| 1. |
- Ambort, Daniel, 1978, et al.
(author)
-
Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin.
- 2012
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 109:15, s. 5645-50
-
Journal article (peer-reviewed)abstract
- 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 Ca(2+) 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 Ca(2+) 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.
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| 2. |
- Ambort, Daniel, 1978, et al.
(author)
-
Perspectives on mucus properties and formation--lessons from the biochemical world.
- 2012
-
In: Cold Spring Harbor perspectives in medicine. - : Cold Spring Harbor Laboratory. - 2157-1422. ; 2:11
-
Research review (peer-reviewed)abstract
- 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.
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| 3. |
- Arike, Liisa, et al.
(author)
-
Protein Turnover in Epithelial Cells and Mucus along the Gastrointestinal Tract Is Coordinated by the Spatial Location and Microbiota
- 2020
-
In: Cell Reports. - : Elsevier BV. - 2211-1247. ; 30:4, s. 1077-1087
-
Journal article (peer-reviewed)abstract
- 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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| 4. |
- Bos, Meike F., et al.
(author)
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Goblet cell interactions reorient bundled mucus strands for efficient airway clearance
- 2023
-
In: PNAS Nexus. - 2752-6542. ; 2:11
-
Journal article (peer-reviewed)abstract
- The respiratory tract of larger animals is cleared by sweeping bundled strands along the airway surface. These bundled strands can be millimetric in length and consist of MUC5B mucin. They are produced by submucosal glands, and upon emerging from these glands, the long axis of the bundled strands is oriented along the cilia-mediated flow toward the oral cavity. However, after release, the bundled strands are found to have turned orthogonal to the flow, which maximizes their clearance potential. How this unexpected reorientation is accomplished is presently not well understood. Recent experiments suggest that the reorientation process involves bundled strands sticking to MUC5AC mucus threads, which are tethered to the goblet cells. Such goblet cells are present in small numbers throughout the airway epithelium. Here, we develop a minimal model for reorientation of bundled mucus strands through adhesive interactions with surface goblet cells. Our simulations reveal that goblet cell interactions can reorient the bundled strands within 10 mm of release-making reorientation on the length scale of the tracheal tube feasible-and can stabilize the orthogonal orientation. Our model also reproduces other experimental observations such as strong velocity fluctuations and significant slow-down of the bundled strand with respect to the cilia-mediated flow. We further provide insight into the strand turning mechanism by examining the effect of strand shape on the impulse exerted by a single goblet cell. We conclude that goblet cell-mediated reorientation is a viable route for bundled strand reorientation, which should be further validated in future experiment.
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| 5. |
- Dolan, Brendan, et al.
(author)
-
Clearance of small intestinal crypts involves goblet cell mucus secretion by intracellular granule rupture and enterocyte ion transport
- 2022
-
In: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 15:752
-
Journal article (peer-reviewed)abstract
- Goblet cells in the small intestinal crypts contain large numbers of mucin granules that are rapidly discharged to clean bacteria from the crypt. Because acetylcholine released by neuronal and nonneuronal cells controls many aspects of intestinal epithelial function, we used tissue explants and organoids to investigate the response of the small intestinal crypt to cholinergic stimulation. The activation of muscarinic acetylcholine receptors initiated a coordinated and rapid emptying of crypt goblet cells that flushed the crypt contents into the intestinal lumen. Cholinergic stimulation induced an expansion of the granule contents followed by intracellular rupture of the mucin granules. The mucus expanded intracellularly before the rupture of the goblet cell apical membrane and continued to expand after its release into the crypt lumen. The goblet cells recovered from membrane rupture and replenished their stores of mucin granules. Mucus secretion from the goblet cells depended on Ca2+ signaling and the expansion of the mucus in the crypt depended on gap junctions and on ion and water transport by enterocytes adjacent to the goblet cells. This distinctive mode of mucus secretion, which we refer to as “expanding secretion,” efficiently cleans the small intestine crypt through coordinated mucus, ion, and fluid secretion by goblet cells and enterocytes.
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| 6. |
- El Hachmane, Michael, et al.
