ABSTRACT
Rationale: Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. Objectives: To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. Methods: STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1ß) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main Results: Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1ß expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1ß exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. Conclusions: STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.
ABSTRACT
Mucins MUC5AC and MUC5B are large glycoproteins that play an essential role in the innate defense of epithelial surfaces and their quantitation in biological samples would be informative about the health status of the tissue/samples they are derived from. However, they are difficult to study and quantify with traditional methods such as ELISA and western blot, due to their size, heterogeneity, and high degree of glycosylation. We successfully implemented a stable isotope labeling mass spectrometry approach for absolute quantification of mucin macromolecules. Here, in detail, we describe this accurate and sensitive liquid chromatography and mass spectrometry (LC-MS) method applied for both MUC5AC and MUC5B quantification in diverse and complex biological samples.
Subject(s)
Glycoproteins , Mucins , Isotope Labeling , Mass Spectrometry , Enzyme-Linked Immunosorbent AssayABSTRACT
Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1ß contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1ß-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1ß-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.
Subject(s)
Bronchiectasis , Cystic Fibrosis , Humans , Bronchioles , Dilatation, Pathologic , Bronchiectasis/genetics , Mucins/metabolism , Interleukin-1beta , Fibrosis , RNA , Mucin 5AC/geneticsABSTRACT
Hyper-secretion and/or hyper-concentration of mucus is a defining feature of multiple obstructive lung diseases, including chronic obstructive pulmonary disease (COPD). Mucus itself is composed of a mixture of water, ions, salt and proteins, of which the gel-forming mucins, MUC5AC and MUC5B, are the most abundant. Recent studies have linked the concentrations of these proteins in sputum to COPD phenotypes, including chronic bronchitis (CB) and acute exacerbations (AE). We sought to determine whether common genetic variants influence sputum mucin concentrations and whether these variants are also associated with COPD phenotypes, specifically CB and AE. We performed a GWAS to identify quantitative trait loci for sputum mucin protein concentration (pQTL) in the Sub-Populations and InteRmediate Outcome Measures in COPD Study (SPIROMICS, n = 708 for total mucin, n = 215 for MUC5AC, MUC5B). Subsequently, we tested for associations of mucin pQTL with CB and AE using regression modeling (n = 822-1300). Replication analysis was conducted using data from COPDGene (n = 5740) and by examining results from the UK Biobank. We identified one genome-wide significant pQTL for MUC5AC (rs75401036) and two for MUC5B (rs140324259, rs10001928). The strongest association for MUC5B, with rs140324259 on chromosome 11, explained 14% of variation in sputum MUC5B. Despite being associated with lower MUC5B, the C allele of rs140324259 conferred increased risk of CB (odds ratio (OR) = 1.42; 95% confidence interval (CI): 1.10-1.80) as well as AE ascertained over three years of follow up (OR = 1.41; 95% CI: 1.02-1.94). Associations between rs140324259 and CB or AE did not replicate in COPDGene. However, in the UK Biobank, rs140324259 was associated with phenotypes that define CB, namely chronic mucus production and cough, again with the C allele conferring increased risk. We conclude that sputum MUC5AC and MUC5B concentrations are associated with common genetic variants, and the top locus for MUC5B may influence COPD phenotypes, in particular CB.
