Intestinal Anti-inflammatory Effects of Outer Membrane Vesicles from Escherichia coli Nissle 1917 in DSS-Experimental Colitis in Mice

Abstract

Escherichia coli Nissle 1917 (EcN) is a probiotic strain with proven efficacy in inducing and maintaining remission of ulcerative colitis. However, the microbial factors that mediate these beneficial effects are not fully known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a direct pathway for delivering selected bacterial proteins and active compounds to the host. In fact, vesicles released by gut microbiota are emerging as key players in signaling processes in the intestinal mucosa. In the present study, the dextran sodium sulfate (DSS)-induced colitis mouse model was used to investigate the potential of EcN OMVs to ameliorate mucosal injury and inflammation in the gut. The experimental protocol involved pre-treatment with OMVs for 10 days before DSS intake, and a 5-day recovery period. Oral administration of purified EcN OMVs (5 μg/day) significantly reduced DSS-induced weight loss and ameliorated clinical symptoms and histological scores. OMVs treatment counteracted altered expression of cytokines and markers of intestinal barrier function. This study shows for the first time that EcN OMVs can mediate the anti-inflammatory and barrier protection effects previously reported for this probiotic in experimental colitis. Remarkably, translation of probiotics to human healthcare requires knowledge of the molecular mechanisms involved in probiotic-host interactions. Thus, OMVs, as a non-replicative bacterial form, could be explored as a new probiotic-derived therapeutic approach, with even lower risk of adverse events than probiotic administration.

Keywords: DSS-experimental colitis; Escherichia coli Nissle 1917; immune modulation; mouse model; outer membrane vesicles; probiotic.

FIGURE 1

Experimental design to evaluate the potential of EcN OMVs to alleviate DSS-induced colitis in mice. The OMV-treated group daily received intragastrically 200 μl of PBS containing 5 μg of EcN OMVs over the 20-day experimental period, whereas the colitic group received 200 μl of PBS. The non-colitic control group was not challenged with DSS and received 200 μl of PBS over the entire experimental period.

FIGURE 2

Negative staining electron microscopy of isolated EcN OMVs. Vesicles were isolated from 1-liter LB culture of EcN and resuspended in a final volume of 0.2 ml PBS. A representative image of OMV samples (1:20 dilution) is shown. Scale bar: 200 nm.

FIGURE 3

EcN OMVs treatment improves clinical signs of DSS-induced colitis in mice. After the 10-day pre-treatment period, mice received 3% DSS in drinking water for 5 days. Mice were sacrificed 5 days later. Mice from the OMVs-treated group (triangles; n = 9) were administered with EcN OMVs (5 μg/day) throughout the experiment, whereas the two other experimental groups, colitic DSS control (squares; n = 9) and non-colitic control (circles; n = 6), received PBS instead. (A) Weight evolution in animals over the DSS-treatment and the recovery period. Values are presented as percentage of the body weight at the beginning of DSS intake (day 0). (B) DAI score of each experimental group from the beginning of the induction of colitis by DSS treatment (day 0) until sacrifice (day 10), calculated as described in the Section “Materials and Methods.” (C) Colon weight/length ratio calculated following resection. Data are expressed as means ± SEM. Different letters indicate significant differences between groups (P < 0.05).

FIGURE 4

EcN OMVs treatment promotes recovery of DSS-induced intestinal injury and inflammation in mice. On day 20, colons were excised and processed for microscopic analysis. (A) Histological images of colonic tissue stained with haematoxylin and eosin showing the effect of EcN OMVs on DSS-induced colitis. Representative images of each experimental group are shown: Control, DSS control and DSS OMVs. In Control, images show the normal appearance of the intact mucosa containing the crypts with goblet cells plenty of their mucin content (arrows). In DSS control, images show changes in the mucosa with areas of ulceration on the epithelial layer (asterisks), reduction of goblet cells with depletion of their mucin content (arrows) and intense inflammatory cell infiltrate (cross). In DSS OMVs, an improvement of the colonic histology is observed, with reduced area of ulceration, mostly in process of recovery (asterisks), presence of goblet cells replenished with their mucin content (arrow) and reduced inflammatory cell infiltrate (cross). (B) Histological scores calculated after microscopic analyses of longitudinal colon sections as described in the Section “Materials and Methods.” Results are expressed as mean ± SEM. Control group (n = 6), DSS control group (n = 9), DSS OMVs group (n = 9). Different letters indicate significant differences between groups (P < 0.05).

FIGURE 5

Effects of EcN OMVs treatment on colonic cytokine expression in DSS colitic mice. (A) Relative mRNA levels of the indicated cytokines measured by RT-qPCR in colonic tissue. Data are presented as fold-change compared to the non-colitic control group (expression value set to 1). (B) IL-10, IL-1β and IL-6 protein levels measured by ELISA in culture supernatants of colonic fragments from mice of each experimental group incubated for 24 h in complete DMEM medium as described in the Section “Materials and Methods.” Data are expressed as mean ± SEM. Control group (n = 6), DSS control group (n = 9), DSS OMVs group (n = 9). Different letters indicate significant differences between groups (P < 0.05).

FIGURE 6

Effects of EcN OMVs treatment on colonic expression of markers of intestinal barrier function in DSS colitic mice. Relative mRNA levels of TFF-3, MMP-9, MMP-2, ZO-1 and occludin were measured by RT-qPCR in colonic tissue. Data are presented as fold-change compared to the non-colitic control group. Control group (n = 6), DSS control group (n = 9), DSS OMVs group (n = 9). Data are presented as mean ± SEM. Different letters indicate significant differences between groups (P < 0.05).

FIGURE 7

Effects of EcN OMVs treatment on colonic COX-2 and iNOS expression. (A) Relative mRNA levels of COX-2 and iNOS measured by RT-qPCR in colonic tissue. Data are presented as fold-change compared to the non-colitic control group. Different letters indicate significant differences between groups (P < 0.05). (B) Western blot analysis of iNOS levels in colonic fragments collected from three mice (lines 1, 2, 3) of each group. Anti-β-actin antibody was used as internal control. Representative images are shown.