Publications by authors named "Gunnar C Hansson"

143 Publications

New generation ENaC inhibitors detach cystic fibrosis airway mucus bundles via sodium/hydrogen exchanger inhibition.

Eur J Pharmacol 2021 Aug 8;904:174123. Epub 2021 May 8.

Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Sweden. Electronic address:

Cystic fibrosis (CF) is a recessive inherited disease caused by mutations affecting anion transport by the epithelial ion channel cystic fibrosis transmembrane conductance regulator (CFTR). The disease is characterized by mucus accumulation in the airways and intestine, but the major cause of mortality in CF is airway mucus accumulation, leading to bacterial colonization, inflammation and respiratory failure. Several drug targets are under evaluation to alleviate airway mucus obstruction in CF and one of these targets is the epithelial sodium channel ENaC. To explore effects of ENaC inhibitors on mucus properties, we used two model systems to investigate mucus characteristics, mucus attachment in mouse ileum and mucus bundle transport in piglet airways. We quantified mucus attachment in explants from CFTR null (CF) mice and tracheobronchial explants from newborn CFTR null (CF) piglets to evaluate effects of ENaC or sodium/hydrogen exchanger (NHE) inhibitors on mucus attachment. ENaC inhibitors detached mucus in the CF mouse ileum, although the ileum lacks ENaC expression. This effect was mimicked by two NHE inhibitors. Airway mucus bundles were immobile in untreated newborn CF piglets but were detached by the therapeutic drug candidate AZD5634 (patent WO, 2015140527). These results suggest that the ENaC inhibitor AZD5634 causes detachment of CF mucus in the ileum and airway via NHE inhibition and that drug design should focus on NHE instead of ENaC inhibition.
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http://dx.doi.org/10.1016/j.ejphar.2021.174123DOI Listing
August 2021

An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.

Science 2021 04;372(6539)

Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, 40530 Gothenburg, Sweden.

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.
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http://dx.doi.org/10.1126/science.abb1590DOI Listing
April 2021

Association between and irritable bowel syndrome with diarrhoea.

Gut 2021 Jun 11;70(6):1117-1129. Epub 2020 Nov 11.

Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

Objective: The incidence of IBS increases following enteric infections, suggesting a causative role for microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. Here, we used metaproteomics to investigate potential associations between mucus-resident microbiota and IBS symptoms.

Design: Mucus samples were prospectively collected from sigmoid colon biopsies from patients with IBS and healthy volunteers, and their microbial protein composition analysed by mass spectrometry. Observations were verified by immunofluorescence, electron microscopy and real-time PCR, further confirmed in a second cohort, and correlated with comprehensive profiling of clinical characteristics and mucosal immune responses.

Results: Metaproteomic analysis of colon mucus samples identified peptides from potentially pathogenic species in a subset of patients with IBS. Using multiple diagnostic methods, mucosal colonisation was detected in a total of 19/62 (31%) patients with IBS from two prospective cohorts, versus 0/31 healthy volunteers (p<0.001). The prevalence of colonisation in IBS with diarrhoea (IBS-D) was 40% in both cohorts (p=0.02 and p=0.006 vs controls). attachment to the colonocyte apical membrane was observed in 20% of patients with IBS and associated with accelerated oro-anal transit, mild mucosal inflammation, mast cell activation and alterations of molecular pathways linked to bacterial uptake and ion-fluid homeostasis. Metronidazole treatment paradoxically promoted relocation into goblet cell secretory granules-possibly representing a novel bacterial strategy to evade antibiotics.

Conclusion: Mucosal colonisation was significantly more common in IBS and associated with distinctive clinical, histological and molecular characteristics. Our observations suggest a role for in the pathogenesis of IBS, particularly IBS-D.
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http://dx.doi.org/10.1136/gutjnl-2020-321466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8108289PMC
June 2021

Obesity-associated microbiota contributes to mucus layer defects in genetically obese mice.

J Biol Chem 2020 11 8;295(46):15712-15726. Epub 2020 Sep 8.

Wallenberg Laboratory, Department of Molecular and Clinical Medicine, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Clinical Physiology, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden.

