Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 43
Filtrar
1.
bioRxiv ; 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-39211176

RESUMEN

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.

2.
bioRxiv ; 2024 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-38370820

RESUMEN

In vitro models play a major role in studying airway physiology and disease. However, the native lung's complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

3.
Am J Physiol Lung Cell Mol Physiol ; 325(6): L765-L775, 2023 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-37847709

RESUMEN

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.


Asunto(s)
Enfermedades Pulmonares , Depuración Mucociliar , Ratones , Humanos , Animales , Depuración Mucociliar/fisiología , Sistema Respiratorio/metabolismo , Moco/metabolismo , Enfermedades Pulmonares/metabolismo , Ratones Noqueados
4.
Front Mol Biosci ; 10: 1221796, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37555015

RESUMEN

Background: Genetic defects in motile cilia cause primary ciliary dyskinesia (PCD), a rare disease with no specific therapeutics. Individuals with PCD often have impaired fertility and laterality defects and universally suffer from upper and lower airway diseases. Chronic rhinosinusitis is a universal feature of PCD, and mucus accumulation and subsequent infections of the sinonasal cavity cause significant morbidity in individuals with PCD. Despite this, there are no approved treatments that specifically target mucus. Objective: The goals of this study were to determine whether computed tomography (CT) imaging could be used to quantify mucus accumulation and whether the use of a mucolytic agent to reduce disulfide cross-links present in mucins would improve the effectiveness of nasal lavage at removing mucus in a murine model of PCD. Methods: Adult mice with a deletion of the axonemal dynein Dnaic1 were imaged using CT scanning to characterize mucus accumulation. The animals were then treated by nasal lavage with saline, with/without the disulfide-reducing agent tris(2-carboxyethyl)phosphine. Post-treatment CT scans were used to quantify improvement in the sinonasal cavity. Results: Mucus accumulation in the nasal cavity was readily quantified by CT. Compared to sham-treated control animals, nasal lavage with/without a mucolytic agent resulted in a significant reduction of accumulated mucus (p < 0.01). Treatment with the mucolytic agent showed a greater reduction of accumulated mucus than treatment with saline alone. Conclusion: The results suggest that inclusion of a mucolytic agent may increase the effectiveness of nasal lavage at reducing mucus burden in PCD.

5.
Sci Transl Med ; 15(699): eabo7728, 2023 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-37285404

RESUMEN

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of ß and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.


Asunto(s)
COVID-19 , Fibrosis Quística , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Pulmón/metabolismo , Moco/metabolismo , Hipoxia/metabolismo
6.
Curr Opin Pharmacol ; 64: 102235, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35576754

RESUMEN

Few human genetic diseases can rely on the availability of as many and as diverse animal models as cystic fibrosis (CF), a multiorgan syndrome caused by functional absence of cystic fibrosis transmembrane regulator (CFTR). The recent development of highly effective CFTR modulator drug therapies simultaneously highlighted the remarkable clinical improvement achievable with these treatments, the lack of therapeutic alternatives for non-responders, and the need to understand the kinetics of disease upon early life/chronic treatment. These advances have rekindled efforts to leverage animal models to address critical knowledge gaps in CF. This article provides a concise overview of the areas of interests for therapeutic intervention in the current CF landscape, focusing on the contributions of in vivo models to understand CF pathogenesis, identify therapeutic windows, and develop novel therapies for all CFTR mutations.


Asunto(s)
Fibrosis Quística , Animales , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Modelos Animales , Mutación
7.
Front Physiol ; 13: 842592, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35356083

