<i>Bifidobacterium longum</i> CCM 7952 Promotes Epithelial Barrier Function and Prevents Acute DSS-Induced Colitis in Strictly Strain-Specific Manner

<div><p>Background</p><p>Reduced microbial diversity has been associated with inflammatory bowel disease (IBD) and probiotic bacteria have been proposed for its prevention and/or treatment. Nevertheless, comparative studies of strains of the same subspecies for specific health benefits are scarce. Here we compared two <i>Bifidobacterium longum</i> ssp. <i>longum </i>strains for their capacity to prevent experimental colitis.</p><p>Methods</p><p>Immunomodulatory properties of nine probiotic bifidobacteria were assessed by stimulation of murine splenocytes. The immune responses to <i>B</i>. <i>longum</i> ssp. <i>longum</i> CCM 7952 (Bl 7952) and CCDM 372 (Bl 372) were further characterized by stimulation of bone marrow-derived dendritic cell, HEK293/TLR2 or HEK293/NOD2 cells. A mouse model of dextran sulphate sodium (DSS)-induced colitis was used to compare their beneficial effects <i>in vivo</i>.</p><p>Results</p><p>The nine bifidobacteria exhibited strain-specific abilities to induce cytokine production. Bl 372 induced higher levels of both pro- and anti-inflammatory cytokines in spleen and dendritic cell cultures compared to Bl 7952. Both strains engaged TLR2 and contain ligands for NOD2. In a mouse model of DSS-induced colitis, Bl 7952, but not Bl 372, reduced clinical symptoms and preserved expression of tight junction proteins. Importantly, Bl 7952 improved intestinal barrier function as demonstrated by reduced FITC-dextran levels in serum.</p><p>Conclusions</p><p>We have shown that Bl 7952, but not Bl 372, protected mice from the development of experimental colitis. Our data suggest that although some immunomodulatory properties might be widespread among the genus <i>Bifidobacterium</i>, others may be rare and characteristic only for a specific strain. Therefore, careful selection might be crucial in providing beneficial outcome in clinical trials with probiotics in IBD.</p></div

Bl 7952 induces upregulation of zonulin-1 and occludin in colon.

<p>Mice were treated with Bl 7952 (n = 10), with Bl 372 (n = 8) or with PBS (n = 10) on ten consecutive days or were left untreated (Naïve; n = 5). Colonic inflammation was induced by addition of 2.5% (w/v) DSS in the drinking water for 7 days. (A) Immunohistochemical detection of occludin and zonulin-1 proteins on representative paraffin-embedded sections of colon. (B) Representative western blotting of occludin and zonulin-1 proteins in the colonic mucosa. Expression of β-actin was used as internal control. (C) Evaluation of intestinal permeability by FITC-dextran. Serum levels of 4.0-kDa FITC-dextran were measured in naïve controls (n = 5), PBS/DSS-treated (n = 5) and Bl 7952/DSS-treated mice (n = 5) 5 hours after its intragastric administration. Data are express as mean ± SEM. Unpaired Student’s t-test was used for comparison of Bl 7952/DSS group <i>vs</i>. control PBS/DSS group (**p < 0.01).</p

Stimulation of bone marrow-derived dendritic cells with Bl 7952 and Bl 372.

<p>Bone marrow-derived dendritic cells (BM-DC) from naïve mice were cultured with formalin-inactivated Bl 7952 or Bl 372 (10⁷ CFU/ml) for 18 h. Ultra-pure lipopolysaccharide from <i>E</i>. <i>coli</i> (LPS, 1 μg/ml) and Pam3CSK4 (PAM3C, 1 μg/ml) were used as positive controls. Untreated cells (M) served as negative control. (A) Expression of CD40, CD80 and CD86 was assessed by means of flow cytometry. BM-DCs were gated as MHCII+CD11c+. Numbers represent florescence units from one representative experiment out of three. (B) Cytokines in cell culture supernatants were determined by ELISA. Results are representative of three repeat experiments. Data are expressed as mean ± SEM. Significant differences between cytokine levels of experimental group to negative control (M) was calculated using One-way ANOVA and Dunnett’s multiple comparison post-hoc test (**p < 0.01, ***p < 0.001).</p

Cytokine production by splenocytes stimulated with inactivated bacteria of different <i>Bifidobacterium</i> strains.

<p>Splenocytes isolated from naïve mice (n = 5) were stimulated with formalin-inactivated bifidobacteria (6 x 10⁷ CFU/ml) for 48 h. Pam3CSK4 (PAM3C, 1μg/ml) was use as a positive control. Non-stimulated splenocytes (Medium) were evaluated as control of basal cytokine levels. Concentration of cytokines in supernatants was determined by multiplex assay. Data are expressed as mean ± SEM. Results are representatives of two repeat experiments. Significant difference to medium was calculated using One-way ANOVA and Dunnett’s multiple comparison post-hoc test *p < 0.05; **p < 0.01.</p><p>Cytokine production by splenocytes stimulated with inactivated bacteria of different <i>Bifidobacterium</i> strains.</p

Activation of TLR2 and NOD2 by Bl 7952 and Bl 372.