The intestinal mucus layer is a physical barrier separating the tremendous number of gut bacteria from the host epithelium. Defects in the mucus layer have been linked to metabolic diseases, but previous studies predominantly investigated mucus function during high-caloric/low-fiber dietary interventions, thus making it difficult to separate effects mediated directly through diet quality from potential obesity-dependent effects. As such, we decided to examine mucus function in mouse models with metabolic disease to distinguish these factors. Here we show that, in contrast to their lean littermates, genetically obese (ob/ob) mice have a defective inner colonic mucus layer that is characterized by increased penetrability and a reduced mucus growth rate. Exploiting the coprophagic behavior of mice, we next co-housed ob/ob and lean mice to investigate if the gut microbiota contributed to these phenotypes. Co-housing rescued the defect of the mucus growth rate, whereas mucus penetrability displayed an intermediate phenotype in both mouse groups. Of note, non-obese diabetic mice with high blood glucose levels displayed a healthy colonic mucus barrier, indicating that the mucus defect is obesity- rather than glucose-mediated. Thus, our data suggest that the gut microbiota community of obesity-prone mice may regulate obesity-associated defects in the colonic mucosal barrier, even in the presence of dietary fiber.
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http://dx.doi.org/10.1074/jbc.RA120.015771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667970PMC
November 2020

Normal murine respiratory tract has its mucus concentrated in clouds based on the Muc5b mucin.

Am J Physiol Lung Cell Mol Physiol 2020 06 29;318(6):L1270-L1279. Epub 2020 Apr 29.

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, , , and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 µm/s compared with 20 µm/s for beads not associated with clouds. In mice, Muc5b formed mucus strands attached to the tissue surface, while in mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs.
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http://dx.doi.org/10.1152/ajplung.00485.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347266PMC
June 2020

Mucins and the Microbiome.

Authors:
Gunnar C Hansson

Annu Rev Biochem 2020 06 3;89:769-793. Epub 2020 Apr 3.

Department of Medical Biochemistry, University of Gothenburg, SE 405 30 Gothenburg, Sweden; email:

Generating the barriers that protect our inner surfaces from bacteria and other challenges requires large glycoproteins called mucins. These come in two types, gel-forming and transmembrane, all characterized by large, highly -glycosylated mucin domains that are diversely decorated by Golgi glycosyltransferases to become extended rodlike structures. The general functions of mucins on internal epithelial surfaces are to wash away microorganisms and, even more importantly, to build protective barriers. The latter function is most evident in the large intestine, where the inner mucus layer separates the numerous commensal bacteria from the epithelial cells. The host's conversion of MUC2 to the outer mucus layer allows bacteria to degrade the mucin glycans and recover the energy content that is then shared with the host. The molecular nature of the mucins is complex, and how they construct the extracellular complex glycocalyx and mucus is poorly understood and a future biochemical challenge.
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http://dx.doi.org/10.1146/annurev-biochem-011520-105053DOI Listing
June 2020

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps.

Anal Biochem 2020 05 25;597:113668. Epub 2020 Mar 25.

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden. Electronic address:

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.
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http://dx.doi.org/10.1016/j.ab.2020.113668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184670PMC
May 2020

Membrane mucins of the intestine at a glance.

J Cell Sci 2020 03 13;133(5). Epub 2020 Mar 13.

Dept. Medical Biochemistry, University of Gothenburg, Box 440, 40530 Gothenburg, Sweden

Membrane mucins cover most mucosal surfaces throughout the human body. The intestine harbors complex population of microorganisms (the microbiota) and numerous exogenous molecules that can harm the epithelium. In the colon, where the microbial burden is high, a mucus barrier forms the first line of defense by keeping bacteria away from the epithelial cells. In the small intestine where the mucus layer is less organized, microbes are kept at bay by peristalsis and antimicrobial peptides. Additionally, a dense glycocalyx consisting of extended and heavily glycosylated membrane mucins covers the surface of enterocytes. Whereas many aspects of mucosal barriers are being discovered, the function of membrane mucins remains a largely overlooked topic, mainly because we lack the necessary reagents and experimental animal models to investigate these large glycoproteins. In this Cell Science at a Glance article and accompanying poster, we highlight central concepts of membrane mucin biology and the role of membrane mucins as integral components of intestinal mucosal barriers. We also present the current consensus concerning the role of membrane mucins in host-microbe interactions. Moreover, we discuss how regulatory circuits that govern membrane mucins in the healthy gut display strong overlap with pathways that are perturbed during chronic inflammation. Finally, we review how dysregulation of intestinal membrane mucins may contribute to human diseases, such as inflammation and cancer.
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http://dx.doi.org/10.1242/jcs.240929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075048PMC
March 2020

Protein Turnover in Epithelial Cells and Mucus along the Gastrointestinal Tract Is Coordinated by the Spatial Location and Microbiota.