Subject(s)
Mucins , Pulmonary Disease, Chronic Obstructive , Humans , Mucins/genetics , Mucins/metabolism , Sputum/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Mucus/metabolism , PhenotypeABSTRACT
The dynamics describing the vicious cycle characteristic of cystic fibrosis (CF) lung disease, initiated by stagnant mucus and perpetuated by infection and inflammation, remain unclear. Here we determine the effect of the CF airway milieu, with persistent mucoobstruction, resident pathogens, and inflammation, on the mucin quantity and quality that govern lung disease pathogenesis and progression. The concentrations of MUC5AC and MUC5B were measured and characterized in sputum samples from subjects with CF (N = 44) and healthy subjects (N = 29) with respect to their macromolecular properties, degree of proteolysis, and glycomics diversity. These parameters were related to quantitative microbiome and clinical data. MUC5AC and MUC5B concentrations were elevated, 30- and 8-fold, respectively, in CF as compared with control sputum. Mucin parameters did not correlate with hypertonic saline, inhaled corticosteroids, or antibiotics use. No differences in mucin parameters were detected at baseline versus during exacerbations. Mucin concentrations significantly correlated with the age and sputum human neutrophil elastase activity. Although significantly more proteolytic cleavages were detected in CF mucins, their macromolecular properties (e.g., size and molecular weight) were not significantly different than control mucins, likely reflecting the role of S-S bonds in maintaining multimeric structures. No evidence of giant mucin macromolecule reflecting oxidative stress-induced cross-linking was found. Mucin glycomic analysis revealed significantly more sialylated glycans in CF, and the total abundance of nonsulfated O-glycans correlated with the relative abundance of pathogens. Collectively, the interaction of mucins, pathogens, epithelium, and inflammatory cells promotes proteomic and glycomic changes that reflect a persistent mucoobstructive, infectious, and inflammatory state.
Subject(s)
Cystic Fibrosis , Cystic Fibrosis/pathology , Humans , Inflammation , Mucin 5AC , Mucin-5B , Mucus , Proteomics , Respiratory System/pathologyABSTRACT
Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.
Subject(s)
Cystic Fibrosis , Animals , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Mucus/metabolism , Respiratory System/metabolism , Sputum/metabolism , SwineABSTRACT
The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus rich in mucin glycoproteins. Abnormal mucus accumulation is a cardinal feature of chronic respiratory diseases, but the relationship between mucus and pathogens during exacerbations is poorly understood. We identified elevations in airway mucin 5AC (MUC5AC) and MUC5B concentrations during spontaneous and experimentally induced chronic obstructive pulmonary disease (COPD) exacerbations. MUC5AC was more sensitive to changes in expression during exacerbation and was therefore more predictably associated with viral load, inflammation, symptom severity, decrements in lung function, and secondary bacterial infections. MUC5AC was functionally related to inflammation, as Muc5ac-deficient (Muc5ac-/-) mice had attenuated RV-induced (RV-induced) airway inflammation, and exogenous MUC5AC glycoprotein administration augmented inflammatory responses and increased the release of extracellular adenosine triphosphate (ATP) in mice and human airway epithelial cell cultures. Hydrolysis of ATP suppressed MUC5AC augmentation of RV-induced inflammation in mice. Therapeutic suppression of mucin production using an EGFR antagonist ameliorated immunopathology in a mouse COPD exacerbation model. The coordinated virus induction of MUC5AC and MUC5B expression suggests that non-Th2 mechanisms trigger mucin hypersecretion during exacerbations. Our data identified a proinflammatory role for MUC5AC during viral infection and suggest that MUC5AC inhibition may ameliorate COPD exacerbations.
Subject(s)
Mucin 5AC , Pulmonary Disease, Chronic Obstructive , Adenosine Triphosphate/metabolism , Animals , Disease Models, Animal , Humans , Inflammation/metabolism , Mice , Mucin 5AC/genetics , Mucin 5AC/metabolism , Mucin-5B/genetics , Mucin-5B/metabolism , Mucus/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/virology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathologyABSTRACT
Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.