(author)
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Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1
- 2018
-
In: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 475, s. 691-704
-
Journal article (peer-reviewed)abstract
- In the present study, we have applied ratiometric measurements of intracellular Ca2+ concentrations ([Ca2+](i)) to show that extracellularly applied ATP (adenosine triphosphate) (100 mM) stimulates store-operated Ca2+ entry (SOCE) in 3T3-L1 adipocytes. ATP produced a rapid increase in [Ca2+](i) consisting of an initial transient elevation followed by a sustained elevated phase that could be observed only in the presence of extracellular Ca2+. Gene expression data and [Ca2+](i) recordings with uridine-50-triphosphate or with the phospholipase C (PLC) inhibitor U73122 demonstrated the involvement of purinergic P2Y2 receptors and the PLC/inositol trisphosphate pathway. The [Ca2+](i) elevation produced by reintroduction of a Ca2+-containing intracellular solution to adipocytes exposed to ATP in the absence of Ca2+ was diminished by known SOCE antagonists. The chief molecular components of SOCE, the stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1), were detected at the mRNA and protein level. Moreover, SOCE was largely diminished in cells where STIM1 and/or ORAI1 had been silenced by small interfering (si) RNA. We conclude that extracellular ATP activates SOCE in white adipocytes, an effect predominantly mediated by STIM1 and ORAI1.
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| 7. |
- Ermund, Anna, et al.
(author)
-
Assembly, Release, and Transport of Airway Mucins in Pigs and Humans
- 2018
-
In: Annals of the American Thoracic Society. - 1546-3222. ; 15
-
Journal article (peer-reviewed)abstract
- 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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| 8. |
- Ermund, Anna
(author)
-
Bioactive Lipids in Nociception
- 2008
-
Doctoral thesis (other academic/artistic)abstract
- This thesis focuses on bioactive lipids as (1) metabolites of the widely used antipyretic and analgesic drug acetaminophen and (2) activators of the ion channel TRPV1, an important downstream target for inflammatory mediators, in the phospholipase C (PLC)/TRPV1 signaling pathway. Evidence is presented for a fatty acid amide hydrolase (FAAH)-dependent fatty acid conjugation of p-aminophenol, a known acetaminophen metabolite, to form the potent TRPV1 activator AM404 in the central nervous system. We show that acetaminophen is able to reduce the content of prostanoids in brain and kidney, but this effect is independent of FAAH. While ibuprofen produces a similar reduction as acetaminophen of the PGE2 level in brain, only acetaminophen displays antinociceptive activity in animal tests of acute non-inflammatory pain, indicating that other mechanisms than central cyclooxygenase inhibition must be sought to explain the analgesic effect of acetaminophen in these tests. Knowledge of the mechanisms of action of acetaminophen may help to develop analogs with improved analgesic activity. Many TRP ion channels, including TRPV1, are regulated by PLC-coupled surface receptors. After PLC-dependent cleavage of phosphatidylinositol bisphosphate into diacylglycerol (DAG) and inositoltrisphosphate, diacylglycerol may be further metabolized to monoacyl-glycerols, such as 2-arachidonoylglycerol (2-AG) and 2-oleoylglycerol by the action of DAG lipase. 2-Arachidonoylglycerol and 1-AG activate both native TRPV1 on sensory nerve fibers in rodent mesenteric arteries and heterologously expressed rat and human TRPV1. The effects of 2-AG and the metabolically stable analog noladin ether were almost absent in TRPV1 gene-deficient mice. Stimulation of isolated rat dorsal root ganglia with the inflammatory mediators bradykinin and ATP led to an increase in the level of 2-AG, whereas the levels of anandamide and 2-oleoylglycerol were unaffected. Extensive metabolism of d8-2-AG and d5-1-AG, in contrast to d8-anandamide, was demonstrated in homogenates of rat mesenteric arteries. The monoacylglycerol lipase inhibitor MAFP prevented the metabolism of these lipids and enhanced the TRPV1-mediated vascular effects of 2-AG and 1-AG, but not anandamide. The existence of a regulated biosynthesis and enzymatic degradation of 2-AG and 1-AG in TRPV1-containing tissues is compatible with a physiological role for these monoacylglycerols as messengers in the PLC/TRPV1 signaling cascade. Bioactive lipids, with TRPV1 activity, can be formed via drug metabolism and after stimulation of PLC-coupled surface receptors.
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| 9. |
- Ermund, Anna, et al.