<p>Human embryonic kidney cells (HEK293) stably transfected with an expression vector for human TLR2 (293-hTLR2/CD14) or with NOD2 (293-hNOD2) were stimulated with formalin-inactivated Bl 7952 or Bl 372 for 20 h. Stimulation was performed at concentrations of 10⁶, 10⁷ or 10⁸ CFU/ml. Cells stimulated with ultra-pure lipopolysaccharide from <i>E</i>. <i>coli</i> (LPS, 1 μg/ml) or untreated cells (M) were used as negative controls. Cells stimulated with Pam3CSK4 (PAM3C, 1 μg/ml) or muramyl dipeptide (MDP, 1 μg/ml) were used as positive controls for TLR2 or NOD2, respectively. Data are expressed as one representative experiment out of three.</p

Gut microbiota comparison of co-housed mice.

<p>Principal Component Analysis (PCA) plot based on DGGE profiles of 16S rRNA gene PCR derived amplicons of feces samples collected from mice in the co-housing experiment at nine weeks of age. Germ-free (GF) mice were conventionalized at one week of age (red balls) or at three weeks of age (yellow balls) by co-housing them with specific pathogen-free (SPF) mice (green balls). No differences in gut microbiota composition were detected between the SPF and co-housed ex-GF mice. DGGE: Denaturing Gradient Gel Electrophoresis.</p

A germ-free postnatal period alters systemic mononuclear cell populations.

<p>Distribution of cell populations in spleen (A) and mesenteric lymph node (B) sampled from mice in the co-housing experiment were measured by flow cytometry. Germ-free (GF) mice were conventionalized at one week of age (1W) or at three weeks of age (3W) by co-housing them with Specific Pathogen Free mice (SPF) raised in our barrier. A group of mice remained germ-free (GF). Percentages of NK cells (CD3⁻CD49b⁺), NKT cells (CD3⁺CD49b⁺), CD69⁺ and CD103⁺ T cells (CD3⁺CD4⁺) and IFN-γ producing T cells (CD3⁺CD4⁺) are illustrated. Lymphocytes were cultivated for 4 hrs with 50 ng/mL PMA and 750 ng/mL Ionomycin (Sigma-Aldrich) in the presence of BD GolgiStop before cells were harvested and intracellular IFN-γ stained. The mean is illustrated. * represents <i>p</i><0.05, ** <0.01, *** <0.001 compared with SPF mice. PMA: Phorbol 12-Myristate 13-Acetate.</p

Bl 7952 strain downregulated the secretion of pro-inflammatory cytokines in mesenteric lymph node cells of mice with DSS-induced colitis.

<p>Spontaneous production of anti-inflammatory IL-10 (A) or pro-inflammatory cytokines TNF-α (B) and IFN-γ (C) were analysed by multiplex assay in supernatants of mesenteric lymph node cells incubated with media only for 48 h. Data are expressed as mean ± SEM of untreated (n = 5), PBS/DSS (n = 10), Bl 7952/DSS (n = 10) or Bl 372/DSS (n = 8) mice. Unpaired Student’s t-test was used for comparison of experimental groups <i>vs</i>. PBS/DSS groups (*p < 0.05).</p

Impact of Bl 7952 and Bl 372 on DSS-induced colitis.

<p>(A) Mice were treated with Bl 7952 (n = 10), with Bl 372 (n = 8) or with PBS (n = 10) on ten consecutive days. Naïve animals (n = 5) were left untreated. Colonic inflammation was induced by the addition of 2.5% (w/v) DSS in the drinking water for 7 days. (B) Disease activity index and (C) colon length were evaluated at the end of the experiment. (D) Body weight of mice was evaluated throughout the experiment and the values are expressed as percentage of change of the initial value measured before DSS administration. Changes in colonic mucosa after DSS-treatment are shown on representative histological sections of healthy untreated mice (Naïve), mice treated with PBS (PBS/DSS), Bl 7952 (Bl 7952/DSS) or Bl 372 (Bl 372/DSS). The sections were stained by H&E (E) to address the degree of inflammation and by alcian blue (F) to show the changes in production of mucus in colonic tissue. Graphs show mean ± SEM and represent one out of two experiments. Unpaired Student’s t-test was used for comparison of experimental groups <i>vs</i>. control PBS/DSS group (*p < 0.05, ***p < 0.001).</p

Microbial inoculation at three weeks of age results in pro-inflammatory tuning of the immune system.

<p>Cytokines were measured in mesenteric lymph node (MLN) cell culture supernatants and analyzed by use of FlowCytomix Multiplex Th1/Th2 10plex. MLN were isolated from specific pathogen-free (SPF) mice (SPF), germ-free (GF) mice (GF) and ex-GF mice inoculated with a caecal microbiota suspension at one (1W) or three weeks (3W) of age. The lymphocytes were stimulated for 24 hrs with 1 µg/mL lipopolysaccharide (LPS) from <i>Escherichia coli</i> O26:B6 before the supernatants were used for analysis. Only cytokines with a significant difference among groups are illustrated. Error bars represent the SEM. * represents <i>p</i><0.05 compared with SPF mice.</p