i> L15. n = 2 independent experiments. D. HT29-19A cells were treated with TNFα±<i>Bb</i>-CM. Total proteins were separated by SDS-PAGE and revealed with antibodies to phospho-IκB-α, IκBα and β-actin. <i>Bb</i>-CM inhibited the TNFα-induced phosphorylation of IκB-α as early as 5 min and IκB-α degradation after 15 min. n = 3 independent experiments.</p

<i>Bb</i> and its soluble factors dampen TNFα-induced cytokine secretion in epithelial cells.

<p>A. After 4 hour-incubation of HT29-19A cells with TNFα±<i>Bb</i> (MOI 100) or <i>Bb</i>-CM, epithelial cell supernatants were tested by the Raybio® human cytokine array V (One representative array of two experiments). Secretion of chemokines (CXCL1, CXCL8, CCL4) and of other inflammatory molecules was inhibited in the presence of <i>Bb</i> and <i>Bb</i>-CM as judged by densitometric analysis. B. Long lasting inhibition (up to 7 hours) of CXCL8 secretion by <i>Bb</i>-CM (∼50%) was observed using ELISA. n = 7, *<i>p</i><0.01 <i>vs</i> TNFα. C–D. HT29-19A cell monolayers were treated (4 hours) from the basolateral compartment with TNFα together with <i>Bb</i> or commensal bacteria (<i>B.breve</i> ATCC 15698, <i>L.rhamnosus</i> 10863, <i>E.rectale</i> L15) placed in the apical compartment at increasing MOI. As quantified by ELISA, <i>Bb</i> dose-dependently inhibited TNFα-induced CXCL8 secretion, in contrast to other bacteria. n = 11, †<i>p</i><0.0001 ; *<i>p</i><0.001 vs TNFα.</p

<i>Bb</i> preserves the integrity of HT29-19A epithelial cells.

<p>Filter-grown HT29-19A monolayers were treated with 10 ng/ml TNFα with or without live <i>Bb</i> 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 <i>Bb</i>, 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 <i>Bb</i> at MOI 100, no M30 labeling was observed and ZO-1 distribution was preserved. Results are representative of three independent experiments.</p