Topical TMPRSS2 inhibition prevents SARS-CoV-2 infection in differentiated primary human airway cells

AbstractBackgroundThere are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection.MethodsThe likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection.ResultsWe first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection.ConclusionThese experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.

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Antidiabetic Effects of Bisamide Derivative of Dicarboxylic Acid in Metabolic Disorders

International Journal of Molecular Sciences ◽

10.3390/ijms21030991 ◽

2020 ◽

Vol 21 (3) ◽

pp. 991 ◽

Cited By ~ 1

Author(s):

Angelina Vladimirovna Pakhomova ◽

Vladimir Evgenievich Nebolsin ◽

Olga Victorovna Pershina ◽

Vyacheslav Andreevich Krupin ◽

Lubov Alexandrovna Sandrikina ◽

...

Keyword(s):

Metabolic Syndrome ◽

Stem Cells ◽

Dicarboxylic Acid ◽

Metabolic Disorders ◽

Biological Effects ◽

Treatment Options ◽

The Metabolic Syndrome ◽

Pancreatic Stem Cells ◽

The Impact

In clinical practice, the metabolic syndrome can lead to multiple complications, including diabetes. It remains unclear which component of the metabolic syndrome (obesity, inflammation, hyperglycemia, or insulin resistance) has the strongest inhibitory effect on stem cells involved in beta cell regeneration. This makes it challenging to develop effective treatment options for complications such as diabetes. In our study, experiments were performed on male C57BL/6 mice where metabolic disorders have been introduced experimentally by a combination of streptozotocin-treatment and a high-fat diet. We evaluated the biological effects of Bisamide Derivative of Dicarboxylic Acid (BDDA) and its impact on pancreatic stem cells in vivo. To assess the impact of BDDA, we applied a combination of histological and biochemical methods along with a cytometric analysis of stem cell and progenitor cell markers. We show that in mice with metabolic disorders, BDDA has a positive effect on lipid and glucose metabolism. The pancreatic restoration was associated with a decrease of the inhibitory effects of inflammation and obesity factors on pancreatic stem cells. Our data shows that BDDA increases the number of pancreatic stem cells. Thus, BDDA could be used as a new compound for treating complication of the metabolic syndrome such as diabetes.

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Benzisothiazolinone upregulates the MUC5AC expression via ERK1/2, p38, and NF-κB pathways in airway epithelial cells

Toxicology Research ◽

10.1039/c9tx00135b ◽

2019 ◽

Vol 8 (5) ◽

pp. 704-710

Author(s):

Soyoung Kwak ◽

Yoon Seok Choi ◽

Hyung Gyun Na ◽

Chang Hoon Bae ◽

Si-Youn Song ◽

...

Keyword(s):

Epithelial Cells ◽

Inflammatory Diseases ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Mucus Hypersecretion ◽

Human Airway ◽

Airway Diseases ◽

Extracellular Signal ◽

Human Airway Epithelial Cells

Abstract Mucus plays an important role in protecting the respiratory tract from irritants. However, mucus hypersecretion is a major indicator of airway diseases. 1,2-Benzisothiazolin-3-one (BIT), as a microbicide, induces asthmatic inflammation. Therefore, we focused on the effects of BIT-related mucin secretion in airway epithelial cells. Our in vivo study showed increased mucus and MUC5AC expressions in the bronchioles of mice that inhaled BIT. For investigating the signaling pathways, we performed experiments in human airway epithelial cells. BIT induced the MUC5AC expression and significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The specific inhibitors of ERK1/2, p38, and NF-κB blocked the BIT-induced MUC5AC expression. Therefore, these results suggest that BIT induces the MUC5AC expression via the ERK1/2, p38, and NF-κB pathways in human airway epithelial cells, which may be involved in mucus hypersecretion associated with airway inflammatory diseases.

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Functional analysis of the impact of ORMDL3 expression on inflammation and activation of the unfolded protein response in human airway epithelial cells

Allergy Asthma & Clinical Immunology ◽

10.1186/1710-1492-9-4 ◽

2013 ◽

Vol 9 (1) ◽

Cited By ~ 29

Author(s):

Karolynn J Hsu ◽

Stuart E Turvey

Keyword(s):

Functional Analysis ◽

Epithelial Cells ◽

Unfolded Protein Response ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Unfolded Protein ◽

Human Airway ◽

The Unfolded Protein Response ◽

Protein Response ◽

The Impact

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Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.1.535-540.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 535-540 ◽

Cited By ~ 27

Author(s):

Robert W. Walters ◽

Dongsheng Duan ◽

John F. Engelhardt ◽

Michael J. Welsh

Keyword(s):

Calcium Phosphate ◽

Gene Transfer ◽

Mouse Lung ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway

ABSTRACT Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaPicoprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.

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Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells

Cells ◽

10.3390/cells9092137 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2137

Author(s):

Vinciane Saint-Criq ◽

Livia Delpiano ◽

John Casement ◽

Jennifer C. Onuora ◽

JinHeng Lin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Cell Lineage ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Functional Responses ◽

Human Airway ◽

The Impact ◽

Cftr Modulators ◽

Human Airway Epithelial Cells

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications.