Subject(s)
Lung Diseases , Mucins , Adult , Animals , Female , Humans , Lung/metabolism , Lung Diseases/metabolism , Mice , Mucin 5AC/genetics , Mucin-5B/genetics , Mucins/metabolism , Mucociliary Clearance/genetics , Mucus/metabolismABSTRACT
BACKGROUND: We previously described the contributions of increased total airway mucin concentrations to the pathogenesis and diagnosis of the chronic bronchitic component of chronic obstructive pulmonary disease (COPD). Here, we investigated the relative contribution of each of the major airway gel-forming mucins, MUC5AC and MUC5B, to the initiation, progression, and early diagnosis of airways disease in COPD. METHODS: SPIROMICS was a multicentre, observational study in patients aged 40-80 years recruited from six clinical sites and additional subsites in the USA. In this analysis, MUC5AC and MUC5B were quantitated by stable isotope-labelled mass spectrometry in induced sputum samples from healthy never-smokers, ever-smokers at risk for COPD, and ever-smokers with COPD. Participants were extensively characterised using results from questionnaires, such as the COPD assessment test (CAT) and St George's Respiratory Questionnaire; quantitative CT, such as residual volume/total lung capacity ratio (RV/TLC) and parametric response mapping-functional small airway disease (PRM-fSAD); and pulmonary function tests, such as FEV1, forced vital capacity (FVC), and forced expiratory flow, midexpiratory phase (FEF25-75%). Absolute concentrations of both MUC5AC and MUC5B were related to cross-sectional (baseline, initial visit) and 3-year follow-up longitudinal data, including lung function, small airways obstruction, prospective acute exacerbations, and smoking status as primary outcomes. This study is registered with ClinicalTrials.gov (NCT01969344). FINDINGS: This analysis included 331 participants (mean age 63 years [SEM 9·40]), of whom 40 were healthy never-smokers, 90 were at-risk ever-smokers, and 201 were ever-smokers with COPD. Increased MUC5AC concentrations were more reliably associated with manifestations of COPD than were MUC5B concentrations, including decreased FEV1 and FEF25-75%, and increased prospective exacerbation frequency, RV/TLC, PRM-fSAD, and COPD assessment scores. MUC5AC concentrations were more reactive to cigarette smoke exposure than were MUC5B concentrations. Longitudinal data from 3-year follow-up visits generated a multivariate-adjusted odds ratio for two or more exacerbations of 1·24 (95% CI 1·04-1·47, p=0·015) for individuals with high baseline MUC5AC concentration. Increased MUC5AC, but not MUC5B, concentration at baseline was a significant predictor of FEV1, FEV1/FVC, FEF25-75%, and CAT score decline during the 3-year follow-up. Moreover, current smokers in the at-risk group showed raised MUC5AC concentrations at initial visits and decreased lung function over 3 years. By contrast, former smokers in the at-risk group showed normal MUC5AC concentrations at the initial visit and preserved lung function over 3 years. INTERPRETATION: These data indicate that increased MUC5AC concentration in the airways might contribute to COPD initiation, progression, exacerbation risk, and overall pathogenesis. Compared with MUC5B, greater relative changes in MUC5AC concentrations were observed as a function of COPD severity, and MUC5AC concentration seems to be an objective biomarker to detect disease in at-risk and pre-COPD individuals. These data suggest that MUC5AC-producing pathways could be potential targets for future therapeutic strategies. Thus, MUC5AC could be a novel biomarker for COPD prognosis and for testing the efficacy of therapeutic agents. FUNDING: National Institutes of Health; National Heart, Lung, and Blood Institute.
Subject(s)
Mucin 5AC , Mucin-5B , Pulmonary Disease, Chronic Obstructive , Adult , Aged , Aged, 80 and over , Cross-Sectional Studies , Disease Progression , Forced Expiratory Volume , Humans , Lung , Middle Aged , Mucin 5AC/analysis , Mucin-5B/analysis , Prospective Studies , Pulmonary Disease, Chronic Obstructive/complicationsABSTRACT
Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.
Subject(s)
Bronchiectasis/drug therapy , Bronchiectasis/physiopathology , Erythromycin/therapeutic use , Mucus/chemistry , Respiratory System/physiopathology , Sputum/chemistry , Aged , Cohort Studies , Female , Humans , Male , Middle Aged , Mucus/microbiology , Queensland , Sputum/microbiologyABSTRACT
Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.