(author)
-
Hyper-osmolarity and calcium chelation: Effects on cystic fibrosis mucus
- 2015
-
In: European Journal of Pharmacology. - : Elsevier BV. - 0014-2999. ; 764, s. 109-117
-
Journal article (peer-reviewed)abstract
- A non-functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) leads to the disease cystic fibrosis (CF). Although the CFTR is expressed in multiple organs, pulmonary disease is the major cause of illness and death in patients with CF. Stagnant mucus, causing airway obstruction, bacterial overgrowth, persistent inflammation and tissue destruction characterizes the disease, but how the defect in CFTR function is coupled to the mucus phenotype is still controversial. We have recently shown that bicarbonate ions passing through CFTR are necessary for proper unfolding of the MUC2 mucin, thus highlighting the importance of bicarbonate ion transport via the CFTR and the ability of these ions to raise the pH and chelate calcium bound to the mucin as the important steps in forming normal mucus. In order to find potential CF treatments and expand our knowledge about the usefulness of bicarbonate as an active ingredient in formulations to alleviate mucus plugging, we used an Ussing-type chamber and explants from the F508del-CFTR mutant mouse ileum to test the effect of calcium chelators on mucus attachment, either in isolation or in combination with osmolytes such as mannitol or hypertonic saline. We found that increasing the concentration of bicarbonate, both alone or in combination with increased osmolarity of the solution, detached the otherwise attached CF mucus. (C) 2015 Elsevier B.V. All rights reserved.
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| 10. |
- Ermund, Anna, et al.
(author)
-
Hypertonic saline releases the attached small intestinal cystic fibrosis mucus.
- 2015
-
In: Clinical and experimental pharmacology & physiology. - : Wiley. - 1440-1681 .- 0305-1870. ; 42:1, s. 69-75
-
Journal article (peer-reviewed)abstract
- Hypertonic saline inhalation has become a cornerstone in the treatment of cystic fibrosis (CF), but its effect on CF mucus is still not understood. In CF, mucus stagnates in the airways, causing mucus plugging, and forming a substrate for bacterial invasion. Using horizontal Ussing-type chambers to allow easy access to the tissue, we have recently shown that the small intestinal mucus of CF mice is attached to the epithelium and not freely movable as opposed to normal mucus, thus pointing to a similarity between the CF mucus in the ileum and airways. In the same type of system, we investigated how hypertonic saline affects mucus thickness, attachment and penetrability to fluorescent beads the size of bacteria in ileal explants from the cystic fibrosis transmembrane conductance regulator mutant (ΔF508) mouse, in order to characterize how this common therapy affects mucus properties. Hypertonic saline (1.75-5%) detached the mucus from the epithelium, but the mucus remained impenetrable to beads the size of bacteria. This approach might be used to test other mucolytic interventions in CF.
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| 11. |
- Ermund, Anna, et al.
(author)
-
Mucus properties and goblet cell quantification in mouse, rat and human ileal Peyer's patches.
- 2013
-
In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:12
-
Journal article (peer-reviewed)abstract
- Peyer's patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.
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| 12. |
- Ermund, Anna, et al.
(author)
-
Mucus threads from surface goblet cells clear particles from the airways
- 2021
-
In: Respiratory Research. - : Springer Science and Business Media LLC. - 1465-993X. ; 22:1
-
Journal article (peer-reviewed)abstract
- Background The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. Methods The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. Results We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. Conclusions These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.
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| 13. |
- Ermund, Anna, et al.
(author)
-
OligoG CF-5/20 normalizes cystic fibrosis mucus by chelating calcium.
- 2017
-
In: Clinical and experimental pharmacology & physiology. - : Wiley. - 1440-1681 .- 0305-1870. ; 44:6, s. 639-647
-
Journal article (peer-reviewed)abstract
- The goal of this study was to determine whether the guluronate (G) rich alginate OligoG CF-5/20 (OligoG) could detach cystic fibrosis (CF) mucus by calcium chelation, which is also required for normal mucin unfolding. Since bicarbonate secretion is impaired in CF, leading to insufficient mucin unfolding and thereby attached mucus and since bicarbonate has the ability to bind calcium, we hypothesized that the calcium chelating property of OligoG would lead to detachment of CF mucus. Indeed, OligoG could compete with the N-terminus of the MUC2 mucin for calcium binding as shown by microscale thermophoresis. Further, effects on mucus thickness and attachment induced by OligoG and other alginate fractions of different length and composition were evaluated in explants of CF mouse ileum mounted in horizontal Ussing-type chambers. OligoG at 1.5% caused effective detachment of CF mucus and the most potent alginate fraction tested, the poly-G fraction of about 12 residues, had similar potency compared to OligoG whereas mannuronate-rich (M) polymers had minimal effect. In conclusion, OligoG binds calcium with appropriate affinity without any overt harmful effect on the tissue and can be exploited for treating mucus stagnation. This article is protected by copyright. All rights reserved.
