scholarly journals Transcriptomic profile of cystic fibrosis airway epithelial cells undergoing repair

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Alice Zoso ◽  
Aderonke Sofoluwe ◽  
Marc Bacchetta ◽  
Marc Chanson
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Primary Cultures ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Molecular Signature ◽  
Airway Epithelial ◽  
Transcriptomic Profile ◽  
Different Cell Types

Abstract Pathological remodeling of the airway epithelium is commonly observed in Cystic Fibrosis (CF). The different cell types that constitute the airway epithelium are regenerated upon injury to restore integrity and maintenance of the epithelium barrier function. The molecular signature of tissue repair in CF airway epithelial cells has, however, not well been investigated in primary cultures. We therefore collected RNA-seq data from well-differentiated primary cultures of bronchial human airway epithelial cells (HAECs) of CF (F508del/F508del) and non-CF (NCF) origins before and after mechanical wounding, exposed or not to flagellin. We identified the expression changes with time of repair of genes, the products of which are markers of the different cell types that constitute the airway epithelium (basal, suprabasal, intermediate, secretory, goblet and ciliated cells as well as ionocytes). Researchers in the CF field may benefit from this transcriptomic profile, which covers the initial steps of wound repair and revealed differences in this process between CF and NCF cultures.

Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C243-C251 ◽  
Author(s):  
M. E. Egan ◽  
E. M. Schwiebert ◽  
W. B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Incubation Temperature ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Cl Channel ◽  
Channel Expression

When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF)-affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells.

A method for measuring Cl efflux from dispersed cells of airway epithelium

1995 ◽  
Vol 78 (3) ◽  
pp. 1197-1202 ◽  
Author(s):  
T. Ohrui ◽  
B. Q. Shen ◽  
R. J. Mrsny ◽  
J. H. Widdicombe
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Room Temperature ◽  
Primary Cultures ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Isolated Cells ◽  
T84 Cells ◽  
Tracheal Epithelial Cells ◽  
3T3 Fibroblasts

This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3′,5′-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals β2-Adrenoceptor signaling in airway epithelial cells promotes eosinophilic inflammation, mucous metaplasia, and airway contractility

2017 ◽  
Vol 114 (43) ◽  
pp. E9163-E9171 ◽  
Author(s):  
Long P. Nguyen ◽  
Nour A. Al-Sawalha ◽  
Sergio Parra ◽  
Indira Pokkunuri ◽  
Ozozoma Omoluabi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Smooth Muscle Cells ◽  
Airway Epithelial ◽  
Eosinophilic Inflammation ◽  
Mucus Production ◽  
Asthma Phenotype ◽  
Transgenic Expression

The mostly widely used bronchodilators in asthma therapy are β2-adrenoreceptor (β2AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that β2AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that β2AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of β2AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that β2AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent β2AR ligand shows the receptors are highly expressed in airway epithelium. In β2AR−/− mice, transgenic expression of β2ARs only in airway epithelium is sufficient to rescue IL-13–induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of β-arrestin-2 (βarr-2−/−) attenuates the asthma phenotype as in β2AR−/− mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by β2AR signaling. Together, these results suggest β2ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the β2AR involves βarr-2. These results identify β2AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells.

scholarly journals TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Claudia Bilodeau ◽  
Sharareh Shojaie ◽  
Olivia Goltsis ◽  
Jinxia Wang ◽  
Daochun Luo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Es Cells ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Basal Cells ◽  
Airway Epithelial ◽  
Multipotent Cells ◽  
Cell Source ◽  
Different Cell Types ◽  
Airway Cells

AbstractThe use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

NF-κB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

2005 ◽  
Vol 288 (3) ◽  
pp. L471-L479 ◽  
Author(s):  
Theresa Joseph ◽  
Dwight Look ◽  
Thomas Ferkol
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Cell Cultures ◽  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Airway Epithelial

