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Sample records for airway epithelial repair

  1. Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair.

    PubMed

    Schiller, Katherine R; Maniak, Peter J; O'Grady, Scott M

    2010-11-01

    The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airway epithelial cell line (Calu-3) of serous gland origin. Measurements of wound repair were performed using continuous impedance sensing to determine the time course for wound closure. Control experiments showed that the increase in impedance corresponding to cell migration into the wound was blocked by treatment with the actin polymerization inhibitor, cytochalasin D. Time lapse imaging revealed that NHBE and Calu-3 cell wound closure was dependent on cell migration, and that movement occurred as a collective sheet of cells. Selective inhibition of CFTR activity with CFTR(inh)-172 or short hairpin RNA silencing of CFTR expression produced a significant delay in wound repair. The CF cell line UNCCF1T also exhibited significantly slower migration than comparable normal airway epithelial cells. Inhibition of CFTR-dependent anion transport by treatment with CFTR(inh)-172 slowed wound closure to the same extent as silencing CFTR protein expression, indicating that ion transport by CFTR plays a critical role in migration. Moreover, morphologic analysis of migrating cells revealed that CFTR inhibition or silencing significantly reduced lamellipodia protrusion. These findings support the conclusion that CFTR participates in airway epithelial wound repair by a mechanism involving anion transport that is coupled to the regulation of lamellipodia protrusion at the leading edge of the cell.

  2. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  3. Understanding Cellular Mechanisms Underlying Airway Epithelial Repair: Selecting the Most Appropriate Animal Models

    PubMed Central

    Yahaya, B.

    2012-01-01

    Understanding the mechanisms underlying the process of regeneration and repair of airway epithelial structures demands close characterization of the associated cellular and molecular events. The choice of an animal model system to study these processes and the role of lung stem cells is debatable since ideally the chosen animal model should offer a valid comparison with the human lung. Species differences may include the complex three-dimensional lung structures, cellular composition of the lung airway as well as transcriptional control of the molecular events in response to airway epithelium regeneration, and repair following injury. In this paper, we discuss issues related to the study of the lung repair and regeneration including the role of putative stem cells in small- and large-animal models. At the end of this paper, the author discuss the potential for using sheep as a model which can help bridge the gap between small-animal model systems and humans. PMID:23049478

  4. Ineffective correction of PPARγ signaling in cystic fibrosis airway epithelial cells undergoing repair.

    PubMed

    Bou Saab, J; Bacchetta, M; Chanson, M

    2016-09-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) represents a potential target to treat airway mucus hypersecretion in cystic fibrosis (CF). We aimed to determine if PPARγ is altered in CF human airway epithelial cells (HAECs), if PPARγ contributes to mucin expression and HAEC differentiation, and if PPARγ ligand therapy corrects the CF phenotype. To this end, well-differentiated CF and NCF HAEC primary cultures were wounded to monitor the expression of key genes involved in PPARγ activation and mucus homeostasis, and to evaluate the effect of a PPARγ agonist, at different times of repair. Hydroxyprostaglandin dehydrogenase (HPGD) converts prostaglandin E2 to 15-keto PGE2 (15kPGE2), an endogenous PPARγ ligand. Interestingly, PPARγ and HPGD expression dramatically decreased in CF HAECs. These changes were accompanied by an increase in the expression of MUC5B. The correlation between PPARγ and MUC5B was confirmed in an airway epithelial cell line after CFTR knock-down. Exposure of HAECs to 15kPGE2 did not correct the CF phenotype but revealed a defect in the process of basal cell (BC) differentiation. The HPGD/PPARγ axis is deregulated in primary HAEC cultures from CF patients, which may impact the maturation of BCs to differentiated luminal cells. Importantly, PPARγ therapy was inefficient in correcting the CF defect. PMID:27484450

  5. Abnormal epithelial structure and chronic lung inflammation after repair of chlorine-induced airway injury

    PubMed Central

    Mo, Yiqun; Chen, Jing; Humphrey, David M.; Fodah, Ramy A.; Warawa, Jonathan M.