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| 14. |
- Ermund, Anna, et al.
(author)
-
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.
- 2013
-
In: American journal of physiology. Gastrointestinal and liver physiology. - : American Physiological Society. - 1522-1547 .- 0193-1857. ; 305:5
-
Journal article (peer-reviewed)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.
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| 15. |
- Ermund, Anna, et al.
(author)
-
The mucus bundles responsible for airway cleaning are retained in cystic fibrosis and by cholinergic stimulation
- 2018
-
In: European Respiratory Journal. - : European Respiratory Society (ERS). - 0903-1936 .- 1399-3003. ; 52:2
-
Journal article (peer-reviewed)abstract
- 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. Pseudomonas aeruginosa 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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| 16. |
- Ermund, Anna, et al.
(author)
-
The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin
- 2017
-
In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 492:3, s. 331-337
-
Journal article (peer-reviewed)abstract
- 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. (C) 2017 The Authors. Published by Elsevier Inc.
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| 17. |
- Fakih, Dalia, et al.
(author)
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Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs
- 2018
-
In: Jci Insight. - : American Society for Clinical Investigation. - 2379-3708. ; 3:17
-
Journal article (peer-reviewed)abstract
- 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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| 18. |
- Fakih, Dalia, et al.
(author)
-
Normal murine respiratory tract has its mucus concentrated in clouds based on the Muc5b mucin
- 2020
-
In: American Journal of Physiology-Lung Cellular and Molecular Physiology. - : American Physiological Society. - 1040-0605 .- 1522-1504. ; 318:6
-
Journal article (peer-reviewed)abstract
- 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 naive C57BL/6, Muc5b(-/-), Muc5ac(-/-), 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 mu m/s compared with 20 mu m/s for beads not associated with clouds. In Muc5ac(-/-) mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b(-/-) 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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| 19. |
- Giorgetti, Melania, et al.
(author)
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New generation ENaC inhibitors detach cystic fibrosis airway mucus bundles via sodium/hydrogen exchanger inhibition
- 2021
-
In: European Journal of Pharmacology. - : Elsevier BV. - 0014-2999. ; 904
-
Journal article (peer-reviewed)abstract
- 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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| 20. |
- Gustafsson, Jenny K, 1981, et al.
(author)
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An ex vivo method for studying mucus formation, properties and thickness in human colonic biopsies and mouse small and large intestinal explants.
- 2012
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In: American journal of physiology. Gastrointestinal and liver physiology. - : American Physiological Society. - 1522-1547 .- 0193-1857. ; 302:4, s. 430-438
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Journal article (peer-reviewed)abstract
- The colon mucus layers minimize the contact between the luminal flora and the epithelial cells and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Materials and methods: Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side and mucus thickness was measured using a micropipette. Mucus thickness, adhesion and growth rate was recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. Results: In mouse ileum the mucus could be removed by gentle aspiration, whereas in colon about 40 µm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 µm/h) and mouse (100 µm/h) colon, but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 µm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this set up also allows for studies of the highly dynamic mucus system under well controlled conditions.
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| 21. |
- Gustafsson, Jenny K, 1981, et al.
(author)
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Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype.
- 2012
-
In: The Journal of experimental medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 209:7, s. 1263-72
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Journal article (peer-reviewed)abstract
- Cystic fibrosis (CF) is caused by a nonfunctional chloride and bicarbonate ion channel (CF transmembrane regulator [CFTR]), but the link to the phenomenon of stagnant mucus is not well understood. Mice lacking functional CFTR (CftrΔ508) have no lung phenotype but show similar ileal problems to humans. We show that the ileal mucosa in CF have a mucus that adhered to the epithelium, was denser, and was less penetrable than that of wild-type mice. The properties of the ileal mucus of CF mice were normalized by secretion into a high concentration sodium bicarbonate buffer (~100 mM). In addition, bicarbonate added to already formed CF mucus almost completely restored the mucus properties. This knowledge may provide novel therapeutic options for CF.