Cell Rep 2020 01 28;30(4):1077-1087.e3. Epub 2020 Jan 28.

Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden. Electronic address:

The gastrointestinal tract is covered by a single layer of epithelial cells that, together with the mucus layers, protect the underlying tissue from microbial invasion. The epithelium has one of the highest turnover rates in the body. Using stable isotope labeling, high-resolution mass spectrometry, and computational analysis, we report a comprehensive dataset of the turnover of more than 3,000 and the expression of more than 5,000 intestinal epithelial cell proteins, analyzed under conventional and germ-free conditions across five different segments in mouse intestine. The median protein half-life is shorter in the small intestine than in the colon. Differences in protein turnover rates along the intestinal tract can be explained by distinct physiological and immune-related functions between the small and large intestine. An absence of microbiota results in an approximately 1 day longer protein half-life in germ-free animals.
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http://dx.doi.org/10.1016/j.celrep.2019.12.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996021PMC
January 2020

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2-processing properties.

J Biol Chem 2019 11 29;294(45):17075-17089. Epub 2019 Sep 29.

Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, SE-405 30 Gothenburg, Sweden

Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.
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http://dx.doi.org/10.1074/jbc.RA119.009940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851300PMC
November 2019

The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function.

J Exp Med 2019 11 16;216(11):2602-2618. Epub 2019 Aug 16.

Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled , detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.
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http://dx.doi.org/10.1084/jem.20190679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829596PMC
November 2019

The human transmembrane mucin MUC17 responds to TNFα by increased presentation at the plasma membrane.

Biochem J 2019 08 22;476(16):2281-2295. Epub 2019 Aug 22.

Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden

Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended -glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNFα resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic , indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.
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http://dx.doi.org/10.1042/BCJ20190180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705488PMC
August 2019

Potential roles of gut microbiome and metabolites in modulating ALS in mice.

Nature 2019 08 22;572(7770):474-480. Epub 2019 Jul 22.

Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder, in which the clinical manifestations may be influenced by genetic and unknown environmental factors. Here we show that ALS-prone Sod1 transgenic (Sod1-Tg) mice have a pre-symptomatic, vivarium-dependent dysbiosis and altered metabolite configuration, coupled with an exacerbated disease under germ-free conditions or after treatment with broad-spectrum antibiotics. We correlate eleven distinct commensal bacteria at our vivarium with the severity of ALS in mice, and by their individual supplementation into antibiotic-treated Sod1-Tg mice we demonstrate that Akkermansia muciniphila (AM) ameliorates whereas Ruminococcus torques and Parabacteroides distasonis exacerbate the symptoms of ALS. Furthermore, Sod1-Tg mice that are administered AM are found to accumulate AM-associated nicotinamide in the central nervous system, and systemic supplementation of nicotinamide improves motor symptoms and gene expression patterns in the spinal cord of Sod1-Tg mice. In humans, we identify distinct microbiome and metabolite configurations-including reduced levels of nicotinamide systemically and in the cerebrospinal fluid-in a small preliminary study that compares patients with ALS with household controls. We suggest that environmentally driven microbiome-brain interactions may modulate ALS in mice, and we call for similar investigations in the human form of the disease.
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http://dx.doi.org/10.1038/s41586-019-1443-5DOI Listing
August 2019

Normal Calcium-Activated Anion Secretion in a Mouse Selectively Lacking TMEM16A in Intestinal Epithelium.

Front Physiol 2019 13;10:694. Epub 2019 Jun 13.

Centro de Estudios Científicos (CECs), Valdivia, Chile.

Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with and/or intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal knockout and double mutants with dual and intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by silencing as was observed when was deleted from mouse intestine. silencing neither affected animal survival nor modified the lethality observed in the intestinal -null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.
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http://dx.doi.org/10.3389/fphys.2019.00694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585864PMC
June 2019

Mucus Architecture and Near-Surface Swimming Affect Distinct Salmonella Typhimurium Infection Patterns along the Murine Intestinal Tract.

Cell Rep 2019 05;27(9):2665-2678.e3

Institute for Microbiology, ETH Zürich, 8093 Zürich, Switzerland. Electronic address:

Mucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis.
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http://dx.doi.org/10.1016/j.celrep.2019.04.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547020PMC
May 2019

Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis.

Gut 2019 12 26;68(12):2142-2151. Epub 2019 Mar 26.

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

Objective: The colonic inner mucus layer protects us from pathogens and commensal-induced inflammation, and has been shown to be defective in active UC. The aim of this study was to determine the underlying compositional alterations, their molecular background and potential contribution to UC pathogenesis.

