Henrik Sjövall

The gastrointestinal mucus system in health and disease

Mucins—large, highly glycosylated proteins—are important for the luminal protection of the gastrointestinal tract. Enterocytes have their apical surface covered by transmembrane mucins and goblet cells produce the secreted gel-forming mucins that form mucus. The small intestine has a single unattached mucus layer, which in cystic fibrosis becomes attached, accounting for the intestinal manifestations of this disease. The stomach and colon have two layers of mucus; the inner layer is attached and the outer layer is less dense and unattached. In the colon, the outer mucus layer is the habitat for commensal bacteria. The inner mucus layer is impervious to bacteria and is renewed every hour by surface goblet cells. The crypt goblet cells have the ability to restitute the mucus layer by secretion, for example after an ischaemic challenge. Proteases of certain parasites and some bacteria can cleave mucins and dissolve the mucus as part of their pathogenicity. The inner mucus layer can, however, also become penetrable to bacteria by several other mechanisms, including aberrations in the immune system. When bacteria reach the epithelial surface, the immune system is activated and inflammation is triggered. This mechanism might occur in some types of ulcerative colitis.

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.

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.

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (approximately 5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS-agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive beta-elimination and analyzed by nanoLC/mass spectrometry and MS(n). The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Gal beta 1-3/4-GlcNAc beta 1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were -Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.

T-Cell Activation in Patients With Irritable Bowel Syndrome

OBJECTIVES:Irritable bowel syndrome (IBS) has been found to be associated with low-grade immune activation in a subset of patients. We therefore investigated blood and colonic T-cell activity in IBS patients.METHODS:Blood samples were initially obtained from 74 IBS patients and 30 controls. Supplementary blood samples, to confirm data, were taken from another cohort (26 patients and 14 controls). In addition, colonic biopsies were taken from a third cohort (11 patients and 10 controls). Peripheral blood and colonic mononuclear cells were stimulated with anti-CD3/CD28 antibodies. Proliferation, cytokine secretion, and T-cell phenotype were investigated. IBS symptom severity was assessed.RESULTS:IBS patients displayed an activated phenotype with increased frequencies of blood T cells expressing CD69 and integrin β7/HLA-DR. Anti-CD3/CD28-stimulated blood and colonic T cells from IBS patients proliferated less than T cells from controls. IBS patients had an increased polyclonally stimulated T-cell secretion of IL-1β, which also weakly correlated with increased bowel habit dissatisfaction. Furthermore, despite normal frequencies of CD25high T cells in the blood of IBS patients, lower blood CD25high T-cell frequencies were modestly correlated with more bowel habit dissatisfaction and increased total IBS symptom severity.CONCLUSIONS:IBS patients have an increased frequency of activated T cells, demonstrated by the expression of activation markers and reduced proliferation in response to restimulation in vitro. The increased level of T-cell activation is consistent with the hypothesis of low-grade immune activation in IBS and may also be involved in symptom generation in IBS.

Functional CD4+CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity

Background: Factors determining the extension and degree of inflammation in the colonic mucosa of patients with ulcerative colitis (UC) are largely unknown, but CD4+CD25high regulatory T cells (Tregs) have been implicated to play an important role in suppressing inflammation. Therefore, the aims of this study were to determine whether colonic Tregs have suppressive effects on colonic effector T cells in UC and to analyze the association between segmental colonic Treg distribution and disease activity. Materials and Methods: The suppressive activity of colonic CD4+CD25high Tregs from patients with active UC was determined in coculture assays measuring proliferation and cytokine production. The frequency of Tregs and the expression of the Treg marker FOXP3 were analyzed with flow cytometry and RT‐PCR in isolated cells and the whole mucosa from patients with active and inactive disease, as well as healthy mucosa. Results: Colonic CD4+CD25high T cells from patients with UC suppressed the proliferation and cytokine secretion of colonic effector CD4+ T cells. Healthy controls but not patients with UC had lower Treg frequencies in the sigmoid than in the ascending colon. Patients with UC with active disease had increased frequency of colonic Tregs. The frequency of Tregs was positively correlated with colonic disease activity and serum C‐reactive protein. Conclusions: Colonic CD4+CD25high Tregs are able to suppress colonic effector T cell activity in vitro, and the Treg frequency in the inflamed intestine increases with disease activity in patients with active UC. This suggests that Tregs may be outnumbered by other inflammatory cells or that their suppressive activity may be influenced by the in vivo environment.

Small intestinal motility disturbances and bacterial overgrowth in patients with liver cirrhosis and portal hypertension

OBJECTIVES:Altered small bowel motility and a high prevalence of small intestinal bacterial overgrowth (SIBO) has been observed in patients with liver cirrhosis. Our aim was to explore the relationship between motility abnormalities, portal hypertension, and SIBO.METHODS:Twenty-four patients with liver cirrhosis were included. Twelve had portal hypertension (PH) and 12 had liver cirrhosis (LC) alone. Child-Pugh score was the same in the groups. Antroduodenojejunal pressure recordings were performed, and noninvasive variceal pressure measurements were undertaken. Thirty-two healthy volunteers served as a reference group. Bacterial cultures were obtained from jejunal aspirates.RESULTS:The PH group had a higher proportion of individual pressure waves that were retrograde in the proximal duodenum during phase II (52% vs 13% vs 8% of propagated contractions; p < 0.001) as well as postprandially (49% vs 18% vs 13%; p < 0.01) compared with LC and controls, respectively. Long clusters were more common in PH than in controls (9.1 ± 2.1 vs 4.9 ± 0.8; p < 0.05), and a higher motility index in phase III in the proximal and distal duodenum was seen in the PH as compared with the other groups. The mean variceal pressure was 21 ± 1 mm Hg. Motor abnormalities were not correlated to the level of variceal pressure. Thirty-three percent of the patients in the PH group but none in the LC group had SIBO.CONCLUSIONS:Abnormal small bowel motility and SIBO is common in patients with liver cirrhosis with concomitant portal hypertension. Portal hypertension per se might be significantly related to small bowel abnormalities observed in patients with liver cirrhosis.

Altered O‐glycosylation profile of MUC2 mucin occurs in active ulcerative colitis and is associated with increased inflammation

Background: The MUC2 mucin organizes the two mucus layers in the colon. This mucin carries a large number of O‐glycans that are assumed to be attachment sites for the commensal flora found in the outer mucus layer. Methods: Single biopsies from the sigmoid colon of controls (25) and patients with inactive (13) or active (15) ulcerative colitis (UC) were collected during routine colonoscopy. The insoluble MUC2 mucin was prepared and separated by gel electrophoresis, its relative amount estimated, its O‐glycans released, and glycans analyzed by novel sensitive glycomics chromatography / mass spectrometry providing information on glycan structures and relative abundances. The glycosylation pattern was related to the degree of mucosal inflammation and clinical severity of the disease. Results: The relative abundance of MUC2 showed high individual variability. Two major glycan profiles were found; a normal pattern in the control and inactive UC patients and an aberrant profile in patients with active colitis with an increase in a subset of the smaller glycans and a decrease of several complex glycans. The magnitude of this phenomenon was significantly related to both the degree of inflammation in the biopsies and also to some extent the severity of disease course. The aberrant profile was further shown to be reversible upon remission. Conclusions: In the majority of the active UC patients MUC2 mucin has an altered glycan profile as compared to inactive UC and control patients. Patients with strong alterations in the glycan pattern tended to have a more severe disease course. (Inflamm Bowel Dis 2011)