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| 22. |
- Hoang, Oanh N, et al.
(author)
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Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.
- 2022
-
In: American journal of respiratory and critical care medicine. - 1073-449X .- 1535-4970. ; 206:9, s. 1081-1095
-
Journal article (peer-reviewed)abstract
- Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1β and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.
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| 23. |
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| 24. |
- Jabbar, Karolina S., et al.
(author)
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Association between Brachyspira and irritable bowel syndrome with diarrhoea
- 2021
-
In: Gut. - : BMJ. - 0017-5749 .- 1468-3288. ; 70, s. 1117-1129
-
Journal article (peer-reviewed)abstract
- 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 Brachyspira species in a subset of patients with IBS. Using multiple diagnostic methods, mucosal Brachyspira 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 Brachyspira colonisation in IBS with diarrhoea (IBS-D) was 40% in both cohorts (p=0.02 and p=0.006 vs controls). Brachyspira 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 Brachyspira relocation into goblet cell secretory granules-possibly representing a novel bacterial strategy to evade antibiotics. Conclusion: Mucosal Brachyspira colonisation was significantly more common in IBS and associated with distinctive clinical, histological and molecular characteristics. Our observations suggest a role for Brachyspira in the pathogenesis of IBS, particularly IBS-D. © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
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| 25. |
- Jakobsson, Hedvig E, et al.
(author)
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The composition of the gut microbiota shapes the colon mucus barrier.
- 2015
-
In: EMBO reports. - : EMBO. - 1469-3178 .- 1469-221X. ; 16, s. 164-177
-
Journal article (peer-reviewed)abstract
- Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria-which is comparable to what we observed in free-living wild mice-whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.
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| 26. |
- Johansson, Malin E V, 1971, et al.
(author)
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Composition and functional role of the mucus layers in the intestine.
- 2011
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In: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 68, s. 3635-3641
-
Research review (peer-reviewed)abstract
- 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.
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| 27. |
- Johansson, Malin E V, 1971, et al.
(author)
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Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization
- 2015
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In: Cell Host & Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 18:5, s. 582-592
-
Journal article (peer-reviewed)abstract
- The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus organization of GF mice was similar to that of conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required 5 weeks to be normally detached and colonic inner mucus 6 weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until 3 weeks, when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization.
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| 28. |
- Ljungholm, Penny L., et al.
(author)
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The anion exchanger slc26a3 regulates colonic mucus expansion during steady state and in response to prostaglandin E2, while Cftr regulates de novo mucus release in response to carbamylcholine
- 2024
-
In: PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY. - 0031-6768 .- 1432-2013.
-
Journal article (peer-reviewed)abstract
- The intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E-2 (PGE(2)). The broad-spectrum anion transport inhibitor 4,4 '-diisothiocyanatostilbene-2,2 '-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE(2) stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE(2) stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE(2) with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE(2) activates additional anion transport processes that help release mucus from intestinal goblet cells.
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| 29. |
- Mallet, Christophe, et al.
(author)
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TRPV1 in Brain Is Involved in Acetaminophen-Induced Antinociception
- 2010
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:9
-
Journal article (peer-reviewed)abstract
- Background: Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular overthe- counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV1) in vitro. Pharmacological activation of TRPV1 in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV1 in the brain contributes to the analgesic effect of acetaminophen. Methodology/Principal Findings: Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV1 knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E-2 (PGE(2)) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV1-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV1 in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test. Conclusions: This study shows that TRPV1 in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV1 in the brain.
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| 30. |
- Movahed Rad, Pouya, et al.
(author)
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Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists.
- 2005
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In: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 280:46, s. 38496-38504
-
Journal article (peer-reviewed)abstract
- The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB1 receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV1) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV1 on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV1 gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV1. Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB1 receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV1 and the cannabinoid CB1 receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV1 and thus may play a role as endogenous TRPV1 modulators.
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| 31. |
- Pelaseyed, Thaher, 1979, et al.
(author)
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Carbachol-induced MUC17 endocytosis is concomitant with NHE3 internalization and CFTR membrane recruitment in enterocytes.