Design: In this single-centre case-control study, sigmoid colon biopsies were obtained from patients with UC with ongoing inflammation (n=36) or in remission (n=28), and from 47 patients without colonic disease. Mucus samples were collected from biopsies ex vivo, and their protein composition analysed by nanoliquid chromatography-tandem mass spectrometry. Mucus penetrability and goblet cell responses to microbial stimulus were assessed in a subset of patients.

Results: The core mucus proteome was found to consist of a small set of 29 secreted/transmembrane proteins. In active UC, major structural mucus components including the mucin MUC2 (p<0.0001) were reduced, also in non-inflamed segments. Active UC was associated with decreased numbers of sentinel goblet cells and attenuation of the goblet cell secretory response to microbial challenge. Abnormal penetrability of the inner mucus layer was observed in a subset of patients with UC (12/40; 30%). Proteomic alterations in penetrable mucus samples included a reduction of the SLC26A3 apical membrane anion exchanger, which supplies bicarbonate required for colonic mucin barrier formation.

Conclusion: Core mucus structural components were reduced in active UC. These alterations were associated with attenuation of the goblet cell secretory response to microbial challenge, but occurred independent of local inflammation. Thus, mucus abnormalities are likely to contribute to UC pathogenesis.
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http://dx.doi.org/10.1136/gutjnl-2018-317571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872445PMC
December 2019

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals.

Sci Rep 2018 11 30;8(1):17503. Epub 2018 Nov 30.

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.
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http://dx.doi.org/10.1038/s41598-018-35499-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269512PMC
November 2018

Assembly, Release, and Transport of Airway Mucins in Pigs and Humans.

Ann Am Thorac Soc 2018 11;15(Suppl 3):S159-S163

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

The respiratory system is protected from inhaled particles and microbes by the mucociliary system. This system differs between animal species, where pigs and humans have numerous submucosal glands. The polymer-forming mucin, MUC5B, is packed in a highly organized way in granules of the mucus-secreting cells in the glands. Upon secretion, the packed MUC5B is flushed out by a chloride- and bicarbonate-rich fluid from the cystic fibrosis transmembrane conductance regulator-expressing serosal cells located at the most distal part of the gland. The bicarbonate raises the pH and removes calcium from the N terminus of MUC5B, allowing the mucin to be pulled out into a linear polymer. Thousands of such polymers gather in bundles in the submucosal gland duct, and these bundles appear at the opening of the glands. They are moved by the beating cilia, and sweep over the airway surface and are patchily coated with the MUC5AC mucin from the surface goblet cells. The movement of these bundles is controlled by the MUC5AC mucin attachment/detachment to the goblet cells. Thus, higher animals with submucosal glands and large diameters of the proximal airways are efficiently cleaned by the thick mucus bundles sweeping the airway surface and moving particles and bacteria toward the larynx.
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http://dx.doi.org/10.1513/AnnalsATS.201804-238AWDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855098PMC
November 2018

Dietary destabilisation of the balance between the microbiota and the colonic mucus barrier.

Gut Microbes 2019 25;10(2):246-250. Epub 2018 Sep 25.

a Department of Medical Biochemistry , University of Gothenburg , Gothenburg , Sweden.

It has long been acknowledged that dietary fibres are important to maintain a healthy gut. Over the past decade, several studies have shown that loss of complex polysaccharides from the Western diet has resulted in alterations to our colonic microbiota. The concurrent increase in the incidence of inflammatory bowel disease in the Western world has driven us to explore the potential mechanistic link between diet, the microbiota and the host defence systems that normally prevent inflammation. Using mice fed a low fibre Western-style diet and robust live tissue analytical methods we have now provided evidence that this diet impairs the colonic inner mucus layer that normally separates bacteria from host cells. Western societies urgently need to develop their understanding of the molecular mechanisms of the diet-microbiota-mucus axis and its implications for inflammatory diseases.
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http://dx.doi.org/10.1080/19490976.2018.1513765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546334PMC
August 2019

Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs.

JCI Insight 2018 09 6;3(17). Epub 2018 Sep 6.

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.
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http://dx.doi.org/10.1172/jci.insight.120994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171804PMC
September 2018

Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity.

EBioMedicine 2018 Jul 7;33:134-143. Epub 2018 Jun 7.

Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, 40530 Gothenburg, Sweden. Electronic address:

Many epithelial surfaces of the body are covered with protective mucus, and disrupted mucus homeostasis is coupled to diseases such as ulcerative colitis, helminth infection, cystic fibrosis, and chronic obstructive lung disease. However, little is known how a balanced mucus system is maintained. By investigating the involvement of proteases in colonic mucus dynamics we identified metalloprotease activity to be a key contributor to mucus expansion. The effect was mediated by calcium-activated chloride channel regulator 1 (CLCA1) as application of recombinant CLCA1 on intestinal mucus in freshly dissected tissue resulted in increased mucus thickness independently of ion and mucus secretion, but dependent on its metallohydrolase activity. Further, CLCA1 modulated mucus dynamics in both human and mouse, and knock-out of CLCA1 in mice was compensated for by cysteine proteases. Our results suggest that CLCA1 is involved in intestinal mucus homeostasis by facilitating processing and removal of mucus to prevent stagnation. In light of our findings, we suggest future studies to investigate if upregulation of CLCA1 in diseases associated with mucus accumulation could facilitate removal of mucus in an attempt to maintain homeostasis.
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http://dx.doi.org/10.1016/j.ebiom.2018.05.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085540PMC
July 2018

The mucus bundles responsible for airway cleaning are retained in cystic fibrosis and by cholinergic stimulation.

Eur Respir J 2018 08 30;52(2). Epub 2018 Aug 30.

Dept of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden.

The beneficial effect of anticholinergic therapy for chronic lung diseases such as chronic obstructive pulmonary disease (COPD) is well documented, although cholinergic stimulation paradoxically inhibits liquid absorption, increases ciliary beat frequency and increases airway surface liquid transport.Using pig tracheobronchial explants, we quantified basal mucus transport before as well as after incubation with the clinically used antimuscarinic compound ipratropium bromide (Atrovent) and stimulation with acetylcholine.As expected, surface liquid transport was increased by acetylcholine and carbachol. In contrast, the mucus bundles secreted from the submucosal glands normally transported on the cilia were stopped from moving by acetylcholine, an effect inhibited by ipratropium bromide. Interestingly, in pigs lacking a functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel, the mucus bundles were almost immobile. As in wild-type pigs, CF surface liquid transport increased after carbachol stimulation. The stagnant CF mucus bundles were trapped on the tracheal surface attached to the surface goblet cells. bacteria were moved by the mucus bundles in wild-type but not CF pigs.Acetylcholine thus uncouples airway surface liquid transport from transport of the surface mucus bundles as the bundles are dynamically inhibited by acetylcholine and the CFTR channel, explaining initiation of CF and COPD, and opening novel therapeutic windows.
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http://dx.doi.org/10.1183/13993003.00457-2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247115PMC
August 2018

Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.

J Biol Chem 2018 04 13;293(15):5746-5754. Epub 2018 Feb 13.

From the Department of Medical Biochemistry, University of Gothenburg, 40530 Gothenburg, Sweden,

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.
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http://dx.doi.org/10.1074/jbc.RA117.001014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900763PMC
April 2018

Bifidobacteria or Fiber Protects against Diet-Induced Microbiota-Mediated Colonic Mucus Deterioration.

Cell Host Microbe 2018 01 21;23(1):27-40.e7. Epub 2017 Dec 21.

Wallenberg Laboratory and Sahlgrenska Center for Cardiovascular and Metabolic Research, Department of Molecular and Clinical Medicine, Institute of Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden; Novo Nordisk Foundation Center for Basic Metabolic Research, Section for Metabolic Receptology and Enteroendocrinology, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Diet strongly affects gut microbiota composition, and gut bacteria can influence the colonic mucus layer, a physical barrier that separates trillions of gut bacteria from the host. However, the interplay between a Western style diet (WSD), gut microbiota composition, and the intestinal mucus layer is less clear. Here we show that mice fed a WSD have an altered colonic microbiota composition that causes increased penetrability and a reduced growth rate of the inner mucus layer. Both barrier defects can be prevented by transplanting microbiota from chow-fed mice. In addition, we found that administration of Bifidobacterium longum was sufficient to restore mucus growth, whereas administration of the fiber inulin prevented increased mucus penetrability in WSD-fed mice. We hypothesize that the presence of distinct bacteria is crucial for proper mucus function. If confirmed in humans, these findings may help to better understand diseases with an affected mucus layer, such as ulcerative colitis.
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http://dx.doi.org/10.1016/j.chom.2017.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764785PMC
January 2018

Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB.

