scholarly journals The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 555-566 ◽  
Author(s):  
Verónica Regueiro ◽  
Miguel A. Campos ◽  
Jaume Pons ◽  
Sebastián Albertí ◽  
José A. Bengoechea
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Inflammatory Response ◽  
Nuclear Translocation ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Wild Type ◽  
Cellular Activation ◽  
Epithelial Cell Surface

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-κB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.

scholarly journals Tumor-Specific, Replication-Competent Adenovirus Vectors Overexpressing the Adenovirus Death Protein

2000 ◽  
Vol 74 (13) ◽  
pp. 6147-6155 ◽  
Author(s):  
Konstantin Doronin ◽  
Karoly Toth ◽  
Mohan Kuppuswamy ◽  
Peter Ward ◽  
Ann E. Tollefson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nude Mice ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Tumor Formation ◽  
Airway Epithelial ◽  
Wild Type ◽  
Normal Tissues ◽  
Lung Carcinoma Cells ◽  
Efficient Replication

ABSTRACT We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G0 to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1− E3−replication-defective vector expressing β-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10−4 PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.

scholarly journals The BH3-only protein Bik/Blk/Nbk inhibits nuclear translocation of activated ERK1/2 to mediate IFNγ-induced cell death

2008 ◽  
Vol 183 (3) ◽  
pp. 429-439 ◽  
Author(s):  
Yohannes A. Mebratu ◽  
Burton F. Dickey ◽  
Chris Evans ◽  
Yohannes Tesfaigzi
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Cathepsin B ◽  
Messenger Rna ◽  
Prolonged Exposure ◽  
Nuclear Translocation ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Wild Type ◽  
Extracellular Regulated Kinase

IFNγ induces cell death in epithelial cells, but the mediator for this death pathway has not been identified. In this study, we find that expression of Bik/Blk/Nbk is increased in human airway epithelial cells (AECs [HAECs]) in response to IFNγ. Expression of Bik but not mutant BikL61G induces and loss of Bik suppresses IFNγ-induced cell death in HAECs. IFNγ treatment and Bik expression increase cathepsin B and D messenger RNA levels and reduce levels of phospho–extracellular regulated kinase 1/2 (ERK1/2) in the nuclei of bik+/+ compared with bik−/− murine AECs. Bik but not BikL61G interacts with and suppresses nuclear translocation of phospho-ERK1/2, and suppression of ERK1/2 activation inhibits IFNγ- and Bik-induced cell death. Furthermore, after prolonged exposure to allergen, hyperplastic epithelial cells persist longer, and nuclear phospho-ERK is more prevalent in airways of IFNγ−/− or bik−/− compared with wild-type mice. These results demonstrate that IFNγ requires Bik to suppress nuclear localization of phospho-ERK1/2 to channel cell death in AECs.

scholarly journals Attenuated lncRNA NKILA Enhances the Secretory Function of Airway Epithelial Cells Stimulated by Mycoplasma pneumoniae via NF-κB

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Fengxia Zhang ◽  
Jiamin Zhang ◽  
Feng Liu ◽  
Yao Zhou ◽  
Yun Guo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mycoplasma Pneumoniae ◽  
Noncoding Rna ◽  
Nuclear Translocation ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Regulatory Function ◽  
Airway Epithelial ◽  
Secretory Function ◽  
Tnf Α

The secretory function of airway epithelial cells is important in the pathogenesis of Mycoplasma pneumoniae pneumonia (MPP). To investigate the regulatory function of NKILA (nuclear factor-κB (NF-κB) interacting long noncoding RNA (lncRNA)) in MPP, we first detected NKILA as well as the concentration of interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of children with MPP. Then, NKILA was knocked down in epithelial cells to investigate its effect on their secretory function. The results suggested that NKILA was downregulated in children with MPP, while IL-8 and TNF-α levels increased. Knockdown of NKILA in vitro promoted the inflammatory effects of Mycoplasma pneumoniae (MP) in epithelial A549 and BEAS-2B cells. Knockdown of NKILA promoted inhibitor of κBα (IκBα) phosphorylation and degradation, and NF-κB p65 nuclear translocation. Furthermore, RNA immunoprecipitation showed that NKILA could physically bind to IκBα in MP-treated A549 cells. Collectively, our data demonstrated that attenuation of NKILA enhances the effects of MP-stimulated secretory functions of epithelial cells via regulation of NF-κB signaling.

scholarly journals Klebsiella pneumoniae Capsule Polysaccharide Impedes the Expression of β-Defensins by Airway Epithelial Cells

2010 ◽  
Vol 78 (12) ◽  
pp. 5352-5352
Author(s):  
David Moranta ◽  
Verónica Regueiro ◽  
Catalina March ◽  
Enrique Llobet ◽  
Javier Margareto ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Airway Epithelial Cells ◽  
Airway Epithelial

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.

