Acidic Submucosal Gland pH and Elevated Protein Concentration Produce Abnormal Cystic Fibrosis Mucus.
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ABSTRACT:
SUBMITTER: Xie Y
PROVIDER: S-EPMC7484121 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cystic fibrosis (CF) is a recessive genetic disease that is characterised by airway mucus plugging and reduced mucus clearance. There are currently alternative hypotheses that attempt to describe the abnormally viscous and elastic mucus that is a hallmark of CF airways disease, including: 1) loss of CF transmembrane regulator (CFTR)-dependent airway surface volume (water) secretion, producing mucus hyperconcentration-dependent increased viscosity, and 2) impaired bicarbonate secretion by CFTR, producing acidification of airway surfaces and increased mucus viscosity.A series of experiments was conducted to determine the contributions of mucus concentration versus pH to the rheological properties of airway mucus across length scales from the nanoscopic to macroscopic.For length scales greater than the nanoscopic, i.e. those relevant to mucociliary clearance, the effect of mucus concentration dominated over the effect of airway acidification.Mucus hydration and chemical reduction of disulfide bonds that connect mucin monomers are more promising therapeutic approaches than alkalisation.
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Project description:BackgroundChronic rhinosinusitis (CRS) may be initiated by innately impaired host defense mechanisms that predispose the upper airways to infection. Recent evidence suggests tethering of submucosal gland mucus strands represents an inciting event within cystic fibrosis (CF) airways, occurring prior to onset of chronic infection. Submucosal gland hypertrophy and defective mucociliary clearance (MCC) are present in actively inflamed sinuses, but mucus strand velocity may also be affected as a secondary event, further contributing to chronic disease. The objective of this study is to assess whether mucus strand velocity is decreased in patients with CRS.MethodsMucosal explants from patients with and without CRS were submerged in Ringer's solution mixed with fluorescent nanospheres. Methacholine was then added, and videos demonstrating strand growth and detachment were generated from a time-lapse of Z-stack images using a multiphoton confocal microscope. Dynamic mucus strands were identified and individual velocities quantified with the MTrackJ plug-in of ImageJ.ResultsFifteen patients met criteria for ex vivo analysis of mucus strand velocities (CRS, n = 9 vs controls, n = 6). Mucus strands were recorded (pixels/second) streaming from the submucosal gland openings. Average mucus strand velocities were significantly decreased in patients with CRS (1.53 ± 0.67 vs controls, 4.86 ± 1.68 pixels/second; p < 0.001).ConclusionThis study is the first to report evidence of abnormal mucus strand velocity from submucosal glands in diseased sinonasal mucosa. Future pharmacologic studies targeting this critical component of MCC are warranted.
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Project description:Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.
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Project description:Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non-CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HCO3- concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator-dependent alkalinization to abnormal CF ASL. In addition, we found that increasing Ca2+ concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing Ca2+ concentration, and/or altering electrostatic interactions in ASL might benefit early CF.
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Project description:SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.
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Project description:In cystic fibrosis (CF), a lack of functional CF transmembrane conductance regulator (CFTR) chloride channels causes defective secretion by submucosal glands (SMGs), leading to persistent bacterial infection that damages airways and necessitates tissue repair. SMGs are also important niches for slow-cycling progenitor cells (SCPCs) in the proximal airways, which may be involved in disease-related airway repair. Here, we report that calcitonin gene-related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. Since CGRP-expressing neuroendocrine cells reside in bronchiolar SCPC niches, we hypothesized that the glandular SCPC niche may be dysfunctional in CF. Consistent with this hypothesis, CFTR-deficient mice failed to maintain glandular SCPCs following airway injury. In wild-type mice, CGRP levels increased following airway injury and functioned as an injury-induced mitogen that stimulated SMG progenitor cell proliferation in vivo and altered the proliferative potential of airway progenitors in vitro. Components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation in a non-cell-autonomous manner. These findings demonstrate that CGRP-dependent pathways for CFTR activation are abnormally upregulated in CF SMGs and that this sustained mitogenic signal alters properties of the SMG progenitor cell niche in CF airways. This discovery may have important implications for injury/repair mechanisms in the CF airway.
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Project description:Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO3- secretory pathway that was defective in CF: (i). ASL [HCO3-] was higher in NL than CF ASL; (ii). activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii). NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent HCO3- secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.
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Project description:Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans. In order to address this deficiency, ASL pH was measured in vivo in children using a novel luminescent technology integrated with fibre-optic probes. Here we show that ASL pH in children with CF is similar to that of children without CF. Findings were supported by highly controlled direct pH measurements in primary human airway epithelial cell culture models, which also suggest that the potential ASL pH gradient produced by defective apical ion transport is balanced out by paracellular shunting of acid/base. Thus, reduced baseline ASL pH is unlikely to be an important pathobiological factor in early CF lung disease.
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Project description:Morbidity and mortality in cystic fibrosis (CF) are due not only to abnormal epithelial cell function, but also to an abnormal immune response. We have shown previously that macrophages lacking CF transmembrane conductance regulator (CFTR), the gene mutated in CF, contribute significantly to the hyperinflammatory response observed in CF. In this study, we show that lack of functional CFTR in murine macrophages causes abnormal TLR4 subcellular localization. Upon LPS stimulation, CFTR macrophages have prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment. This abnormal TLR4 trafficking leads to increased LPS-induced activation of the NF-?B, MAPK, and IFN regulatory factor-3 pathways and decreased TLR4 degradation, which affects downregulation of the proinflammatory state. In addition to primary murine cells, mononuclear cells isolated from CF patients demonstrate similar defects in response to LPS. Moreover, specific inhibition of CFTR function induces abnormal TLR4 trafficking and enhances the inflammatory response of wild-type murine cells to LPS. Thus, functional CFTR in macrophages influences TLR4 spatial and temporal localization and perturbs LPS-mediated signaling in both murine CF models and patients with CF.
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Project description:BACKGROUND: Cap polyposis is a rare disease characterised by mucoid and bloody diarrhoea, with polyps covered by a cap of mucoid and fibrinopurulent exudate. The pathogenesis is not known. AIMS: To pour some light on cap polyposis pathogenesis, by examining the mucus of patients and analysing the expression of five mucin genes, MUC2, MUC3, MUC4, MUC5AC, and MUC5B. PATIENT AND METHODS: The study was performed on biopsy specimens taken from a patient with recurrent cap polyposis. Histochemical examination, electron microscopy, and mRNA in situ hybridisation were used. RESULTS: The mucus of cap polyposis differed in three respects from that of normal adult colon: abnormal ultrastructure of the mucus in the goblet cells, predominance of non-sulphated mucins, abnormal expression of the MUC4, MUC3, and MUC5AC genes. CONCLUSIONS: Most of these abnormalities have been reported for other pathological situations, suggesting that the abnormalities observed in the mucus of this patient with cap polyposis are probably secondary phenomena rather than primary. However, the mucin abnormalities detected, which reflect deregulation of the expression of three apomucin genes, abnormal glycosylation, and abnormalities of the secretion process, are also probably involved in the clinical manifestations of cap polyposis.
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