Cell Rep 2017 Nov;21(8):2090-2103

Institute of Biochemistry, University of Kiel, Kiel, Germany. Electronic address:

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.
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http://dx.doi.org/10.1016/j.celrep.2017.10.087DOI Listing
November 2017

Highly Accurate Identification of Cystic Precursor Lesions of Pancreatic Cancer Through Targeted Mass Spectrometry: A Phase IIc Diagnostic Study.

J Clin Oncol 2018 02 22;36(4):367-375. Epub 2017 Nov 22.

Karolina S. Jabbar, Liisa Arike, and Gunnar C. Hansson, University of Gothenburg; Karolina S. Jabbar and Riadh Sadik, Sahlgrenska University Hospital, Gothenburg, Sweden; and Caroline S. Verbeke, University of Oslo, Oslo, Norway.

Purpose Pancreatic cystic lesions are common incidental findings on imaging, but up to half may be forerunners of pancreatic cancer. Therefore, accurate differential diagnosis is crucial for correct patient management. Unfortunately, currently available diagnostic methods cannot robustly identify premalignant and malignant pancreatic cystic lesions. Methods Cyst fluid samples obtained by routine endoscopic ultrasound-guided aspiration were used for the analyses. In a cohort of 24 patients, eight biomarker candidates for malignant potential and high-grade dysplasia/cancer were identified by an explorative proteomic approach. Subsequently, a quantitative analysis, using 30 heavy-labeled peptides from the biomarkers and parallel reaction monitoring mass spectrometry, was devised, tested in a training cohort of 80, and prospectively evaluated in a validation cohort of 68 patients. End points were surgical pathology diagnosis/clinical follow-up. Diagnostic assessments were blinded to mass spectrometry results. Results The optimal set of markers for detecting malignant potential was a panel of peptides from mucin-5AC and mucin-2, which could discriminate premalignant/malignant lesions from benign with an accuracy of 97% (95% CI, 89% to 99%) in the validation cohort. This result compared favorably with the accuracy of standard analyses: cyst fluid carcinoembryonic antigen (61%; 95% CI, 46% to 74%; P < .001) and cytology (84%; 95% CI, 71% to 92%; P = .02). A combination of proteins mucin-5AC and prostate stem-cell antigen could identify high-grade dysplasia/cancer with an accuracy of 96% (95% CI, 90% to 99%), and detected 95% of malignant/severely dysplastic lesions, compared with 35% and 50% for carcinoembryonic antigen and cytology ( P < .001 and P = .003, respectively). Conclusion Targeted mass spectrometry analysis of just three cyst fluid biomarkers provides highly accurate identification and assessment of cystic precursors to pancreatic adenocarcinoma. Additional studies should determine whether the method can facilitate timely cancer diagnosis, successful intervention, and prevention.
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http://dx.doi.org/10.1200/JCO.2017.73.7288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805478PMC
February 2018

Progress in understanding mucus abnormalities in cystic fibrosis airways.

J Cyst Fibros 2018 03 23;17(2S):S35-S39. Epub 2017 Sep 23.

Institute of Anatomy, University of Lübeck, 23562 Lübeck, Germany; Airway Research Center North (ARCN), German Center for Lung Research (DZL), Germany.

Normal airways below the carina maintain an essentially sterile environment via a multi-pronged innate defence system that includes mucus clearance via mucociliary clearance and cough, multiple antimicrobials and cellular components including macrophages and neutrophils. In cystic fibrosis (CF), loss of CFTR function compromises these defences, and with present standard of care virtually all people with CF eventually develop mucus accumulation, plugging and chronic infections. This review focuses on how mucus is affected by CFTR loss.
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http://dx.doi.org/10.1016/j.jcf.2017.09.003DOI Listing
March 2018

The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin.

Biochem Biophys Res Commun 2017 10 30;492(3):331-337. Epub 2017 Aug 30.

Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden. Electronic address:

To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.
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http://dx.doi.org/10.1016/j.bbrc.2017.08.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596833PMC
October 2017

Bacteria Tell Us How to Protect Our Intestine.

Cell Host Microbe 2017 Jul;22(1):3-4

Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden. Electronic address:

The inner colon mucus layer capacity to separate bacteria from the epithelium is dependent on bacterial colonizers signaling to the host epithelium. In this issue of Cell Host & Microbe, Wlodarska et al. (2017) demonstrate that the mucin-utilizing Peptostreptococcus russellii protects the host from inflammatory disease via metabolite signals.
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http://dx.doi.org/10.1016/j.chom.2017.06.011DOI Listing
July 2017