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Delivery of a mucin domain enriched in cysteine residues strengthens the intestinal mucous barrier - PubMed

  • ️Thu Jan 01 2015

Delivery of a mucin domain enriched in cysteine residues strengthens the intestinal mucous barrier

Valérie Gouyer et al. Sci Rep. 2015.

Abstract

A weakening of the gut mucous barrier permits an increase in the access of intestinal luminal contents to the epithelial cells, which will trigger the inflammatory response. In inflammatory bowel diseases, there is an inappropriate and ongoing activation of the immune system, possibly because the intestinal mucus is less protective against the endogenous microflora. General strategies aimed at improving the protection of the intestinal epithelium are still missing. We generated a transgenic mouse that secreted a molecule consisting of 12 consecutive copies of a mucin domain into its intestinal mucus, which is believed to modify the mucus layer by establishing reversible interactions. We showed that the mucus gel was more robust and that mucin O-glycosylation was altered. Notably, the gut epithelium of transgenic mice housed a greater abundance of beneficial Lactobacillus spp. These modifications were associated with a reduced susceptibility of transgenic mice to chemically induced colitis. Furthermore, transgenic mice cleared faster Citrobacter rodentium bacteria which were orally given and mice were more protected against bacterial translocation induced by gavage with adherent-invasive Escherichia coli. Our data show that delivering the mucin CYS domain into the gut lumen strengthens the intestinal mucus blanket which is impaired in inflammatory bowel diseases.

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Figures

Figure 1
Figure 1. The transgene is secreted into intestinal mucus.

(a). Schematic map of the two DNA fragments that were developed and employed for transgenic (Tg) mice generation and deduced peptide organization. The three exons (exons 1, 2 and 3) are indicated by boxes and numbered. White boxes represent the 5′ and 3′UTR regions. (b). Representative immunohistochemistry of colonic tissue sections of wild-type (WT) and Tg mice. The Tg product is visualized in green and Muc2 is visualized in red using the UEA1 lectin. Cells were counterstained with Hoechst 33258 (blue). (c). Representative IHC of colonic tissue sections of the two Tg mouse lines. Cells were counterstained either with propidium iodide in red (Tg208) or with Hoechst 33258 in blue (Tg222). Muc2 is visualized using an anti-Muc2 antibody in green (Tg208) or in red (Tg222). The colonic mucus was conserved in the two Tg mouse lines. Lu, lumen. (d). The distribution (%) of fluorescent beads loaded at the mucus surface into the colonic mucus in wild-type (WT; n = 12) and Tg (n = 11) mice was studied by confocal microscopy, which showed that the Tg mucus was less penetrable.

Figure 2
Figure 2. Modifications of goblet cells.

(a). Some mucus granules were more vacuolated in the colon of transgenic (Tg) mice. (b). The ileum of Tg mice harbored a higher number of goblet cells, as counted on AB-PAS tissue sections. (c). Electron microscopic analysis showing that the colon of transgenic (Tg) mice harbored goblet cells with the same average surface, but with fewer mucus granules compared with wild-type (WT) mice. Close examination showed that some mucus granules were merged.

Figure 3
Figure 3. Modification of ileal mucin glycosylation.

(a). Nano-electrospray-mass spectrometry of oligosaccharides from wild-type (WT) and transgenic (Tg) ileal mucins acquired in the negative ion mode. The oligosaccharide compositions of major peaks are indicated as 5 numbers separated by comma for Hex, HexNAc, Fuc, NeuAc, and SO3, respectively. (b). Colocalization (in white) of Muc2 (in green) and UEA1 (in red) is shown and is expressed as the ratio of UEA1/Muc2 fluorescence. Cells were counterstained with Hoechst 33258 (blue).

Figure 4
Figure 4. The microbiota differs between WT and Tg mice.

(a). The total cultivable flora of the ileum from wild-type (WT; n = 8) and transgenic (Tg, n = 11) mice showed a higher load of Lactobacilli for Tg mice. (b). Identification of Lactobacillus ssp. by MS showed an alteration of the Lactobacillus composition in both the colon and ileum between the two genotypes.

Figure 5
Figure 5. The transgene protects against dextran sodium sulfate (DSS)-induced colitis.

(a). Wild-type (WT; n = 11) and transgenic (Tg, n = 9) mice were treated with 2.5% DSS or phosphate-buffered saline (PBS) for 5 days, and then received water for 7 days. Body mass was scored daily. (b). Histological score was calculated at day 12. (c). Proliferating cell nuclear antigen (PCNA) immunostaining of colon sections and its quantification showed an increase in epithelial cell proliferation in Tg mice.

Figure 6
Figure 6. The transgene protects against bacterial challenges.

(a). Wild-type (WT; n = 14) and transgenic (Tg, n = 11) mice were infected by oral gavage with 100 μL of an overnight culture of C. rodentium (~3.6 × 109 CFU) and sacrificed at 10 dpi. Feces were collected from live mice at 3, 5, and 10 dpi and C. rodentium was counted. (b). The colon weight/length ratio was calculated at 10 dpi. (c). WT (11) and Tg (8) mice were infected by oral gavage with 100 μL of an overnight culture of the clinical AIEC strain 06362 containing ~1 × 109 CFU bacteria on days 1 and 2, and were sacrificed on day 7. Liver and spleen homogenates were serially diluted in PBS and plated to determine the translocation of the total cultivable bacteria.

Comment in

  • Biological modeling of mucus to modulate mucus barriers.

    Desseyn JL, Gouyer V, Gottrand F. Desseyn JL, et al. Am J Physiol Gastrointest Liver Physiol. 2016 Feb 15;310(4):G225-7. doi: 10.1152/ajpgi.00274.2015. Epub 2015 Dec 10. Am J Physiol Gastrointest Liver Physiol. 2016. PMID: 26660538

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