The Administration of Escherichia coli Nissle 1917 Ameliorates Development of DSS-Induced Colitis in Mice

Abstract

The beneficial effects of probiotics on immune-based pathologies such as inflammatory bowel disease (IBD) have been well reported. However, their exact mechanisms have not been fully elucidated. Few studies have focused on the impact of probiotics on the composition of the colonic microbiota. The aim of the present study was to correlate the intestinal anti-inflammatory activity of the probiotic Escherichia coli Nissle 1917 (EcN) in the dextran sodium sulfate (DSS) model of mouse colitis with the changes induced in colonic microbiota populations. EcN prevented the DSS-induced colonic damage, as evidenced by lower disease activity index (DAI) values and colonic weight/length ratio, when compared with untreated control mice. The beneficial effects were confirmed biochemically, since the probiotic treatment improved the colonic expression of different cytokines and proteins involved in epithelial integrity. In addition, it restored the expression of different micro-RNAs (miR-143, miR-150, miR-155, miR-223, and miR-375) involved in the inflammatory response that occurs in colitic mice. Finally, the characterization of the colonic microbiota by pyrosequencing showed that the probiotic administration was able to counteract the dysbiosis associated with the intestinal inflammatory process. This effect was evidenced by an increase in bacterial diversity in comparison with untreated colitic mice. The intestinal anti-inflammatory effects of the probiotic EcN were associated with an amelioration of the altered gut microbiome in mouse experimental colitis, especially when considering bacterial diversity, which is reduced in these intestinal conditions. Moreover, this probiotic has shown an ability to modulate expression levels of miRNAs and different mediators of the immune response involved in gut inflammation. This modulation could also be of great interest to understand the mechanism of action of this probiotic in the treatment of IBD.

Keywords: DSS colitis; intestinal microbiota; microRNA; probiotic; pyrosequencing.

FIGURE 1

Effect of Escherichia coli Nissle 1917 (EcN) in DSS-induced colitis model. (A,B) Disease activity index (DAI) values and weight evolution in DSS-colitic mice over the 26-day experimental period. Statistical significance among groups was evaluated by one-way ANOVA followed by Dunnett’s test. ^∗p < 0.05 vs. DSS-colitic group. (C) Colonic weight/length ratio, expressed as means ± SEM. Non-colitic group (n = 10), DSS-colitic (n = 10), and EcN (n = 10). Statistical significance among groups was evaluated by one-way ANOVA followed by the Tukey’s test. Bars with different letters are significantly different (p < 0.05). Non-colitic: untreated healthy group (n = 10); DSS-colitic: untreated DSS-induced colitic group (n = 10) and EcN: DSS-induced colitic group treated with probiotic (n = 10).

FIGURE 2

Escherichia coli Nissle 1917 (EcN) treatment promotes recovery of DSS-induced intestinal injury and inflammation in mice. (A) Histological images of colonic tissue stained with hematoxylin and eosin showing the effect of EcN on DSS-induced colitis. Representative images of each experimental group are shown: non-colitic, DSS-colitic, and EcN. In non-colitic mice, the images show the normal appearance of the intact mucosa containing the crypts with goblet cells full of mucin. In DSS-colitic group, the images show changes in the mucosa with areas of ulceration on the epithelial layer, in addition to a lower number of goblet cells which were depleted in mucin and an intense inflammatory cell infiltrate. In EcN group, an improvement of the colonic histology is found, with a reduced area of ulceration, mostly in process of recovery, presence of goblet cells replenished with their mucin content and reduced inflammatory cell infiltrate. (B) Histological scores calculated after microscopic analyses of longitudinal colon sections. Results are expressed as mean ± SEM. Statistical significance among groups was evaluated by one-way ANOVA followed by the Tukey’s test. Different letters denote significant differences between groups (p < 0.05). Non-colitic: untreated healthy group (n = 10); DSS-colitic: untreated DSS-induced colitic group (n = 10) and EcN: DSS-induced colitic group treated with probiotic (n = 10).

FIGURE 3

Biochemical evaluation of the effects of Escherichia coli Nissle 1917 (EcN); mRNA expression of cytokines (A) IL-1β, (B) IL-12, (C) TGF-β, and (D) ICAM-1 was quantified by real-time PCR, and fold changes are expressed as means ± SEM. Non-colitic group (n = 10), DSS-colitic group (n = 10), and EcN group (n = 10). Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Bars with different letters are significantly different (p < 0.05). Non-colitic: untreated healthy group (n = 10); DSS-colitic: untreated DSS-induced colitic group (n = 10) and EcN: DSS-induced colitic group treated with probiotic (n = 10).

FIGURE 4

Biochemical evaluation of the effects of Escherichia coli Nissle 1917 (EcN); mRNA expression of epithelial integrity proteins, (A) MUC-2, (B) MUC-3, (C) occludin (OCLN), and (D) zonula occludens-1 (ZO-1), was quantified by real-time PCR, and fold changes are expressed as means ± SEM. Non-colitic group (n = 10), DSS-colitic group (n = 10), and EcN group (n = 10). Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Bars with different letters are significantly different (p < 0.05). Non-colitic: untreated healthy group (n = 10); DSS-colitic: untreated DSS-induced colitic group (n = 10) and EcN: DSS-induced colitic group treated with probiotic (n = 10).

FIGURE 5

Biochemical evaluation of the effects of Escherichia coli Nissle 1917 (EcN); the expression of (A) miR-150, (B) miR-155, (C) miR-223, (D) miR-143, and (E) miR-375 was quantified by real-time PCR. Fold changes are expressed as means ± SEM. Non-colitic group (n = 10), DSS-colitic group (n = 10), and EcN group (n = 10). Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Bars with different letters are significantly different (p < 0.05). Non-colitic: untreated healthy group (n = 10); DSS-colitic: untreated DSS-induced colitic group (n = 10) and EcN: DSS-induced colitic group treated with probiotic (n = 10).

FIGURE 6

Comparison of microbiota composition between non-colitic group (n = 3), DSS-colitic group (n = 4), and Escherichia coli Nissle 1917 (EcN) group (n = 3). (A) Phylum breakdown of the most abundant bacterial communities in the different groups. Results were expressed as the mean ± SEM of the proportion of sequences and compared by one-way ANOVA followed by Tukey’s test. (B) The Firmicutes/Bacteriodetes ratio (F/B ratio) was calculated as a biomarker of gut dysbiosis. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Bars with different letters are significantly different (p < 0.05).

FIGURE 7

(A) Principal component analysis plot based on Bray–Curtis distances, calculated on the metagenomic table of fecal samples of the different groups [non-colitic (n = 3), DSS-colitic (n = 4), and Escherichia coli Nissle 1917 (EcN; n = 3)]. (B) Mean percentage of sequences of the most important genera in DSS-induced colitis model. Results were shown as the means ± SEM and compared by one-way ANOVA followed by Tukey’s test. Bars with different letters are significantly different (p < 0.05).

FIGURE 8

Estimation of the phylogenetic diversity of the gut microbiota found in the different experimental groups; non-colitic (n = 3), DSS-colitic (n = 4), and Escherichia coli Nissle 1917 (EcN; n = 3) using the (A) Chao richness, (B) Shannon diversity, and (C) Pielou evenness. The values are means, and error bars with different letters are significantly different (p < 0.05). Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Non-colitic: untreated healthy group (n = 3); DSS-colitic: untreated DSS-induced colitic group (n = 4) and EcN: DSS-induced colitic group treated with probiotic (n = 3).