Subject(s)
Mucus/chemistry , Potassium/analysis , Rheology/methods , Sodium/analysis , Trachea/chemistry , Adult , Aged , Aged, 80 and over , Cations, Monovalent , Female , Healthy Volunteers , Humans , Intubation, Intratracheal , Male , Middle Aged , Polysaccharides/classification , Polysaccharides/isolation & purification , Potassium/metabolism , Proteins/classification , Proteins/isolation & purification , Sodium/metabolism , Sputum/chemistry , Trachea/physiologyABSTRACT
Airway epithelium structure/function can be altered by local inflammatory/immune signals, and this process is called epithelial remodeling. The mechanism by which this innate response is regulated, which causes mucin/mucus overproduction, is largely unknown. Exosomes are nanovesicles that can be secreted and internalized by cells to transport cellular cargo, such as proteins, lipids, and miRNA. The objective of this study was to understand the role exosomes play in airway remodeling through cell-cell communication. We used two different human airway cell cultures: primary human tracheobronchial (HTBE) cells, and a cultured airway epithelial cell line (Calu-3). After intercellular exosomal transfer, comprehensive proteomic and genomic characterization of cell secretions and exosomes was performed. Quantitative proteomics and exosomal miRNA analysis profiles indicated that the two cell types are fundamentally distinct. HTBE cell secretions were typically dominated by fundamental innate/protective proteins, including mucin MUC5B, and Calu-3 cell secretions were dominated by pathology-associated proteins, including mucin MUC5AC. After exosomal transfer/intake, approximately 20% of proteins, including MUC5AC and MUC5B, were significantly altered in HTBE secretions. After exosome transfer, approximately 90 miRNAs (â¼4%) were upregulated in HTBE exosomes, whereas Calu-3 exosomes exhibited a preserved miRNA profile. Together, our data suggest that the transfer of exosomal cargo between airway epithelial cells significantly alters the qualitative and quantitative profiles of airway secretions, including mucin hypersecretion, and the miRNA cargo of exosomes in target cells. This finding indicates that cellular information can be carried between airway epithelial cells via exosomes, which may play an important role in airway biology and epithelial remodeling.
Subject(s)
Airway Remodeling/physiology , Bronchi/cytology , Cell Communication/physiology , Epithelial Cells/metabolism , Extracellular Vesicles/metabolism , Cell Line , Cells, Cultured , Culture Media/metabolism , Epithelial Cells/cytology , Exosomes/metabolism , Extracellular Vesicles/genetics , Gene Expression , Humans , MicroRNAs , Mucin 5AC/metabolism , Mucin-5B/metabolism , Proteins/analysis , Proteins/metabolismABSTRACT
RATIONALE: MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. OBJECTIVES: To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). METHODS: Multiple airway regions from 16 nonsmoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/colocalization were assessed by RNA in situ hybridization and immunohistochemistry in five lungs with histologically healthy airways. Droplet digital PCR and cell cultures were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. MEASUREMENTS AND MAIN RESULTS: Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA in situ hybridization revealed MUC5AC and MUC5B were colocalized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-copositive cells dominated distal regions. CONCLUSIONS: In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium and glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP-positive secretory cells in superficial airway epithelia.
Subject(s)
Lung/diagnostic imaging , Lung/physiology , Mucin 5AC/analysis , Mucin-5B/analysis , Protein Transport/physiology , Respiratory Physiological Phenomena , HumansABSTRACT
RATIONALE: Cystic fibrosis (CF) airways disease produces a mucoobstructive lung phenotype characterized by airways mucus plugging, epithelial mucous cell metaplasia/hyperplasia, chronic infection, and inflammation. Simultaneous biochemical and functional in vivo studies of mucin synthesis and secretion from CF airways are not available. In vitro translational models may quantitate differential CF versus normal mucin and fluid secretory responses to infectious/inflammatory stimuli. OBJECTIVES: We tested the hypothesis that CF airways exhibit defective epithelial fluid, but not mucin, secretory responses to bacterial/inflammatory host products. METHODS: Well-differentiated primary human bronchial epithelial cultures were exposed to supernatant from mucopurulent material (SMM) from human CF airways as a test of bacterial/inflammatory host product stimulus. Human bronchial epithelia (HBE) with normal CF transmembrane conductance regulator function were compared with ΔF508/ΔF508 CF HBE. MEASUREMENTS AND MAIN RESULTS: Acute (up to 60 min) SMM exposure promoted mucin secretion, but mucins were degraded by the proteolytic enzymes present in SMM. Chronic SMM exposure induced upregulation of mucin synthesis and storage and generated absolute increases in basal and stimulated mucin release in normal and CF cultures. These responses were similar in normal and CF cultures. In contrast, SMM produced a coordinated CF transmembrane conductance regulator-mediated Cl- secretory response in normal HBE, but not in CF HBE. The absence of the fluid secretory response in CF produced quantitatively more dehydrated mucus. CONCLUSIONS: Our study reveals the interplay between regulation of mucin and fluid secretion rates in inflamed versus noninflamed conditions and why a hyperconcentrated mucus is produced in CF airways.