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MUC1 contributes to goblet cell metaplasia and MUC5AC expression in response to cigarette smoke in vivo

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00049.2019 ◽

2020 ◽

Vol 319 (1) ◽

pp. L82-L90 ◽

Cited By ~ 3

Author(s):

Kosuke Kato ◽

Eugene H. Chang ◽

Yin Chen ◽

Wenju Lu ◽

Marianne M. Kim ◽

...

Keyword(s):

Cigarette Smoke ◽

Goblet Cell ◽

Airway Epithelial Cells ◽

Apical Surface ◽

Intracellular Compartment ◽

Egfr Activation ◽

Intermediate Cells ◽

Goblet Cell Metaplasia

Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.

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Effects of xenon gas on human airway epithelial cells during hyperoxia and hypothermia

Journal of Neonatal-Perinatal Medicine ◽

10.3233/npm-190364 ◽

2020 ◽

Vol 13 (4) ◽

pp. 469-476

Author(s):

Y. Zhu ◽

J.J. Mosko ◽

A. Chidekel ◽

M.R. Wolfson ◽

T.H. Shaffer

Keyword(s):

Epithelial Cells ◽

Total Protein ◽

Cell Function ◽

Airway Epithelial Cells ◽

Control Group ◽

Apical Surface ◽

Airway Epithelial ◽

Human Airway ◽

Immunofluorescence Localization ◽

Human Airway Epithelial Cells

BACKGROUND: Hypothermia with xenon gas has been used to reduce brain injury and disability rate after perinatal hypoxia-ischemia. We evaluated xenon gas therapy effects in an in vitro model with or without hypothermia on cultured human airway epithelial cells (Calu-3). METHODS: Calu-3 monolayers were grown at an air-liquid interface and exposed to one of the following conditions: 1) 21% FiO2 at 37°C (control); 2) 45% FiO2 and 50% xenon at 37°C; 3) 21% FiO2 and 50% xenon at 32°C; 4) 45% FiO2 and 50% xenon at 32°C for 24 hours. Transepithelial resistance (TER) measurements were performed and apical surface fluids were collected and assayed for total protein, IL-6, and IL-8. Three monolayers were used for immunofluorescence localization of zonula occludens-1 (ZO-1). The data were analyzed by one-way ANOVA. RESULTS: TER decreased at 24 hours in all treatment groups. Xenon with hyperoxia and hypothermia resulted in greatest decrease in TER compared with other groups. Immunofluorescence localization of ZO-1 (XY) showed reduced density of ZO-1 rings and incomplete ring-like staining in the 45% FiO2– 50% xenon group at 32°C compared with other groups. Secretion of total protein was not different among groups. Secretion of IL-6 in 21% FiO2 with xenon group at 32°C was less than that of the control group. The secretion of IL-8 in 45% FiO2 with xenon at 32°C was greater than that of other groups. CONCLUSION: Hyperoxia and hypothermia result in detrimental epithelial cell function and inflammation over 24-hour exposure. Xenon gas did not affect cell function or reduce inflammation.

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Extracellular Vesicle-Mediated siRNA Delivery, Protein Delivery, and CFTR Complementation in Well-Differentiated Human Airway Epithelial Cells

Genes ◽

10.3390/genes11040351 ◽

2020 ◽

Vol 11 (4) ◽

pp. 351 ◽

Cited By ~ 1

Author(s):

Brajesh K. Singh ◽

Ashley L. Cooney ◽

Sateesh Krishnamurthy ◽

Patrick L. Sinn

Keyword(s):

Epithelial Cells ◽

Airway Epithelial Cells ◽

Target Cells ◽

Airway Epithelial ◽

Long Distance ◽

Human Airway ◽

Well Differentiated ◽

Human Airway Epithelial Cells

Extracellular vesicles (EVs) are a class of naturally occurring secreted cellular bodies that are involved in long distance cell-to-cell communication. Proteins, lipids, mRNA, and miRNA can be packaged into these vesicles and released from the cell. This information is then delivered to target cells. Since EVs are naturally adapted molecular messengers, they have emerged as an innovative, inexpensive, and robust method to deliver therapeutic cargo in vitro and in vivo. Well-differentiated primary cultures of human airway epithelial cells (HAE) are refractory to standard transfection techniques. Indeed, common strategies used to overexpress or knockdown gene expression in immortalized cell lines simply have no detectable effect in HAE. Here we use EVs to efficiently deliver siRNA or protein to HAE. Furthermore, EVs can deliver CFTR protein to cystic fibrosis donor cells and functionally correct the Cl− channel defect in vitro. EV-mediated delivery of siRNA or proteins to HAE provides a powerful genetic tool in a model system that closely recapitulates the in vivo airways.

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Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer

Journal of Virology ◽

10.1128/jvi.74.8.3852-3858.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3852-3858 ◽

Cited By ~ 238

Author(s):

Joseph Zabner ◽

Michael Seiler ◽

Robert Walters ◽

Robert M. Kotin ◽

Wendy Fulgeras ◽

...

Keyword(s):

Gene Transfer ◽

Virus Type ◽

Target Cells ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway ◽

Human Airway Epithelia

ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

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