RESUMEN

As the nasal cavity is the portal of entry for inspired air in mammals, this region is exposed to the highest concentration of inhaled particulate matter and pathogens, which must be removed to keep the lower airways sterile. Thus, one might expect vigorous removal of these substances via mucociliary clearance (MCC) in this region. We have investigated the rate of MCC in the murine nasal cavity compared to the more distal airways (trachea). The rate of MCC in the nasal cavity (posterior nasopharynx, PNP) was ∼3-4× greater than on the tracheal wall. This appeared to be due to a more abundant population of ciliated cells in the nasal cavity (∼80%) compared to the more sparsely ciliated trachea (∼40%). Interestingly, the tracheal ventral wall exhibited a significantly lower rate of MCC than the tracheal posterior membrane. The trachealis muscle underlying the ciliated epithelium on the posterior membrane appeared to control the surface architecture and likely in part the rate of MCC in this tracheal region. In one of our mouse models (Bpifb1 KO) exhibiting a 3-fold increase in MUC5B protein in lavage fluid, MCC particle transport on the tracheal walls was severely compromised, yet normal MCC occurred on the tracheal posterior membrane. While a blanket of mucus covered the surface of both the PNP and trachea, this mucus appeared to be transported as a blanket by MCC only in the PNP. In contrast, particles appeared to be transported as discrete patches or streams of mucus in the trachea. In addition, particle transport in the PNP was fairly linear, in contrast transport of particles in the trachea often followed a more non-linear route. The thick, viscoelastic mucus blanket that covered the PNP, which exhibited ∼10-fold greater mass of mucus than did the blanket covering the surface of the trachea, could be transported over large areas completely devoid of cells (made by a breach in the epithelial layer). In contrast, particles could not be transported over even a small epithelial breach in the trachea. The thick mucus blanket in the PNP likely aids in particle transport over the non-ciliated olfactory cells in the nasal cavity and likely contributes to humidification and more efficient particle trapping in this upper airway region.

8.
Life (Basel) ; 11(5)2021 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-34064654

RESUMEN

Mucociliary clearance (MCC) is a dominant component of pulmonary host defense. In health, the periciliary layer (PCL) is optimally hydrated, thus acting as an efficient lubricant layer over which the mucus layer moves by ciliary force. Airway surface dehydration and production of hyperconcentrated mucus is a common feature of chronic obstructive lung diseases such as cystic fibrosis (CF) and chronic bronchitis (CB). Mucus hydration is driven by electrolyte transport activities, which in turn are regulated by airway epithelial purinergic receptors. The activity of these receptors is controlled by the extracellular concentrations of ATP and its metabolite adenosine. Vesicular and conducted pathways contribute to ATP release from airway epithelial cells. In this study, we review the evidence leading to the identification of major components of these pathways: (a) the vesicular nucleotide transporter VNUT (the product of the SLC17A9 gene), the ATP transporter mediating ATP storage in (and release from) mucin granules and secretory vesicles; and (b) the ATP conduit pannexin 1 expressed in non-mucous airway epithelial cells. We further illustrate that ablation of pannexin 1 reduces, at least in part, airway surface liquid (ASL) volume production, ciliary beating, and MCC rates.

10.
Am J Respir Crit Care Med ; 203(10): 1275-1289, 2021 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-33321047

RESUMEN

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Estudios de Casos y Controles , Técnicas de Cultivo de Célula , Humanos
11.
JCI Insight ; 6(1)2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33232302

RESUMEN

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Animales , Sistemas CRISPR-Cas , Fibrosis Quística/patología , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Femenino , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/fisiopatología , Técnicas de Inactivación de Genes , Humanos , Masculino , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Sistema Respiratorio/patología , Sistema Respiratorio/fisiopatología , Distribución Tisular , Transcriptoma
12.
Am J Respir Cell Mol Biol ; 61(3): 312-321, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-30896965

RESUMEN

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.


Asunto(s)
Proteínas de Unión al ADN/genética , Síndrome de Kartagener/genética , Depuración Mucociliar/genética , Fenotipo , Animales , Axonema/genética , Cilios/genética , Humanos , Ratones , Mutación/genética , Eliminación de Secuencia/genética
13.
Am J Hum Genet ; 104(2): 229-245, 2019 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-30665704

RESUMEN

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.


Asunto(s)
Cilios/patología , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/fisiopatología , Proteínas de Microfilamentos/deficiencia , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas de Xenopus/deficiencia , Animales , Trastornos de la Motilidad Ciliar/patología , Modelos Animales de Enfermedad , Exones/genética , Femenino , Eliminación de Gen , Genes Letales , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Fenotipo , Rotación , Xenopus/embriología , Xenopus/genética , Proteínas de Xenopus/genética
14.
Am J Respir Crit Care Med ; 199(2): 171-180, 2019 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-30212240

RESUMEN

RATIONALE: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. OBJECTIVES: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. METHODS: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. MEASUREMENTS AND MAIN RESULTS: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in ßENaC-overexpressing mice, whereas NAC did not (n = 6-24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. CONCLUSIONS: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.


