Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis - PubMed (original) (raw)

Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis

Malin E V Johansson et al. Gut. 2014 Feb.

Abstract

Objective: The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)?

Methods and results: The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10(-/-) mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls.

Conclusions: Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology.

Keywords: BACTERIAL TRANSLOCATION; MUCINS; MUCOSAL BARRIER; MUCOSAL PATHOLOGY; MUCUS.

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Figures

Figure 1

Figure 1

Localisation of bacteria in the inner mucus layer of colon in different mouse colitis models. Fixed colon sections with preserved mucus were immunostained for Muc2 (green) and bacteria as detected by fluorescence in situ hybridisation with general bacterial 16S probes (red) and DNA stained using 4’,6-diamidino-2-phenylindole (DAPI; blue). Wild-type (WT) was compared with the spontaneous colitis models with different disrupted genes (Muc2−/−, C1galT1−/−, Slc9a3−/−, Tlr5−/− and IL-10−/−). Mice were treated with 3% dextran sodium sulfate (DSS) for 12 h or 5 days. Doubled-headed arrows show the inner mucus layer not always free of bacteria. Arrows point to bacteria close to the epithelial cells. Scale bars are 20 µm. Infl., inflamed; Non-infl., non-inflamed.

Figure 2

Figure 2

Bacteria penetration and inflammation in different colitis models. (A) Bacteria penetration of the inner mucus was scored for IL-10−/− (n=5) and wild-type (WT; n=5) mice, with Muc2−/−, which have high numbers of bacteria in contact with the epithelium, as comparison (n=3). Data are presented as mean±SEM. Penetration scores for all the genotypes are presented in online

supplementary figure

S1. nd, not detected. (B) Inflammation was monitored as histology scores for IL-10−/− and WT (n=5) mice, with Muc2−/− mice as a severely inflamed comparison (n=3). Data are presented as mean±SEM. Histology scores for all the genotypes are presented in online

supplementary figure

S1. (C) H&E-stained tissue sections corresponding to samples in figure 1. Scale bars in all panels are 100 µm. DSS, 3% dextran sodium sulphate; Infl. inflamed.

Figure 3

Figure 3

Mucus thickness in wild-type (WT) and interleukin 10-deficient (IL-10−/−) mice. (A) In vivo measurements of the initial mucus thickness in WT (n=7) and IL-10−/− (n=5) mice. The total mucus thickness (Total) was measured followed by aspiration of the mucus and measurement of the remaining mucus thickness (Adherent). (B) Ex vivo measurements of the increase in total mucus thickness over time in WT (n=5) and IL-10−/− (n=6) mice. (C) Number of goblet cells per upper crypt in WT (n=5) and IL-10−/− (n=5) mice. (D) Area of the goblet cell theca in anti-MUC2C3-stained colon section of WT (n=5) and IL-10−/− (n=5) mice. Data are presented as mean±SEM, and the two-tailed Mann–Whitney U test was used to compare the mucus thickness in WT and IL-10−/− mice.

Figure 4

Figure 4

Mucus penetrability in wild-type (WT; n=5) and interleukin 10-deficient (IL-10−/−; n=5) mice. (A) Representative Z-stack projections with the respective normalised intensity plots. Scale bars 100 µm. (B) Percentage of the total bead intensity in close proximity to the epithelial surface (40 µm). Differences between the groups were analysed using a two-tailed Mann–Whitney U test. (C) Relation between penetrable and impenetrable mucus. The black part of the bar represents impenetrable mucus and the green part mucus containing the 2 µm beads (ie, penetrable).

Figure 5

Figure 5

Mucus penetrability in human colonic biopsy samples from controls (n=12) and patients with ulcerative colitis (Mayo 0, n=17, Mayo 1–3, n=11). (A) Representative Z-stack projections with the respective normalised intensity plots. Scale bars 100 µm. (B) Percentage of total bead intensity in close proximity to the epithelial surface (<120 µm). Differences between groups were analysed using the Kruskal–Wallis test with Dunns’ correction for multiple comparisons (primary analysis p=0.0009 for the 2 µm beads and p=0.0004 for the 0.5 µm beads). (C) Percentage of beads in relation to total mucus thickness of individual patients. Numbers refer to patient numbers given in table 1. (D) Relation between penetrable and impenetrable mucus of the 2 µm green beads. C, control; M0 IP, patients with Mayo endoscopic score 0 and impenetrable mucus; M0 P, Mayo score 0 and penetrable mucus (patients number 1, 2 and 12); M1–3, Mayo score 1–3.

Figure 6

Figure 6

Mucus-filled goblet cells and bacteria in fixed human sigmoid biopsy samples. Human colon biopsy samples were Carnoy fixed to preserve the mucus and immunostained for MUC2 in combination with 4’,6-diamidino-2-phenylindole (DAPI) for DNA in nuclei and bacteria. (A) The number of goblet cells per upper crypt was determined in fixed and MU2-stained sections from patients with ulcerative colitis (UC) and controls. (B) The goblet cell theca area was measured in fixed and MU2-stained sections from patients with UC and controls. Data are presented as mean±SEM, and the Kruskal Wallis test with Dunn's correction for multiple comparisons was used to compare the UC patients with the control group. Ctrl, control; M0 IP, patients with endoscopic Mayo score 0 and impenetrable mucus; M0 P, Mayo score 0 and penetrable mucus (patients number 1, 2 and 12); M1–3, Mayo score 1–3. (C) Sections from human sigmoid biopsy samples stained for MUC2 (green) and DAPI (blue). (a) A biopsy specimen collected and directly fixed from sigmoid colon of a control patient without preceding laxative treatment. (b) A biopsy specimen from a control patient included in the penetrability study who was pretreated with laxatives before colonoscopy. (c) Biopsy specimen from patient with UC pretreated with laxative and with a Mayo endoscopic score of 2 at colonoscopy. Pictures to the right only show the DAPI staining. Bacteria (arrows) are found on the outer surface of the mucus in control patients (a and b). Bacteria are found inside the inner mucus and close to the epithelium in the patient with active UC (c). Some detached cells can be observed (arrowhead). (D) H&E-stained tissue each corresponding to parts a–c in (C). Scale bars are 10 µm (C) and 100 µm (D).

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