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-κB (NF-κB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-κB in cells isolated from five CF (ΔF508/ΔF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-κB subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-κB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-κB inhibitor IκBα were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-κB to nuclei of primary CF epithelial cell cultures, but intranuclear IκBα may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

scholarly journals A transcription factor network represses CFTR gene expression in airway epithelial cells

2018 ◽  
Vol 475 (7) ◽  
pp. 1323-1334 ◽  
Author(s):  
Michael J. Mutolo ◽  
Shih-Hsing Leir ◽  
Sara L. Fossum ◽  
James A. Browne ◽  
Ann Harris
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Positive Impact ◽  
Regulatory Element ◽  
Airway Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Cftr Gene ◽  
Mrna Levels ◽  
Airway Epithelial

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the inherited disorder cystic fibrosis (CF). Lung disease is the major cause of CF morbidity, though CFTR expression levels are substantially lower in the airway epithelium than in pancreatic duct and intestinal epithelia, which also show compromised function in CF. Recently developed small molecule therapeutics for CF are highly successful for one specific CFTR mutation and have a positive impact on others. However, the low abundance of CFTR transcripts in the airway limits the opportunity for drugs to correct the defective substrate. Elucidation of the transcriptional mechanisms for the CFTR locus has largely focused on intragenic and intergenic tissue-specific enhancers and their activating trans-factors. Here, we investigate whether the low CFTR levels in the airway epithelium result from the recruitment of repressive proteins directly to the locus. Using an siRNA screen to deplete ∼1500 transcription factors (TFs) and associated regulatory proteins in Calu-3 lung epithelial cells, we identified nearly 40 factors that upon depletion elevated CFTR mRNA levels more than 2-fold. A subset of these TFs was validated in primary human bronchial epithelial cells. Among the strongest repressors of airway expression of CFTR were Krüppel-like factor 5 and Ets homologous factor, both of which have pivotal roles in the airway epithelium. Depletion of these factors, which are both recruited to an airway-selective cis-regulatory element at −35 kb from the CFTR promoter, improved CFTR production and function, thus defining novel therapeutic targets for enhancement of CFTR.

scholarly journals Pulmonary fibrosis distal airway epithelia are dynamically and structurally dysfunctional

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ian T. Stancil ◽  
Jacob E. Michalski ◽  
Duncan Davis-Hall ◽  
Hong Wei Chu ◽  
Jin-Ah Park ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Airway Epithelium ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Chronic Lung Diseases ◽  
Distal Airway ◽  
Signaling Axis

AbstractThe airway epithelium serves as the interface between the host and external environment. In many chronic lung diseases, the airway is the site of substantial remodeling after injury. While, idiopathic pulmonary fibrosis (IPF) has traditionally been considered a disease of the alveolus and lung matrix, the dominant environmental (cigarette smoking) and genetic (gain of function MUC5B promoter variant) risk factor primarily affect the distal airway epithelium. Moreover, airway-specific pathogenic features of IPF include bronchiolization of the distal airspace with abnormal airway cell-types and honeycomb cystic terminal airway-like structures with concurrent loss of terminal bronchioles in regions of minimal fibrosis. However, the pathogenic role of the airway epithelium in IPF is unknown. Combining biophysical, genetic, and signaling analyses of primary airway epithelial cells, we demonstrate that healthy and IPF airway epithelia are biophysically distinct, identifying pathologic activation of the ERBB-YAP axis as a specific and modifiable driver of prolongation of the unjammed-to-jammed transition in IPF epithelia. Furthermore, we demonstrate that this biophysical state and signaling axis correlates with epithelial-driven activation of the underlying mesenchyme. Our data illustrate the active mechanisms regulating airway epithelial-driven fibrosis and identify targets to modulate disease progression.

scholarly journals Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadzeya Marozkina ◽  
Laura Smith ◽  
Yi Zhao ◽  
Joe Zein ◽  
James F. Chmiel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Nitrogen Oxides ◽  
Airway Epithelium ◽  
Airflow Obstruction ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
Human Airway Epithelial Cells

AbstractEndothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

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