Hydrogen Sulfide Inhibits TMPRSS2 in Human Airway Epithelial Cells: Implications for SARS-CoV-2 Infection

The COVID-19 pandemic has now affected around 190 million people worldwide, accounting for more than 4 million confirmed deaths. Besides ongoing global vaccination, finding protective and therapeutic strategies is an urgent clinical need. SARS-CoV-2 mostly infects the host organism via the respiratory system, requiring angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enter target cells. Therefore, these surface proteins are considered potential druggable targets. Hydrogen sulfide (H2S) is a gasotransmitter produced by several cell types and is also part of natural compounds, such as sulfurous waters that are often inhaled as low-intensity therapy and prevention in different respiratory conditions. H2S is a potent biological mediator, with anti-oxidant, anti-inflammatory, and, as more recently shown, also anti-viral activities. Considering that respiratory epithelial cells can be directly exposed to H2S by inhalation, here we tested the in vitro effects of H2S-donors on TMPRSS2 and ACE2 expression in human upper and lower airway epithelial cells. We showed that H2S significantly reduces the expression of TMPRSS2 without modifying ACE2 expression both in respiratory cell lines and primary human upper and lower airway epithelial cells. Results suggest that inhalational exposure of respiratory epithelial cells to natural H2S sources may hinder SARS-CoV-2 entry into airway epithelial cells and, consequently, potentially prevent the virus from spreading into the lower respiratory tract and the lung.

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Crystalline silica particles cause rapid NLRP3-dependent mitochondrial depolarization and DNA damage in airway epithelial cells

Particle and Fibre Toxicology ◽

10.1186/s12989-020-00370-2 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Rongrong Wu ◽

Johan Högberg ◽

Mikael Adner ◽

Patricia Ramos-Ramírez ◽

Ulla Stenius ◽

...

Keyword(s):

Dna Damage ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Silica Particles ◽

Airway Epithelial ◽

Respiratory Epithelial Cells ◽

Damage Response ◽

Exposure Levels ◽

Respiratory Epithelial

Abstract Background Respirable crystalline silica causes lung carcinomas and many thousand future cancer cases are expected in e.g. Europe. Critical questions are how silica causes genotoxicity in the respiratory epithelium and if new cases can be avoided by lowered permissible exposure levels. In this study we investigate early DNA damaging effects of low doses of silica particles in respiratory epithelial cells in vitro and in vivo in an effort to understand low-dose carcinogenic effects of silica particles. Results We find DNA damage accumulation already after 5–10 min exposure to low doses (5 μg/cm2) of silica particles (Min-U-Sil 5) in vitro. DNA damage was documented as increased levels of γH2AX, pCHK2, by Comet assay, AIM2 induction, and by increased DNA repair (non-homologous end joining) signaling. The DNA damage response (DDR) was not related to increased ROS levels, but to a NLRP3-dependent mitochondrial depolarization. Particles in contact with the plasma membrane elicited a Ser198 phosphorylation of NLRP3, co-localization of NLRP3 to mitochondria and depolarization. FCCP, a mitochondrial uncoupler, as well as overexpressed NLRP3 mimicked the silica-induced depolarization and the DNA damage response. A single inhalation of 25 μg silica particles gave a similar rapid DDR in mouse lung. Biomarkers (CC10 and GPRC5A) indicated an involvement of respiratory epithelial cells. Conclusions Our findings demonstrate a novel mode of action (MOA) for silica-induced DNA damage and mutagenic double strand breaks in airway epithelial cells. This MOA seems independent of particle uptake and of an involvement of macrophages. Our study might help defining models for estimating exposure levels without DNA damaging effects.

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Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00030.2004 ◽

2004 ◽

Vol 287 (1) ◽

pp. L143-L152 ◽

Cited By ~ 139

Author(s):

Shawn J. Skerrett ◽

H. Denny Liggitt ◽

Adeline M. Hajjar ◽

Robert K. Ernst ◽

Samuel I. Miller ◽

...