Subject(s)
Cystic Fibrosis/metabolism , Fluid Therapy , Lung/metabolism , Mucins/biosynthesis , Respiratory Mucosa/metabolism , Cell Culture Techniques , Cystic Fibrosis/pathology , Enzyme-Linked Immunosorbent Assay , Epithelium/metabolism , Epithelium/pathology , Humans , Lung/pathology , Mucins/metabolism , Polymerase Chain Reaction , Respiratory Mucosa/pathologyABSTRACT
RATIONALE: E-cigarettes have become increasingly popular and little is known about their potential adverse health effects. OBJECTIVES: To determine the effects of e-cigarette use on the airways. METHODS: Induced sputum samples from cigarette smokers, e-cigarette users, and nonsmokers were analyzed by quantitative proteomics, and the total and individual concentrations of mucins MUC5AC and MUC5B were determined by light scattering/refractometry and labeled mass spectrometry, respectively. Neutrophil extracellular trap (NET) formation rates were also determined for the same groups. MEASUREMENTS AND MAIN RESULTS: E-cigarette users exhibited significant increases in aldehyde-detoxification and oxidative stress-related proteins associated with cigarette smoke compared with nonsmokers. The levels of innate defense proteins associated with chronic obstructive pulmonary disease, such as elastase and matrix metalloproteinase-9, were significantly elevated in e-cigarette users as well. E-cigarette users' sputum also uniquely exhibited significant increases in neutrophil granulocyte-related and NET-related proteins, such as myeloperoxidase, azurocidin, and protein-arginine deiminase 4, despite no significant elevation in neutrophil cell counts. Peripheral neutrophils from e-cigarette users showed increased susceptibility to phorbol 12-myristate 13-acetate-induced NETosis. Finally, a compositional change in the gel-forming building blocks of airway mucus (i.e., an elevated concentration of mucin MUC5AC) was observed in both cigarette smokers and e-cigarette users. CONCLUSIONS: Together, our results indicate that e-cigarette use alters the profile of innate defense proteins in airway secretions, inducing similar and unique changes relative to cigarette smoking. These data challenge the concept that e-cigarettes are a healthier alternative to cigarettes.
Subject(s)
Electronic Nicotine Delivery Systems , Immunity, Innate/immunology , Lung/immunology , Mucins/immunology , Neutrophil Activation/immunology , Smoking/immunology , Adult , Chromatography, High Pressure Liquid , Cross-Sectional Studies , Female , Humans , Male , Mass Spectrometry , Middle Aged , Mucins/biosynthesis , Respiratory Mucosa/immunology , Sputum/immunology , Young AdultABSTRACT
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitic and emphysematous components. In one biophysical model, the concentration of mucin on the airway surfaces is hypothesized to be a key variable that controls mucus transport in healthy persons versus cessation of transport in persons with muco-obstructive lung diseases. Under this model, it is postulated that a high mucin concentration produces the sputum and disease progression that are characteristic of chronic bronchitis. METHODS: We characterized the COPD status of 917 participants from the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) using questionnaires administered to participants, chest tomography, spirometry, and examination of induced sputum. Total mucin concentrations in sputum were measured with the use of size-exclusion chromatography and refractometry. In 148 of these participants, the respiratory secreted mucins MUC5AC and MUC5B were quantitated by means of mass spectrometry. Data from chronic-bronchitis questionnaires and data on total mucin concentrations in sputum were also analyzed in an independent 94-participant cohort. RESULTS: Mean (±SE) total mucin concentrations were higher in current or former smokers with severe COPD than in controls who had never smoked (3166±402 vs. 1515±152 µg per milliliter) and were higher in participants with two or more respiratory exacerbations per year than in those with zero exacerbations (4194±878 vs. 2458±113 µg per milliliter). The absolute concentrations of MUC5B and MUC5AC in current or former smokers with severe COPD were approximately 3 times as high and 10 times as high, respectively, as in controls who had never smoked. Receiver-operating-characteristic curve analysis of the association between total mucin concentration and a diagnosis of chronic bronchitis yielded areas under the curve of 0.72 (95% confidence interval [CI], 0.65 to 0.79) for the SPIROMICS cohort and 0.82 (95% CI, 0.73 to 0.92) for the independent cohort. CONCLUSIONS: Airway mucin concentrations may quantitate a key component of the chronic bronchitis pathophysiologic cascade that produces sputum and mediates disease severity. Studies designed to explore total mucin concentrations in sputum as a diagnostic biomarker and therapeutic target for chronic bronchitis appear to be warranted. (Funded by the National Heart, Lung, and Blood Institute and others.).