Asunto(s)
Asma/tratamiento farmacológico , Fibrosis Quística/tratamiento farmacológico , Expectorantes/uso terapéutico , Depuración Mucociliar/efectos de los fármacos , Moco/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Acetilcisteína/uso terapéutico , Animales , Asma/fisiopatología , Fibrosis Quística/fisiopatología , Modelos Animales de Enfermedad , Ditiotreitol/uso terapéutico , Humanos , Técnicas In Vitro , Masculino , Ratones , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología
15.
Sci Rep ; 8(1): 14744, 2018 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-30282981

RESUMEN

Mucociliary clearance (MCC) is the first line of defense in clearing airways. In genetically engineered mice, each component of this system (ciliary beat, mucus, airway surface hydration) can be studied separately to determine its contribution to MCC. Because MCC is difficult to measure in mice, MCC measurements are often omitted from these studies. We report a simple method to measure MCC in mice involving nasal inhalation of aerosolized fluorescent beads and trans-tracheal bead tracking. This method has a number of advantages over existing methods: (1) a small volume of liquid is deposited thus minimally disturbing the airway surface; (2) bead behavior on airways can be visualized; (3) useful for adult or neonatal mice; (4) the equipment is relatively inexpensive and easily obtainable. The type of anesthetic had no significant effect on the rate of MCC, but overloading the airways with beads significantly decreased MCC. In addition, the rate of bead transport was not different in alive (3.11 mm/min) vs recently euthanized mice (3.10 mm/min). A 5-min aerosolization of beads in a solution containing UTP significantly increased the rate of MCC, demonstrating that our method would be of value in testing the role of various pharmacological agents on MCC.


Asunto(s)
Depuración Mucociliar/fisiología , Rociadores Nasales , Imagen Óptica/métodos , Tráquea/fisiología , Administración por Inhalación , Anestésicos por Inhalación , Animales , Colorantes Fluorescentes/química , Masculino , Ratones , Ratones Endogámicos C57BL , Microesferas , Moco/fisiología , Nebulizadores y Vaporizadores , Polietilenglicoles/química , Tráquea/anatomía & histología
16.
Am J Respir Cell Mol Biol ; 59(3): 383-396, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29579396

RESUMEN

Understanding how expression of airway secretory mucins MUC5B and MUC5AC is regulated in health and disease is important to elucidating the pathogenesis of mucoobstructive respiratory diseases. The transcription factor SPDEF (sterile α-motif pointed domain epithelial specific transcription factor) is a key regulator of MUC5AC, but its role in regulating MUC5B in health and in mucoobstructive lung diseases is unknown. Characterization of Spdef-deficient mice upper and lower airways demonstrated region-specific, Spdef-dependent regulation of basal Muc5b expression. Neonatal Spdef-deficient mice exhibited reductions in BAL Muc5ac and Muc5b. Adult Spdef-deficient mice partially phenocopied Muc5b-deficient mice as they exhibited reduced Muc5b in nasopharyngeal and airway epithelia but not in olfactory Bowman glands, 75% incidence of nasopharyngeal hair/mucus plugs, and mild bacterial otitis media, without defective mucociliary clearance in the nasopharynx. In contrast, tracheal mucociliary clearance was reduced in Spdef-deficient mice in the absence of lung disease. To evaluate the role of Spdef in the development and persistence of Muc5b-predominant mucoobstructive lung disease, Spdef-deficient mice were crossed with Scnn1b-transgenic (Scnn1b-Tg) mice, which exhibit airway surface dehydration-induced airway mucus obstruction and inflammation. Spdef-deficient Scnn1b-Tg mice exhibited reduced Muc5ac, but not Muc5b, expression and BAL content. Airway mucus obstruction was not decreased in Spdef-deficient Scnn1b-Tg mice, consistent with Muc5b-dominant Scnn1b disease, but increased airway neutrophilia was observed compared with Spdef-sufficient Scnn1b-Tg mice. Collectively, these results indicate that Spdef regulates baseline Muc5b expression in respiratory epithelia but does not contribute to Muc5b regulation in a mouse model of Muc5b-predominant mucus obstruction caused by airway dehydration.


