Jenny K. Gustafsson

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

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.

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel‐forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyers patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate‐keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103+ type. In addition to the gel‐forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Composition and functional role of the mucus layers in the intestine

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

Bacteria penetrate the inner mucus layer before inflammation in the dextran sulfate colitis model.

Background Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described. Principal Findings When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells. Conclusion DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.

Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype

Ileal mucus in CftrΔ508 mice is more adherent, denser, and less penetrable than that of WT mice, but addition of bicarbonate normalizes the properties of CftrΔ508 mucus.

Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin

MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D′D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

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

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 Peyers patches. Stomach and colon had an inner mucus layer that was adherent to the epithelium. In contrast, the small intestine and Peyers 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.

New developments in goblet cell mucus secretion and function

Goblet cells and their main secretory product, mucus, have long been poorly appreciated; however, recent discoveries have changed this and placed these cells at the center stage of our understanding of mucosal biology and the immunology of the intestinal tract. The mucus system differs substantially between the small and large intestine, although it is built around MUC2 mucin polymers in both cases. Furthermore, that goblet cells and the regulation of their secretion also differ between these two parts of the intestine is of fundamental importance for a better understanding of mucosal immunology. There are several types of goblet cell that can be delineated based on their location and function. The surface colonic goblet cells secrete continuously to maintain the inner mucus layer, whereas goblet cells of the colonic and small intestinal crypts secrete upon stimulation, for example, after endocytosis or in response to acetyl choline. However, despite much progress in recent years, our understanding of goblet cell function and regulation is still in its infancy.

Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins

The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the gastrointestinal tract. To ensure that only mucus was analyzed, we combined collection from explant tissues mounted in perfusion chambers, liquid sample preparation, single-shot mass spectrometry, and specific bioinformatics tools, to characterize the proteome of the murine mucus from stomach to distal colon. With our approach, we identified ∼1,300 proteins in the mucus. We found no differences in the protein composition or abundance between sexes, but there were clear differences in mucus along the tract. Noticeably, mucus from duodenum showed similarities to the stomach, probably reflecting the normal distal transport. Qualitatively, there were, however, fewer differences than might had been anticipated, suggesting a relatively stable core proteome (∼80% of the total proteins identified). Quantitatively, we found significant differences (∼40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of such proteins that correlated with Muc2 (e.g., Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system.

Perspectives on Mucus Properties and Formation—Lessons from the Biochemical World

Our model of the MUC2 mucin shows a well-organized netlike gel that is cross-linked by six different covalent and noncovalent bonds. When the MUC2 mucin is packed in the mucin granule it is organized by an amino-terminal concatenated ring platform formed at high calcium and low pH. This packing allows an ordered release and a normal mucin expansion when calcium is removed and pH increased by bicarbonate. This process is defective in the absence of cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate transport. The expanded secreted mucin is suggested to be self-organizing by properties inherited in the MUC2 mucin and by proteolytic processes.