Subject(s)
Bronchitis, Chronic/diagnosis , Mucins/analysis , Pulmonary Disease, Chronic Obstructive/physiopathology , Respiratory System/chemistry , Sputum/chemistry , Aged , Analysis of Variance , Asthma/physiopathology , Biomarkers/analysis , Bronchitis, Chronic/physiopathology , Disease Progression , Female , Humans , Male , Mass Spectrometry , Middle Aged , Mucin 5AC/analysis , Mucin-5B/analysis , ROC Curve , Smoking/adverse effects , Smoking/physiopathology , Surveys and QuestionnairesABSTRACT
Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation.
Subject(s)
Allergens/immunology , Asthma/immunology , Hypersensitivity/immunology , Inflammation/immunology , Inflammation/pathology , Th2 Cells/immunology , Adult , Asthma/complications , Asthma/pathology , Cytokines , Humans , Hypersensitivity/complications , Hypersensitivity/pathology , Inflammation/complications , Lung/pathology , Mucus/metabolism , Muscle, Smooth/immunology , Muscle, Smooth/pathology , PhenotypeABSTRACT
Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.
Subject(s)
Anti-HIV Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Molecular Dynamics Simulation , Salivary Proline-Rich Proteins/chemistry , src Homology Domains , Humans , Surface Plasmon ResonanceABSTRACT
Chronic lung diseases such as cystic fibrosis, chronic bronchitis, and asthma are characterized by hypersecretion and poor clearance of mucus, which are associated with poor prognosis and mortality. Little is known about the relationship between the biophysical properties of mucus and its molecular composition. The mucins MUC5B and MUC5AC are traditionally believed to generate the characteristic biophysical properties of airway mucus. However, the contribution of hundreds of globular proteins to the biophysical properties of mucus is not clear. Approximately one-third of the total mucus proteome comprises distinct, multi-protein complexes centered around airway mucins. These complexes constitute a discrete entity we call the "mucin interactome". The data suggest that while the majority of these proteins interact with mucins via electrostatic and weak interactions, some interact through very strong hydrophobic and/or covalent interactions. Using reagents that interfere with protein-protein interactions, the complexes can be disassembled, and mucus rheology can be dramatically altered. Using MUC5B-glutathione S-transferase (GST) and MUC5B-galectin-3 as a representative of these interactions, we provide evidence that individual mucin protein interactions can alter the biophysical properties of mucus and modulate the biological function of the protein. We propose that the key mechano- and bio-active functions of mucus depend on the dynamic interactions between mucins and globular proteins. These observations challenge the paradigm that mucins are the only molecules that confer biophysical properties of mucus. These observations may ultimately lead to a greater understanding of the system and guide the development of strategies for more effective interventions using better therapeutic agents.
Subject(s)
Carrier Proteins/metabolism , Immunity, Innate , Immunity, Mucosal , Mucins/metabolism , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Humans , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Protein Interaction Mapping , Protein Interaction Maps , Proteome , Proteomics/methodsABSTRACT
A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.