Effect of defensins on interleukin-8 synthesis in airway epithelial cells

1997 ◽  
Vol 272 (5) ◽  
pp. L888-L896 ◽  
Author(s):  
S. Van Wetering ◽  
S. P. Mannesse-Lazeroms ◽  
M. A. Van Sterkenburg ◽  
M. R. Daha ◽  
J. H. Dijkman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neutrophil Elastase ◽  
Proteinase Inhibitor ◽  
De Novo ◽  
Tissue Injury ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Airway Epithelial

Neutrophils play an important role in inflammatory processes in the lung and may cause tissue injury through, for example, release of proteinases such as neutrophil elastase. In addition to neutrophil elastase, stimulated neutrophils also release small nonenzymatic and cationic polypeptides termed defensins. The aim of the present study was to investigate whether defensins induce interleukin (IL)-8 expression in cells of the A549 lung epithelial cell line and in human primary bronchial epithelial cells (PBEC). Supernatants of defensin-treated A549 cells contained increased neutrophil chemotactic activity (16-fold) that was inhibited by antibodies against IL-8. Concurrently, within 3 and 6 h, defensins significantly increased the IL-8 levels in supernatants of both A549 cells (n = 6, P < 0.05 and P < 0.01, respectively) and PBEC (n = 4, P < 0.001 and P < 0.001, respectively). This defensin-induced increase was fully inhibited by the serine proteinase inhibitor alpha 1-proteinase inhibitor. In addition, defensins also increased IL-8 mRNA levels (12-fold); this increase was dependent on de novo mRNA synthesis and did not require protein synthesis. Furthermore, defensins did not affect IL-8 mRNA stability, indicating that the enhanced IL-8 expression was due to increased transcription. Our findings suggest that defensins, released by stimulated neutrophils, stimulate IL-8 synthesis by airway epithelial cells and thus may mediate the recruitment of additional neutrophils into the airways.

scholarly journals A Type IV Secretion System Contributes to Intracellular Survival and Replication of Burkholderia cenocepacia

2008 ◽  
Vol 76 (12) ◽  
pp. 5447-5455 ◽  
Author(s):  
S. Umadevi Sajjan ◽  
Lisa A. Carmody ◽  
Carlos F. Gonzalez ◽  
John J. LiPuma
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Endocytic Pathway ◽  
Burkholderia Cenocepacia ◽  
Type Iv Secretion System ◽  
Airway Epithelial ◽  
Wild Type ◽  
Type Iv

ABSTRACT Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis (CF). Recent studies indicate that B. cenocepacia survives within macrophages and airway epithelial cells in vitro by evading endosome-lysosome fusion. We investigated the role of a plasmid-encoded type IV secretion system in the intracellular survival, replication, and processing of B. cenocepacia. Both a wild-type strain (K56-2) and its type IV secretion system mutant (designated LC101) entered and replicated in CF airway epithelial cells and monocyte-derived macrophages. However, significantly more intracellular K56-2 than LC101 bacteria were found in both cell types at 24 h postinfection. Colocalization of bacteria with markers of the classical endocytic pathway indicated that although both K56-2 and LC101 reside transiently in early endosomes, a greater proportion of the mutant bacteria are targeted to lysosomal degradation. In contrast, wild-type bacteria escape from the classical endocytic pathway and traffic to the endoplasmic reticulum, where they replicate. Our results show that the intracellular processing of B. cenocepacia is similar in both professional and nonprofessional phagocytes and that a functional plasmid-encoded type IV secretion system contributes to the survival and replication of B. cenocepacia in eukaryotic cells.

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.

scholarly journals Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

2012 ◽  
Vol 303 (2) ◽  
pp. L97-L106 ◽  
Author(s):  
Shilpa Nimishakavi ◽  
Marina Besprozvannaya ◽  
Wilfred W. Raymond ◽  
Charles S. Craik ◽  
Dieter C. Gruenert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Selective Inhibition ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+ channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

Anti-inflammatory effects of resveratrol in lung epithelial cells: molecular mechanisms

2004 ◽  
Vol 287 (4) ◽  
pp. L774-L783 ◽  
Author(s):  
Louise E. Donnelly ◽  
Robert Newton ◽  
Gina E. Kennedy ◽  
Peter S. Fenwick ◽  
Rachel H. F. Leung ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Anti Inflammatory ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Factor Release

Resveratrol (3,4′,5-trihydroxystilbene) is a polyphenolic stilbene found in the skins of red fruits, including grapes, that may be responsible for some of the health benefits ascribed to consumption of red wine. Resveratrol has been shown to have antioxidant properties and can act as an estrogen agonist. This study examined the anti-inflammatory effects of resveratrol on human airway epithelial cells. Resveratrol and the related molecule quercetin, but not deoxyrhapontin, inhibited IL-8 and granulocyte-macrophage colony-stimulating factor release from A549 cells. Neither the estrogen receptor antagonist tamoxifen nor the glucocorticoid antagonist mifepristone altered the inhibitory effect of resveratrol. The mechanism of resveratrol action was investigated further using luciferase reporter genes stably transfected into A549 cells. Resveratrol and quercetin inhibited NF-κB-, activator protein-1-, and cAMP response element binding protein-dependent transcription to a greater extent than the glucocorticosteroid dexamethasone. These compounds also had no significant effect on acetylation or deacetylation of core histones. Resveratrol, but not estradiol or N-acetyl cysteine, inhibited cytokine-stimulated inducible nitric oxide synthase expression and nitrite production (IC50 = 3.6 ± 2.9 μM) in human primary airway epithelial cells. Resveratrol also inhibited granulocyte-macrophage colony-stimulating factor release (IC50 = 0.44 ± 0.17 μM), IL-8 release (IC50 = 4.7 ± 3.3 μM), and cyclooxygenase-2 expression in these cells. This study demonstrates that resveratrol and quercetin have novel nonsteroidal anti-inflammatory activity that may have applications for the treatment of inflammatory diseases.

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