    2014-01-01

    Chlorine is a toxic gas used in a variety of industrial processes and is considered a chemical threat agent. High-level chlorine exposure causes acute lung injury, but the long-term effects of acute chlorine exposure are unclear. Here we characterized chronic pulmonary changes following acute chlorine exposure in mice. A/J mice were exposed to 240 parts per million-hour chlorine or sham-exposed to air. Chlorine inhalation caused sloughing of bronchial epithelium 1 day after chlorine exposure, which was repaired with restoration of a pseudostratified epithelium by day 7. The repaired epithelium contained an abnormal distribution of epithelial cells containing clusters of club or ciliated cells rather than the uniformly interspersed pattern of these cells in unexposed mice. Although the damaged epithelium in A/J mice was repaired rapidly, and minimal airway fibrosis was observed, chlorine-exposed mice developed pneumonitis characterized by infiltration of alveoli with neutrophils and prominent, large, foamy macrophages. Levels of CXCL1/KC, CXCL5/LPS-induced CXC chemokine, granulocyte colony-stimulating factor, and VEGF in bronchoalveolar (BAL) fluid from chlorine-exposed mice showed steadily increasing trends over time. BAL protein levels were increased on day 4 and remained elevated out to day 28. The number of bacteria cultured from lungs of chlorine-exposed mice 4 wk after exposure was not increased compared with sham-exposed mice, indicating that the observed pneumonitis was not driven by bacterial infection of the lung. The results indicate that acute chlorine exposure may cause chronic abnormalities in the lungs despite rapid repair of injured epithelium. PMID:25398987

  6. Abnormal epithelial structure and chronic lung inflammation after repair of chlorine-induced airway injury.

    PubMed

    Mo, Yiqun; Chen, Jing; Humphrey, David M; Fodah, Ramy A; Warawa, Jonathan M; Hoyle, Gary W

    2015-01-15

    Chlorine is a toxic gas used in a variety of industrial processes and is considered a chemical threat agent. High-level chlorine exposure causes acute lung injury, but the long-term effects of acute chlorine exposure are unclear. Here we characterized chronic pulmonary changes following acute chlorine exposure in mice. A/J mice were exposed to 240 parts per million-hour chlorine or sham-exposed to air. Chlorine inhalation caused sloughing of bronchial epithelium 1 day after chlorine exposure, which was repaired with restoration of a pseudostratified epithelium by day 7. The repaired epithelium contained an abnormal distribution of epithelial cells containing clusters of club or ciliated cells rather than the uniformly interspersed pattern of these cells in unexposed mice. Although the damaged epithelium in A/J mice was repaired rapidly, and minimal airway fibrosis was observed, chlorine-exposed mice developed pneumonitis characterized by infiltration of alveoli with neutrophils and prominent, large, foamy macrophages. Levels of CXCL1/KC, CXCL5/LPS-induced CXC chemokine, granulocyte colony-stimulating factor, and VEGF in bronchoalveolar (BAL) fluid from chlorine-exposed mice showed steadily increasing trends over time. BAL protein levels were increased on day 4 and remained elevated out to day 28. The number of bacteria cultured from lungs of chlorine-exposed mice 4 wk after exposure was not increased compared with sham-exposed mice, indicating that the observed pneumonitis was not driven by bacterial infection of the lung. The results indicate that acute chlorine exposure may cause chronic abnormalities in the lungs despite rapid repair of injured epithelium.

  7. Agonist binding to β-adrenergic receptors on human airway epithelial cells inhibits migration and wound repair.

    PubMed

    Peitzman, Elizabeth R; Zaidman, Nathan A; Maniak, Peter J; O'Grady, Scott M

    2015-12-15

    Human airway epithelial cells express β-adrenergic receptors (β-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the β2-AR-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the β-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by β-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that β-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-β1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with β-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in β1-integrin activation in the basal membrane. PMID:26491049

  8. Reduction of DNA mismatch repair protein expression in airway epithelial cells of premenopausal women chronically exposed to biomass smoke.