Asunto(s)
Enfermedades Pulmonares/metabolismo , Mucina 5B/metabolismo , Depuración Mucociliar/fisiología , Proteínas Proto-Oncogénicas c-ets/genética , Animales , Canales Epiteliales de Sodio/genética , Enfermedades Pulmonares/genética , Ratones Transgénicos , Mucina 5B/genética
17.
Am J Physiol Lung Cell Mol Physiol ; 314(2): L318-L331, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29074490

RESUMEN

The epithelial Na+ channel (ENaC) regulates airway surface hydration. In mouse airways, ENaC is composed of three subunits, α, ß, and γ, which are differentially expressed (α > ß > γ). Airway-targeted overexpression of the ß subunit results in Na+ hyperabsorption, causing airway surface dehydration, hyperconcentrated mucus with delayed clearance, lung inflammation, and perinatal mortality. Notably, mice overexpressing the α- or γ-subunit do not exhibit airway Na+ hyperabsorption or lung pathology. To test whether overexpression of multiple ENaC subunits produced Na+ transport and disease severity exceeding that of ßENaC-Tg mice, we generated double (αß, αγ, ßγ) and triple (αßγ) transgenic mice and characterized their lung phenotypes. Double αγENaC-Tg mice were indistinguishable from WT littermates. In contrast, double ßγENaC-Tg mice exhibited airway Na+ absorption greater than that of ßENaC-Tg mice, which was paralleled by worse survival, decreased mucociliary clearance, and more severe lung pathology. Double αßENaC-Tg mice exhibited Na+ transport rates comparable to those of ßENaC-Tg littermates. However, αßENaC-Tg mice had poorer survival and developed severe parenchymal consolidation. In situ hybridization (RNAscope) analysis revealed both alveolar and airway αENaC-Tg overexpression. Triple αßγENaC-Tg mice were born in Mendelian proportions but died within the first day of life, and the small sample size prevented analyses of cause(s) of death. Cumulatively, these results indicate that overexpression of ßENaC is rate limiting for generation of pathological airway surface dehydration. Notably, airway co-overexpression of ß- and γENaC had additive effects on Na+ transport and disease severity, suggesting dose dependency of these two variables.


Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Enfermedades Pulmonares/patología , Neumonía/patología , Mucosa Respiratoria/patología , Animales , Canales Epiteliales de Sodio/genética , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fenotipo , Neumonía/etiología , Neumonía/metabolismo , Mucosa Respiratoria/metabolismo , Transducción de Señal
18.
Genetics ; 207(2): 801-812, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28851744

RESUMEN

Mucus hyper-secretion is a hallmark feature of asthma and other muco-obstructive airway diseases. The mucin proteins MUC5AC and MUC5B are the major glycoprotein components of mucus and have critical roles in airway defense. Despite the biomedical importance of these two proteins, the loci that regulate them in the context of natural genetic variation have not been studied. To identify genes that underlie variation in airway mucin levels, we performed genetic analyses in founder strains and incipient lines of the Collaborative Cross (CC) in a house dust mite mouse model of asthma. CC founder strains exhibited significant differences in MUC5AC and MUC5B, providing evidence of heritability. Analysis of gene and protein expression of Muc5ac and Muc5b in incipient CC lines (n = 154) suggested that post-transcriptional events were important regulators of mucin protein content in the airways. Quantitative trait locus (QTL) mapping identified distinct, trans protein QTL for MUC5AC (chromosome 13) and MUC5B (chromosome 2). These two QTL explained 18 and 20% of phenotypic variance, respectively. Examination of the MUC5B QTL allele effects and subsequent phylogenetic analysis allowed us to narrow the MUC5B QTL and identify Bpifb1 as a candidate gene. Bpifb1 mRNA and protein expression were upregulated in parallel to MUC5B after allergen challenge, and Bpifb1 knockout mice exhibited higher MUC5B expression. Thus, BPIFB1 is a novel regulator of MUC5B.