Keyword(s):

Epithelial Cells ◽

Inflammatory Response ◽

Transgenic Mice ◽

Lung Inflammation ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Respiratory Epithelial Cells ◽

Distal Airway ◽

Tnf Α ◽

Respiratory Epithelial

To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-κB activation in the respiratory epithelium using a mutant IκB-α construct that functioned as a dominant negative inhibitor of NF-κB translocation (dnIκB-α). We developed two lines of transgenic mice in which expression of dnIκB-α was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-κB was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-α, IL-1β, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-α within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-κB activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.

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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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Effects of aqueous extracts of PM10 filters from the Utah Valley on human airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.5.l960 ◽

1999 ◽

Vol 277 (5) ◽

pp. L960-L967 ◽

Cited By ~ 41

Author(s):

Mark W. Frampton ◽

Andrew J. Ghio ◽

James M. Samet ◽

Johnny L. Carson ◽

Jacqueline D. Carter ◽

...

Keyword(s):

Epithelial Cells ◽

Ambient Air ◽

Airway Epithelial Cells ◽

Aqueous Extracts ◽

Airway Epithelial ◽

Steel Mill ◽

Respiratory Epithelial Cells ◽

Lactate Dehydrogenase Release ◽

Oxidant Production ◽

Respiratory Epithelial

We hypothesized that the reduction in hospital respiratory admissions in the Utah Valley during closure of a local steel mill in 1986–1987 was attributable in part to decreased toxicity of ambient air particles. Sampling filters for particulate matter < 10 μm (PM10) were obtained from a Utah Valley monitoring station for the year before ( year 1), during ( year 2), and after ( year 3) the steel mill closure. Aqueous extracts of the filters were analyzed for metal content and oxidant production and added to cultures of human respiratory epithelial (BEAS-2B) cells for 2 or 24 h. Year 2 dust contained the lowest concentrations of soluble iron, copper, and zinc and showed the least oxidant generation. Only dust from year 3 caused cytotoxicity (by microscopy and lactate dehydrogenase release) at 500 μg/ml. Year 1 and year 3, but not year 2, dust induced expression of interleukin-6 and -8 in a dose-response fashion. The effects of ambient air particles on human respiratory epithelial cells vary significantly with time and metal concentrations.

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Visualization of labile zinc and its role in apoptosis of primary airway epithelial cells and cell lines

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.6.l1172 ◽

2000 ◽

Vol 279 (6) ◽

pp. L1172-L1183 ◽

Cited By ~ 63

Author(s):

Ai Q. Truong-Tran ◽

Richard E. Ruffin ◽

Peter D. Zalewski

Keyword(s):

Hydrogen Peroxide ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Zn Deficiency ◽

The Novel ◽

Airway Epithelial ◽

Caspase Activation ◽

Respiratory Epithelial Cells ◽

Upper Respiratory ◽

Respiratory Epithelial

The respiratory epithelium is vulnerable to noxious substances, resulting in the shedding of cells and decreased protection. Zinc (Zn), an antioxidant and cytoprotectant, can suppress apoptosis in a variety of cells. Here we used the novel Zn-specific fluorophore Zinquin to visualize and quantify labile intracellular Zn in respiratory epithelial cells. Zinquin fluorescence in isolated ciliated tracheobronchial epithelial cells and intact epithelium from sheep and pigs revealed an intense fluorescence in the apical and mitochondria-rich cytoplasm below the cilia. Zinquin fluorescence was quenched by the Zn chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and increased by the Zn ionophore pyrithione. We also assessed whether changes in intracellular labile Zn would influence susceptibility of these cells to apoptosis by hydrogen peroxide. Our results confirm that Zn deficiency enhanced hydrogen peroxide-induced caspase activation from 1.24 ± 0.12 to 2.58 ± 0.53 units · μg protein−1· h−1( P ≤ 0.05); Zn supplementation suppressed these effects. These findings are consistent with the hypothesis that Zn protects upper respiratory epithelial cells and may have implications for human asthma where there is hypozincemia and epithelial damage.

