Barrier Protection and Recovery Effects of Gut Commensal Bacteria on Differentiated Intestinal Epithelial Cells In Vitro

Abstract

Alterations in the gut microbiota composition play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) as specific commensal bacterial species are underrepresented in the microbiota of IBD patients. In this study, we examined the therapeutic potential of three commensal bacterial species, Faecalibacterium prausnitzii (F. prausnitzii), Roseburia intestinalis (R. intestinalis) and Bacteroides faecis (B. faecis) in an in vitro model of intestinal inflammation, by using differentiated Caco-2 and HT29-MTX cells, stimulated with a pro-inflammatory cocktail consisting of interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and lipopolysaccharide (LPS). Results obtained in this work demonstrated that all three bacterial species are able to recover the impairment of the epithelial barrier function induced by the inflammatory stimulus, as determined by an amelioration of the transepithelial electrical resistance (TEER) and the paracellular permeability of the cell monolayer. Moreover, inflammatory stimulus increased claudin-2 expression and decreased occludin expression were improved in the cells treated with commensal bacteria. Furthermore, the commensals were able to counteract the increased release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) induced by the inflammatory stimulus. These findings indicated that F. prausnitzii, R. intestinalis and B. faecis improve the epithelial barrier integrity and limit inflammatory responses.

Keywords: Bacteroides faecis; Caco-2 cells; Faecalibacterium prausnitzii; HT29-MTX cells; IBD; Roseburia intestinalis; commensal bacteria; tight junction proteins; transepithelial electrical resistance (TEER).

Figure 1

Effect of F. prausnitzii, B. faecis and R. intestinalis on the transepithelial electrical resistance (TEER) of epithelial cells. Caco-2 (A,C,E) and HT29-MTX (B,D,F) cell monolayers were incubated with all three bacterial species at three different multiplicities of infections (MOIs) in an anaerobic chamber. The transepithelial electrical resistance (TEER) was measured 3 and 6 h after the cells’ exposure to bacteria. Results are represented as the means ±SD (n ≥ 5). Data were analyzed with the Mann–Whitney U-test, * p ≤0.05, ** p ≤0.01, *** p ≤ 0.001; n.s., not statistically significant.

Figure 2

Effect of F. prausnitzii, B. faecis and R. intestinalis individually (A,B) and in combination (C,D) on the transepithelial electrical resistance (TEER) in Caco-2 and HT29-MTX cells monolayers. Both cell lines were treated basolaterally with cytomix + lipopolysaccharide (LPS) for 48 and 10 h, respectively. Cells without any treatment were used as a control. Data are presented as the means ± SD (n ≥ 5). * p ≤ 0.05) cytomix + LPS compared to the control and ^# p ≤ 0.05 treated groups compared to cytomix + LPS, as determined by the Mann–Whitney U test.

Figure 3

Paracellular permeability was determined by the flux of FITC–dextran through the differentiated HT29-MTX (A) and Caco-2 (B) monolayers. Cell monolayers were incubated with cytomix + LPS. After 6 h incubation with bacteria individually and in combination, the flux of 4-KDa FITC–dextran was measured. The control group received culture media. Data are presented as the means ± SD (n = 4). *** p ≤ 0.001 cytokine compared to control and ^# p ≤ 0.05, ^## p ≤ 0.01 treated groups compared to cytokine, as determined by the Mann–Whitney U test.

Figure 4

The secretion of IL-8 and MCP-1 in the inflamed Caco-2 and HT29-MTX monolayers. Epithelial cell monolayers were treated with cytomix + LPS following the incubation with bacteria individually or in combination for 6 h. Culture media sample was collected from the basal compartment. The concentration of IL-8 and MCP-1 secreted by Caco-2 (A,B) and HT29-MTX (C,D) cells were measured by multiplex flow cytometric bead array assay. ** p ≤ 0.01, **** p ≤ 0.0001 compared to the control, ^# p ≤ 0.05, ^### p ≤ 0.001, ^#### p ≤ 0.0001, n.s., no statistical significance compared to cytomix + LPS, as determined by one-way ANOVA and presented as means ± SD (n = 3).

Figure 5

Effect of F. prausnitzii, B. faecis and R. intestinalis alone and in combination on the immunofluorescence localization of tight junction proteins. Both cell line monolayers were incubated with all three bacteria alone and in combination following incubation, cell monolayers were fixed and stained with anti-occludin and claudin-2 antibodies (green) and Hoechst (blue) and imaged by confocal microscopy. Immunofluorescence staining of Occludin in Caco-2 (A) and HT29-MTX (B) cell monolayers. Immunofluorescence staining of claudin-2 in Caco-2 (C) and HT29-MTX (D) cell monolayers. Images are of 40× magnification. Bar = 20 µm.

Figure 6

Effect of F. prausnitzii, B. faecis and R. intestinalis alone and in combination on occludin expression. Representative occludin immunoblot and relative densities of Caco-2 (A,B) and HT29-MTX cells (C,D). The protein bands were quantified through densitometry analysis and normalized to the intensity of the β-actin bands. Data were shown as the mean  ±  SD of three biological replicates (n = 3). * p ≤ 0.05 ** p ≤ 0.01 compared to control group. ^# p ≤ 0.05, ^## p ≤ 0.01, ^### p ≤ 0.001, n.s., no significant compared to the cytomix + LPS group as determined by one-way ANOVA.

Figure 7

Effect of F. prausnitzii, B. faecis and R. intestinalis alone and in combination on claudin-2 expression. Representative claudin-2 immunoblot and the relative densities of Caco-2 (A,B) and HT29-MTX cells (C,D). The protein bands were quantified through densitometry analysis and normalized to the intensity of the β-actin bands. Data were shown as the mean  ±  SD of the three biological replicates (n = 3). ** p ≤ 0.01 *** p ≤ 0.001 compared to the control group. ^# p ≤ 0.05, ^## p ≤ 0.01, ^### p ≤ 0.001, n.s., no significant compared to the cytomix + LPS group as determined by one-way ANOVA.