Asunto(s)
Proteínas Portadoras/metabolismo , Mucina 5AC/metabolismo , Mucina 5B/metabolismo , Sitios de Carácter Cuantitativo , Mucosa Respiratoria/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Mucina 5AC/genética , Mucina 5B/genética
19.
Mucosal Immunol ; 10(2): 395-407, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27435107

RESUMEN

Airway diseases, including cigarette smoke-induced chronic bronchitis, cystic fibrosis, and primary ciliary dyskinesia are associated with decreased mucociliary clearance (MCC). However, it is not known whether a simple reduction in MCC or concentration-dependent mucus adhesion to airway surfaces dominates disease pathogenesis or whether decreasing the concentration of secreted mucins may be therapeutic. To address these questions, Scnn1b-Tg mice, which exhibit airway mucus dehydration/adhesion, were compared and crossed with Muc5b- and Muc5ac-deficient mice. Absence of Muc5b caused a 90% reduction in MCC, whereas Scnn1b-Tg mice exhibited an ∼50% reduction. However, the degree of MCC reduction did not correlate with bronchitic airway pathology, which was observed only in Scnn1b-Tg mice. Ablation of Muc5b significantly reduced the extent of mucus plugging in Scnn1b-Tg mice. However, complete absence of Muc5b in Scnn1b-Tg mice was associated with increased airway inflammation, suggesting that Muc5b is required to maintain immune homeostasis. Loss of Muc5ac had few phenotypic consequences in Scnn1b-Tg mice. These data suggest that: (i) mucus hyperconcentration dominates over MCC reduction alone to produce bronchitic airway pathology; (ii) Muc5b is the dominant contributor to the Scnn1b-Tg phenotype; and (iii) therapies that limit mucin secretion may reduce plugging, but complete Muc5b removal from airway surfaces may be detrimental.


Asunto(s)
Bronquios/fisiología , Bronquitis Crónica/inmunología , Fibrosis Quística/inmunología , Síndrome de Kartagener/inmunología , Mucina 5AC/metabolismo , Mucina 5B/metabolismo , Depuración Mucociliar , Obstrucción de las Vías Aéreas/genética , Animales , Bronquios/patología , Canales Epiteliales de Sodio/genética , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mucina 5AC/genética , Mucina 5B/genética , Depuración Mucociliar/genética , Fumar/efectos adversos
20.
Am J Physiol Lung Cell Mol Physiol ; 310(9): L860-7, 2016 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-26968767

RESUMEN

Respiratory infections are a major cause of morbidity and mortality in the elderly. Previous reports have suggested that mucociliary clearance (MCC) is impaired in older individuals, but the cause is unclear. To unravel the mechanisms responsible for the age-associated decline in MCC, we investigated the MCC system in young (3 mo) and old (2 yr) C57BL/6 mice. We found that old mice had significantly reduced MCC function in both the upper and lower airways compared with young mice. Measurement of bioelectric properties of isolated tracheal and bronchial tissue revealed a significant decrease in Cl(-) secretion, suggesting that the older mice may have a reduced ability to maintain a sufficiently hydrated airway surface for efficient MCC. Ciliary beat frequency was also observed to be reduced in the older animals; however, this reduction was small relative to the reduction in MCC. Interestingly, the level of the major secreted mucin, Muc5b, was found to be reduced in both bronchioalveolar lavage and isolated tracheal tissue. Our previous studies of Muc5b(-/-) mice have demonstrated that Muc5b is essential for normal MCC in the mouse. Furthermore, examination of Muc5b(+/-) and wild-type animals revealed that heterozygous animals, which secrete ∼50% of the wild-type level of Muc5b, also demonstrate a markedly reduced level of MCC, confirming the importance of Muc5b levels to MCC. These results demonstrate that aged mice exhibit a decrease in MCC and suggest that a reduced level of secretion of both Cl(-) and Muc5b may be responsible.


Asunto(s)
Envejecimiento , Mucina 5B/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Cloruros/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Depuración Mucociliar , Tráquea/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA