Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium Faecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice

Abstract

Impaired gut barrier function has been reported in a wide range of diseases and syndromes and in some functional gastrointestinal disorders. In addition, there is increasing evidence that suggests the gut microbiota tightly regulates gut barrier function and recent studies demonstrate that probiotic bacteria can enhance barrier integrity. Here, we aimed to investigate the effects of Lactobacillus rhamnosus CNCM I-3690 on intestinal barrier function. In vitro results using a Caco-2 monolayer cells stimulated with TNF-α confirmed the anti-inflammatory nature of the strain CNCM I-3690 and pointed out a putative role for the protection of the epithelial function. Next, we tested the protective effects of L. rhamnosus CNCM I-3690 in a mouse model of increased colonic permeability. Most importantly, we compared its performance to that of the well-known beneficial human commensal bacterium Faecalibacterium prauznitzii A2-165. Increased colonic permeability was normalized by both strains to a similar degree. Modulation of apical tight junction proteins expression was then analyzed to decipher the mechanism underlying this effect. We showed that CNCM I-3690 partially restored the function of the intestinal barrier and increased the levels of tight junction proteins Occludin and E-cadherin. The results indicate L. rhamnosus CNCM I-3690 is as effective as the commensal anti-inflammatory bacterium F. prausnitzii to treat functional barrier abnormalities.

Keywords: AJs, adherence junctions; CNCM, Collection Nationale de Cultures de Microorganismes; DNBS, DiNitroBenzene Sulfonic; EOS, extremely oxygen sensitive; GVHD, graft-versus-host disease; IBD, inflammatory bowel diseases; IBS, irritable bowel syndrome; LAB, lac; apical junction proteins; lactobacilli; probiotics.

Figure 1.

Protective effect of probiotic lactobacilli strains on intestinal barrier integrity measured by Trans-Epithelial Electrical Resistance (TEER) *P < 0.05. TEER was assayed on Caco-2 cells grown on Transwell. TEER was measured before adding 10⁶ CFU of lactobacilli onto the apical surface for 3h prior to treatment of the basolateral medium with TNF-α (100 ng/ml) for 21 h at 37°C and at the end of TNF-α stimulation. Analysis done by ANOVA Test followed by Student-Newman-Keuls multiple comparison post hoc analysis ***p < 0.001, ****p < 0.0001. The resulting data presented as a ratio. ${\displaystyle Ratio=\frac{TEER Treatment T24/TEER Treatment T0}{TEER Control T24/TEER Control T0}}$

Figure 2.

Percentage of inhibition of NF-κB activation by 3 different lactobacilli strains. *p < 0.05. Immunomodulation was assessed in vitro in stable HT-29 pIgK-luciferase plasmid transfectants. Cells were pre-incubated for 2 hours with each bacterial strain using a MOI of 100 bacteria per cell. After 2 hours of pre-incubation with bacteria, cells were stimulated with TNF-α, 25 ng/well, for 6 hours. After incubation, luminescence was determined on cell lysate. Results are expressed as ratio and percentage of NF-κB activation. Analysis done by ANOVA Test followed by Student-Newman-Keuls multiple comparison post hoc analysis *P < 0.05.

Figure 3.

Measurement of in vivo permeability in DNBS-challenged mice treated with several probiotic strains. For in vivo measurements of gut permeability, animals were orally gavaged with FITC-dextran Control non-inflamed (EtOH-PBS), control inflamed (DNBS-PBS), F. prausnitzii A2-165 strain (DNBS-Fprau), L. rhamnosus CNCM I-3690 strain (DNBS-Lrha) , L. paracasei CNCM I-3689 strain (DNBS-Lpar). Analysis done by Kruskal-Wallis followed by a Dunn's Multiple Comparison Test *P < 0.05 versus DNBS-PBS. n = 16 mice per group.

Figure 4.

Modulation of Claudin family proteins expression. The expression of tight junction proteins was assessed by RT-qPCR on colonic tissue. Control non-inflamed group (EtOH-PBS), control inflamed group (DNBS-PBS), F. prausnitzii A2-165 strain (DNBS-Fprau), L. rhamnosus CNCM I-3690 strain (DNBS-Lrha) , L. paracasei CNCM I-3689 strain (DNBS-Lpar) Analysis done by Kruskal-Wallis followed by a Dunn's Multiple Comparison Test *P < 0.05 vs. DNBS-PBS. n = 6-13 mice per group.

Figure 5.

Modulation of apical-junction proteins expression. The expression of tight junction proteins was assessed by RT-qPCR on colonic tissue. Control non-inflamed group (EtOH-PBS), control inflamed group (DNBS-PBS), F. prausnitzii A2-165 strain (DNBS-Fprau), L. rhamnosus CNCM I-3690 strain (DNBS-Lrha) , L. paracasei CNCM I-3689 strain (DNBS-Lpar) Analysis done by Kruskal-Wallis followed by a Dunn's Multiple Comparison Test *P < 0.05 versus DNBS-PBS. n = 6–13 mice per group.

Figure 6.

Effect on apical junction proteins in a DNBS-induced low-grade inflammation model. Sections of the distal colon were stained for Claudin-4 (red), Fr11 (green), occludin (green) and E-cadherin (red) expression. Nuclei (DAPI; blue). Original magnification X60. Representative images control non-inflamed group (EtOH-PBS), control inflamed group (DNBS-PBS), F. prausnitzii A2-165 strain (DNBS-Fprau), L. rhamnosus CNCM I-3690 strain (DNBS-Lrha), L. paracasei CNCM I-3689 strain (DNBS-Lpar).

Figure 7.

Measurement of colonic cytokine levels. Control non-inflamed group (EtOH-PBS), control inflamed group (DNBS-PBS), F. prausnitzii A2-165 strain (DNBS-Fprau), L. rhamnosus CNCM I-3690 strain (DNBS-Lrha), L. paracasei CNCM I-3689 strain (DNBS-Lpar) Analysis done by Kruskal-Wallis followed by a Dunn's Multiple Comparison Test *P < 0.05 vs. DNBS-PBS. n = 8-16 mice per group.