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Respiratory epithelial cells demonstrate lactoferrin receptors that increase after metal exposure

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.276.6.l933 ◽

1999 ◽

Vol 276 (6) ◽

pp. L933-L940 ◽

Cited By ~ 9

Author(s):

Andrew J. Ghio ◽

Jacqueline D. Carter ◽

Lisa A. Dailey ◽

Robert B. Devlin ◽

James M. Samet

Keyword(s):

Oxidative Stress ◽

Fly Ash ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Metal Exposure ◽

Airway Epithelial ◽

Respiratory Epithelial Cells ◽

Catalytically Active ◽

Oil Fly Ash ◽

Respiratory Epithelial

Human airway epithelial cells can increase expression of both lactoferrin and ferritin after exposure to catalytically active metal. These proteins transport and store metal, with coordination sites fully complexed, and therefore can diminish the oxidative stress. The intracellular transport of lactoferrin results in a transfer of complexed metal to ferritin, where it is stored in a less reactive form. This effort to control the injurious properties of metals would be facilitated by lactoferrin receptors (LfRs) on airway epithelial cells. We tested the hypotheses that 1) LfRs exist on respiratory epithelial cells and 2) exposure to both an air pollution particle, which has abundant concentrations of metals, and individual metal salts increase the expression of LfRs. Before exposure to either the particle or metals, incubation of BEAS-2B cells with varying concentrations of125I-labeled lactoferrin demonstrated lactoferrin binding that was saturable. Measurement of125I-lactoferrin binding after the inclusion of 100 μg/ml of oil fly ash in the incubation medium demonstrated increased binding within 5 min of exposure, which reached a maximal value at 45 min. Inclusion of 1.0 mM deferoxamine in the incubation of BEAS-2B cells with 100 μg/ml of oil fly ash decreased lactoferrin binding. Comparable to the particle, exposure of BEAS-2B cells to either 1.0 mM vanadyl sulfate or 1.0 mM iron (III) sulfate, but not to nickel sulfate, for 45 min elevated LfR activity. We conclude that LfRs on respiratory epithelial cells increased after exposure to metal. LfRs could participate in decreasing the oxidative stress presented to the lower respiratory tract by complexing catalytically active metals.

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Respiratory Viruses Augment the Adhesion of Bacterial Pathogens to Respiratory Epithelium in a Viral Species- and Cell Type-Dependent Manner

Journal of Virology ◽

10.1128/jvi.80.4.1629-1636.2006 ◽

2006 ◽

Vol 80 (4) ◽

pp. 1629-1636 ◽

Cited By ~ 202

Author(s):

Vasanthi Avadhanula ◽

Carina A. Rodriguez ◽

John P. DeVincenzo ◽

Yan Wang ◽

Richard J. Webby ◽

...

Keyword(s):

Influenza Virus ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Respiratory Epithelial Cells ◽

Bronchial Epithelial ◽

Immortalized Cell ◽

Respiratory Epithelial ◽

Primary Bronchial Epithelial Cells ◽

Small Airway Epithelial Cells

ABSTRACT Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.

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Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza a Virus Infection in C57BL/6 Mice

Cells ◽

10.3390/cells10030509 ◽

2021 ◽

Vol 10 (3) ◽

pp. 509 ◽

Cited By ~ 1

Author(s):

Meenakshi Tiwary ◽

Robert J. Rooney ◽

Swantje Liedmann ◽

Kim S. LeMessurier ◽

Amali E. Samarasinghe

Keyword(s):

Epithelial Cells ◽

Influenza A Virus ◽

Early Phase ◽

Influenza A ◽

Airway Epithelial Cells ◽

Epithelial Barrier ◽

Virus Infectivity ◽

Airway Epithelial ◽

In Vivo Models

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

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