- 2013
-
In: American journal of physiology. Cell physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 305:4
-
Journal article (peer-reviewed)abstract
- We have reported that transmembrane mucin MUC17 binds PDZ protein PDZK1, which retains MUC17 apically in enterocytes. MUC17 and transmembrane mucins MUC3 and MUC12 are suggested to build the enterocyte apical glycocalyx. Carbachol (CCh) stimulation of the small intestine results in gel-forming mucin secretion from goblet cells, something that requires adjacent enterocytes to secrete chloride and bicarbonate for proper mucin formation. Surface labeling and confocal imaging demonstrated that apically expressed MUC17 in Caco-2 cells and Muc3(17) in murine enterocytes were endocytosed upon stimulation with CCh. Relocation of MUC17 in response to CCh was specific as MUC3 and MUC12 did not relocate following CCh stimulation. MUC17 colocalized with PDZK1 under basal conditions, while MUC17 relocated to the terminal web and into early endosomes after CCh stimulation. CCh stimulation concomitantly internalized the Na(+/)H(+) exchanger 3 (NHE3) and recruited cystic fibrosis transmembrane conductance regulator (CFTR) to the apical membranes, a process that was important for CFTR-mediated bicarbonate secretion necessary for proper gel-forming mucin unfolding. The reason for the specific internalization of MUC17 is not understood, but it could limit the diffusion barrier for ion secretion caused by the apical enterocyte glycocalyx or alternatively act to sample luminal bacteria. Our results reveal well-orchestrated mucus secretion and trafficking of ion channels and the MUC17 mucin.
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| 32. |
- Pelaseyed, Thaher, 1979, et al.
(author)
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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
- 2014
-
In: Immunological Reviews. - : Wiley. - 0105-2896 .- 1600-065X. ; 260:1, s. 8-20
-
Research review (peer-reviewed)abstract
- 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 Peyer's 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.
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| 33. |
- Rodríguez-Piñeiro, Ana María, et al.
(author)
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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.
- 2013
-
In: American journal of physiology. Gastrointestinal and liver physiology. - : American Physiological Society. - 1522-1547 .- 0193-1857. ; 305:5
-
Journal article (peer-reviewed)abstract
- 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.
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| 34. |
- Rodriguez-Piñeiro, Ana, et al.
(author)
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Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets
- 2023
-
In: Respiratory Research. - : Springer Science and Business Media LLC. - 1465-9921 .- 1465-993X. ; 24
-
Journal article (peer-reviewed)abstract
- Background: The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000–5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. Methods: We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. Results: Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1–4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. Conclusions: These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1–4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.
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| 35. |
- Schütte, André, et al.
(author)
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Microbial-induced meprin β cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus
- 2014
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 111:34, s. 12396-12401
-
Journal article (peer-reviewed)abstract
- The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin β-deficient mice was now found to be attached. Meprin β is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin β was added to the attached mucus of meprin β-deficient mice, the mucus was detached from the epithelium. Similar to meprin β-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin β into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin β to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin β cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin β in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.
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| 36. |
- Trillo-Muyo, Sergio, 1983, et al.
(author)
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Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers
- 2018
-
In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 293:15, s. 5746-5754
-
Journal article (peer-reviewed)abstract
- 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 Ca2+ 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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| 37. |
- Wichert, R., et al.
(author)
-
Mucus Detachment by Host Metalloprotease Meprin beta Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB
- 2017
-
In: Cell Reports. - : Elsevier BV. - 2211-1247. ; 21:8, s. 2090-2103
-
Journal article (peer-reviewed)abstract
- The host metalloprotease meprin beta is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial over-growth. To gain access to the cleavage site in MUC2, meprin b must be proteolytically shed from epithelial cells. Hence, regulation of meprin b shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin b 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 beta and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin beta activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin beta into its active form, impairing meprin beta shedding and its function as a mucus-detaching protease.
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| 38. |
- Wine, J. J., et al.
(author)
-
Progress in understanding mucus abnormalities in cystic fibrosis airways
- 2018
-
In: Journal of Cystic Fibrosis. - : Elsevier BV. - 1569-1993. ; 17:2
-
Journal article (peer-reviewed)abstract
- 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. (C) 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
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| 39. |
- Zygmunt, Peter, et al.
(author)
-
Monoacylglycerols Activate TRPV1 - A Link between Phospholipase C and TRPV1.
- 2013
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:12
-
Journal article (peer-reviewed)abstract
- Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.
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