Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches

Abstract

Colon has been shown to have a two-layered mucus system where the inner layer is devoid of bacteria. However, a complete overview of the mouse gastrointestinal mucus system is lacking. We now characterize mucus release, thickness, growth over time, adhesive properties, and penetrability to fluorescent beads from stomach to distal colon. Colon displayed spontaneous mucus release and all regions released mucus in response to carbachol and PGE2, except the distal colon and domes of Peyer's patches. Stomach and colon had an inner mucus layer that was adherent to the epithelium. In contrast, the small intestine and Peyer's patches had a single mucus layer that was easily aspirated. The inner mucus layer of the distal colon was not penetrable to beads the size of bacteria and the inner layer of the proximal colon was only partly penetrable. In contrast, the inner mucus layer of stomach was fully penetrable, as was the small intestinal mucus. This suggests a functional organization of the intestinal mucus system, where the small intestine has loose and penetrable mucus that may allow easy penetration of nutrients, in contrast to the stomach, where the mucus provides physical protection, and the colon, where the mucus separates bacteria from the epithelium. This knowledge of the mucus system and its organization improves our understanding of the gastrointestinal tract physiology.

Keywords: bacteria; goblet cells; mucin; mucus adhesiveness; mucus thickness.

Figures

Fig. 1.

Colonic mucus forms 2 layers where the inner is a barrier to bacteria. A: explant tissue from the proximal (left) and distal (right) colon mounted in the perfusion chamber with the mucus surface visualized by charcoal and mucus aspirated (Supplemental Videos S1 and S2, first frame in A). B: mucus thickness measured in colon explants from proximal (open bars; n = 10) and distal (solid bars; n = 10). Time 0; initial inner mucus layer. At 60 min, mucus was aspirated and the inner mucus layer thickness measured (after removal). C: mucus thickness measured in proximal (PC) and distal colon (DC), outer loose mucus removed (inner mucus layer, time 0), and mucus thickness measured (20 min). Paired explants, unstimulated (n = 6) or stimulated (n = 6) with carbachol and PGE2 (10 μM of each; arrow); mucus thickness measured every 20 min. The difference between stimulated and unstimulated mucus thickness was significant in the proximal colon (P = 0.04), but not the distal colon (P = 0.59). D: immunostaining of Carnoy-fixed proximal (left) and distal (right) colon for Muc2 (anti-MUC2C3 and Alexa 488 anti-rabbit Ig, green) and DNA (DAPI, blue). Inner mucus layer (i) excludes bacteria (blue), but bacteria are present in the outer mucus layer (o). E: representative confocal Z-stacks of explants (tissue, blue) of proximal colon (n = 7) showing penetrability of mucus to fluorescent beads (red, 0.5 μm; purple, 1 μm; green, 2 μm). E1: between folds. E2: on top of folds. E3: explant stimulated with carbachol and PGE2 (10 μM of each) to induce release of new mucus and show uneven mucus with penetrable and impenetrable region (arrow). F: representative confocal Z-stack visualizing penetrability to fluorescent beads, distal colon. Bar in A = 0.5 mm; bars in D, E, and F = 50 μm.

Fig. 2.

Small intestine has one type of mucus, which is not attached. A: explant tissue from the duodenum (left), jejunum (middle), and ileum (right) mounted and the transparent mucus visualized with charcoal and mucus aspirated showing nonattached mucus (Supplemental Videos S3, S4, and S5, respectively). Mucus in duodenum and jejunum was intermixed with food particles and lipid droplets. B and C: mucus thickness of paired explants from the duodenum, jejunum, and ileum measured (n = 6) every 20 min (B) or stimulated with carbachol and PGE2 (10 μM of each; arrow in C). Stimulation gave a significantly thicker mucus layer in all segments, P = 0.0022, 0.0022, and 0.004 in duodenum, jejunum, and ileum, respectively. After the initial mucus thickness measurement, mucus was removed and remaining mucus thickness was measured (both time 0). D: immunostainings of Carnoy-fixed tissue sections for Muc2 (anti-MUC2C3 and Alexa 488 anti-rabbit Ig, green) and DNA (DAPI, blue). Because of mucus shrinkage caused by fixation and staining, the mucus appears threadlike. E–G: bacterial DNA (blue; arrows) and Muc2 mucin (green) show a few bacteria on the villus tip and between the villi, but no bacteria close to the crypt openings. E: duodenal villus tip. F: jejunal villus tip. G: ileal villus tip. H: fluorescent beads penetrate the mucus in ileum; experiment as in Fig. 1, E and F. Bar in A = 0.5 mm; bar in D1 = 100 μm; bars in D2, D3, and H = 50 μm; bars in E, F, and G = 10 μm.

Fig. 3.

The domes of Peyer's patches are covered by a penetrable mucus layer. A: mucus visualized with charcoal (arrow) on ileal explants containing 4 Peyer's patches. Nonattached mucus demonstrated in the movie (Supplemental Video S6). B: mucus on domes penetrable to fluorescent beads as shown in Fig. 1, E and F. The epithelium (blue) of the dome is convex (viewed slightly tilted) and completely covered by beads. Bar in A = 0.5 mm; bar in B = 50 μm.

Fig. 4.

The stomach has mucus layers that are firmly attached to the epithelium and penetrable to fluorescent beads. A: stomach explants from the corpus, mucus visualized with charcoal were gently aspirated (Supplemental Video S7; initial frame shown in A, left). A second movie recorded to illustrate the firm attachment of the outer layer in the stomach (Supplemental Video S8; initial frame shown in A, right). B: mucus thickness of stomach corpus explants measured in paired experiments (n = 5, ○) or with stimulated with carbachol and PGE2 (10 μM each; ●; arrow). Initial mucus thickness was measured, mucus was removed, and remaining mucus thickness was measured (both time 0). Mucus was significantly thicker in explants after stimulation compared with unstimulated samples, P = 0.0048. C: aspiration of mucus on stomach explants after stimulation of secretion by carbachol and PGE2 mucus (n = 5). D: Karnovsky-fixed, epoxy resin-embedded sections stained with periodic acid-Schiff (left), revealing a thin, striated mucus layer on top of the epithelium (arrow). E: Carnoy-fixed paraffin sections stained with anti-Muc5ac (green) and DNA stain (blue) show Muc5ac-positive goblet cells and mucus. Epithelial surface marked with dotted line. F: fluorescent beads sedimented for 60 min into the mucus as described in Fig. 1, E and F: unstimulated tissue (1), tissue stimulated for 20 min with carbachol and PGE2 (10 μM of each) (2), mucus secreted after 10 min stimulation into an apical pH 3 buffer (3). Bars in A = 0.5 mm; bar in D = 10 μm; bars in E and F = 50 μm.