In vitro 3D culture lung model from expanded primary cystic fibrosis human airway cells

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl− current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

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TMPRSS2 Is the Major Activating Protease of Influenza A Virus in Primary Human Airway Cells and Influenza B Virus in Human Type II Pneumocytes

Journal of Virology ◽

10.1128/jvi.00649-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 29

Author(s):

Hannah Limburg ◽

Anne Harbig ◽

Dorothea Bestle ◽

David A. Stein ◽

Hong M. Moulton ◽

...

Keyword(s):

Influenza A ◽

Influenza B Virus ◽

Influenza B ◽

Virus Infectivity ◽

Type Ii ◽

Proteolytic Activation ◽

Human Airway ◽

Type Ii Pneumocytes ◽

Airway Cells

ABSTRACT Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections. IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro. This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.

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Anex vivocystic fibrosis model recapitulates key clinical aspects of chronicStaphylococcus aureusinfection

10.1101/604363 ◽

2019 ◽

Cited By ~ 1

Author(s):

Esther Sweeney ◽

Marwa M. Hassan ◽

Niamh E. Harrington ◽

Alan R. Smyth ◽

Matthew N. Hurley ◽

...

Keyword(s):

Cystic Fibrosis ◽

Ex Vivo ◽

Lung Model ◽

Clinical Aspects ◽

Small Colony Variant ◽

Tissue Invasion ◽

Disease Characteristics ◽

Small Colony ◽

Clinically Significant

AbstractStaphylococcus aureusis one of the most prevalent organisms isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. Clinical studies are limited in scope by age and health of participants andin vitrostudies are not always able to accurately recapitulate chronic disease characteristics such as the development of small colony variants. Further, animal models also do not fully represent features of clinical disease: in particular, mice are not readily colonized byS. aureusand when infection is established it leads to the formation of abscesses, a phenomenon almost never observed in the human CF lung. Here, we present details of the development of an existingex vivopig lung model of CF infection to investigate the growth ofS. aureus. We show thatS. aureusis able to establish infection and demonstrates clinically significant characteristics including small colony variant phenotype, increased antibiotic tolerance and preferential localisation in mucus. Tissue invasion and the formation of abscesses were not observed, in line with clinical data.

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Intrinsic Abnormalities of Cystic Fibrosis Airway Connective Tissue Revealed by an In Vitro 3D Stromal Model

Cells ◽

10.3390/cells9061371 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1371

Author(s):

Claudia Mazio ◽

Laura S. Scognamiglio ◽

Rossella De Cegli ◽

Luis J. V. Galietta ◽

Diego Di Bernardo ◽

...

Keyword(s):

Cystic Fibrosis ◽

Inflammatory Response ◽

Bacterial Infections ◽

Inflammatory Cells ◽

Lung Model ◽

Lung Dysfunction ◽

Novel Therapeutic Strategies ◽

And Function

Cystic fibrosis is characterized by lung dysfunction involving mucus hypersecretion, bacterial infections, and inflammatory response. Inflammation triggers pro-fibrotic signals that compromise lung structure and function. At present, several in vitro cystic fibrosis models have been developed to study epithelial dysfunction but none of these focuses on stromal alterations. Here we show a new cystic fibrosis 3D stromal lung model made up of primary fibroblasts embedded in their own extracellular matrix and investigate its morphological and transcriptomic features. Cystic fibrosis fibroblasts showed a high proliferation rate and produced an abundant and chaotic matrix with increased protein content and elastic modulus. More interesting, they had enhanced pro-fibrotic markers and genes involved in epithelial function and inflammatory response. In conclusion, our study reveals that cystic fibrosis fibroblasts maintain in vitro an activated pro-fibrotic state. This abnormality may play in vivo a role in the modulation of epithelial and inflammatory cell behavior and lung remodeling. We argue that the proposed bioengineered model may provide new insights on epithelial/stromal/inflammatory cells crosstalk in cystic fibrosis, paving the way for novel therapeutic strategies.

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Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00364.2016 ◽

2017 ◽

Vol 312 (5) ◽

pp. L688-L702 ◽

Cited By ~ 18

Author(s):

Samuel A. Molina ◽

Hannah K. Moriarty ◽

Daniel T. Infield ◽

Barry R. Imhoff ◽

Rachel J. Vance ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Small Molecules ◽

Glucose Uptake ◽

Glucose Transporter ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Airway Epithelia ◽

Human Airway ◽

Airway Cells

Cystic fibrosis-related diabetes is the most common comorbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Because glucose is a nutrient present in the airways of patients with bacterial airway infections and because insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by quantitative PCR, immunoblot, immunofluorescence, and glucose uptake assays. Phosphatidylinositol 3-kinase/protein kinase B (Akt) signaling and cystic fibrosis transmembrane conductance regulator (CFTR) activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed glucose transporter 4 and that application of insulin stimulated cytochalasin B-inhibitable glucose uptake, consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared with that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.

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Glucocorticoid Clearance and Metabolite Profiling in an In Vitro Human Airway Epithelium Lung Model

Drug Metabolism and Disposition ◽

10.1124/dmd.115.066365 ◽

2015 ◽

Vol 44 (2) ◽

pp. 220-226 ◽

Cited By ~ 4

Author(s):

D. Rivera-Burgos ◽

U. Sarkar ◽

A. R. Lever ◽

M. J. Avram ◽

J. R. Coppeta ◽

...

Keyword(s):

Airway Epithelium ◽

Metabolite Profiling ◽

Lung Model ◽

Human Airway ◽

Human Airway Epithelium

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Engineered mutant α-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis–based cell and mice models

Science Advances ◽

10.1126/sciadv.abc5911 ◽

2020 ◽

Vol 6 (47) ◽

pp. eabc5911

Author(s):

Anindit Mukherjee ◽

Kelvin D. MacDonald ◽

Jeonghwan Kim ◽

Michael I. Henderson ◽

Yulia Eygeris ◽

...

Keyword(s):

Cystic Fibrosis ◽

Therapeutic Potential ◽

Lipid Nanoparticles ◽

Sodium Absorption ◽

Airway Surface Liquid ◽

Surface Liquid ◽

Cf Transmembrane Conductance Regulator ◽

Airway Cells

Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.

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Progress in Assessing Air Pollutant Risks from In Vitro Exposures: Matching Ozone Dose and Effect in Human Airway Cells

Toxicological Sciences ◽

10.1093/toxsci/kfu115 ◽

2014 ◽

Vol 141 (1) ◽

pp. 198-205 ◽

Cited By ~ 13

Author(s):

Gary E. Hatch ◽

Kelly E. Duncan ◽

David Diaz-Sanchez ◽

Michael T. Schmitt ◽

Andrew J. Ghio ◽

...

Keyword(s):

Air Pollutant ◽

Human Airway ◽

Ozone Dose ◽

Airway Cells

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In Vitro Analysis of Tobramycin-Treated Pseudomonas aeruginosa Biofilms on Cystic Fibrosis-Derived Airway Epithelial Cells

Infection and Immunity ◽

10.1128/iai.01373-07 ◽

2008 ◽

Vol 76 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 118

Author(s):

Gregory G. Anderson ◽

Sophie Moreau-Marquis ◽

Bruce A. Stanton ◽

George A. O'Toole

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Epithelial Cells ◽

Biofilm Formation ◽

Airway Epithelial Cells ◽

Bacterial Virulence ◽

Model Systems ◽

Virulence Gene Expression ◽

Airway Cells

ABSTRACT P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycin-treated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

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