Pseudomonas aeruginosapyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L94-L103 ◽  
Author(s):  
Yunxia Q. O'Malley ◽  
Krzysztof J. Reszka ◽  
Douglas R. Spitz ◽  
Gerene M. Denning ◽  
Bradley E. Britigan
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Culture Media ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Alternative Source ◽  
Cell Functions

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H2O2formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O2−·. Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.

Inhaled marijuana smoke disrupts mitochondrial energetics in pulmonary epithelial cells in vivo

2006 ◽  
Vol 290 (6) ◽  
pp. L1202-L1209 ◽  
Author(s):  
Theodore A. Sarafian ◽  
Nancy Habib ◽  
Michael Oldham ◽  
Navindra Seeram ◽  
Ru-Po Lee ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitochondrial Function ◽  
A549 Cells ◽  
Cross Flow ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Red Fluorescence ◽  
Tracheal Epithelial Cells

Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8–10 times higher than those measured in blood (75 ± 38 ng/g wet wt tissue vs. 9.2 ± 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.

scholarly journals Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media

2020 ◽  
Author(s):  
Lindsay Broadbent ◽  
Sheerien Manzoor ◽  
Maria Zarcone ◽  
Judit Barabas ◽  
Mike Shields ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Nasal Epithelial Cell ◽  
Human Airway ◽  
Syncytial Virus ◽  
Human Airway Epithelial Cells

AbstractThe culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFNλ1 secreted from PneumaCult compared to Promocell cultures. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells impacts experimental results.

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

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 509 ◽  
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.

scholarly journals PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

2012 ◽  
Vol 302 (7) ◽  
pp. L679-L687 ◽  
Author(s):  
Yong Sung Park ◽  
Erik P. Lillehoj ◽  
Kosuke Kato ◽  
Choon Sik Park ◽  
Kwang Chul Kim
Keyword(s):  
Epithelial Cells ◽  
Culture Media ◽  
A549 Cells ◽  
Luciferase Reporter ◽  
Muc1 Expression ◽  
Mrna Levels ◽  
Airway Epithelial ◽  
Pparγ Agonist ◽  
Tnf Α ◽  
Primary Mouse

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

The wound healing capacity of undifferentiated and differentiated airway epithelial cells in vitro

2018 ◽  
Vol 112 ◽  
pp. 163-168 ◽  
Author(s):  
Cynthia M. Schwartz ◽  
Braedyn A. Dorn ◽  
Selam Habtemariam ◽  
Cynthia L. Hill ◽  
Tendy Chiang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial

scholarly journals In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary-Based Alpha-1-Antitrypsin Deficiency Treatment

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1281
Author(s):  
Shan Guan ◽  
Max Darmstädter ◽  
Chuanfei Xu ◽  
Joseph Rosenecker
Keyword(s):  
Epithelial Cells ◽  
Transfection Efficiency ◽  
Genetic Diseases ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Significant Toxicity ◽  
Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin

In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for alpha-1-antitrypsin deficiency in the future. Here we show that lipoplexes containing A1AT-mRNA prepared in optimum conditions could successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial number of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and could successfully inhibit the enzyme activity of trypsin as well as elastase. Our data indicate that aerosolization of A1AT-mRNA therapy constitutes a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT, while bringing along the unique advantages of IVT-mRNA.

scholarly journals Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells

Nutrients ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 3514
Author(s):  
Yang Cai ◽  
Myrthe S. Gilbert ◽  
Walter J. J. Gerrits ◽  
Gert Folkerts ◽  
Saskia Braber
Keyword(s):  
Epithelial Cells ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Mannheimia Haemolytica ◽  
Epithelial Barrier ◽  
Infection Model ◽  
Airway Epithelial ◽  
Clinical Scores ◽  
Lung Infections ◽  
Anti Inflammatory

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1β concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.

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