    PubMed

    Mukherjee, Bidisha; Dutta, Anindita; Chowdhury, Saswati; Roychoudhury, Sanghita; Ray, Manas Ranjan

    2014-02-01

    Biomass burning is a major source of indoor air pollution in rural India. This study examined whether chronic inhalation of biomass smoke causes change in the DNA mismatch repair (MMR) pathway in the airway cells. For this, airway cells exfoliated in sputum were collected from 72 premenopausal nonsmoking rural women (median age 34 years) who cooked with biomass (wood, dung, crop residues) and 68 control women who cooked with cleaner fuel liquefied petroleum gas (LPG) for the past 5 years or more. The levels of particulate matters with diameters less than 10 and 2.5 μm (PM10 and PM2.5) in indoor air were measured by real-time aerosol monitor. Benzene exposure was monitored by measuring trans,trans-muconic acid (t,t-MA) in urine by high-performance liquid chromatography with ultraviolet detector. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in airway cells were measured by flow cytometry and spectrophotometry, respectively. Immunocytochemical assay revealed lower percentage of airway epithelial cells expressing MMR proteins mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) in biomass-using women compared to LPG-using controls. Women who cooked with biomass had 6.7 times higher level of urinary t,t-MA, twofold increase in ROS generation, and 31 % depletion of SOD. Indoor air of biomass-using households had three times more particulate matters than that of controls. ROS, urinary t,t-MA, and particulate pollution in biomass-using kitchen had negative correlation, while SOD showed positive correlation with MSH2 and MLH1 expression. It appears that chronic exposure to biomass smoke reduces MMR response in airway epithelial cells, and oxidative stress plays an important role in the process.

  9. Tissue Inhibitor of Metalloproteinase-1 Moderates Airway Re-Epithelialization by Regulating Matrilysin Activity

    PubMed Central

    Chen, Peter; McGuire, John K.; Hackman, Robert C.; Kim, Kyoung-Hee; Black, Roy A.; Poindexter, Kurt; Yan, Wei; Liu, Phillip; Chen, Ann J.; Parks, William C.; Madtes, David K.

    2008-01-01

    Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration. PMID:18385523

  10. Airway Trefoil Factor Expression during Naphthalene Injury and Repair

    PubMed Central

    Greeley, Melanie A.; Van Winkle, Laura S.; Edwards, Patricia C.; Plopper, Charles G.

    2010-01-01

    While the role of trefoil factors (TFF) in the maintenance of epithelial integrity in the gastrointestinal tract is well known, their involvement in wound healing in the conducting airway is less well understood. We defined the pattern of expression of TFF1, TFF2, and TFF3 in the airways of mice during repair of both severe (300 mg/kg) and moderate (200 mg/kg) naphthalene-induced Clara cell injury. Quantitative real-time PCR for tff messenger RNA expression and immunohistochemistry for protein expression were applied to airway samples obtained by microdissection of airway trees or to fixed lung tissue from mice at 6 and 24 h and 4 and 7 days after exposure to either naphthalene or an oil (vehicle) control. All three TFF were expressed in normal whole lung and airways. TFF2 was the most abundant and was enriched in airways. Injury of the airway epithelium by 300 mg/kg naphthalene caused a significant induction of tff1 gene expression at 24 h, 4 days, and 7 days. In contrast, tff2 was decreased in the high-dose group at 24 h and 4 days but returned to baseline levels by 7 days. tff3 gene expression was not significantly changed at any time point. Protein localization via immunohistochemistry did not directly correlate with the gene expression measurements. TFF1 and TFF2 expression was most intense in the degenerating Clara cells in the injury target zone at 6 and 24 h. Following the acute injury phase, TFF1 and TFF2 were localized to the luminal apices of repairing epithelial cells and to the adjacent mesenchyme in focal regions that correlated with bifurcations and the bronchoalveolar duct junction. The temporal pattern of increases in TFF1, TFF2, and TFF3 indicate a role in cell death as well as proliferation, migration, and differentiation phases of airway epithelial repair. PMID:19880587

  11. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

    PubMed

    Gomperts, Brigitte N; Belperio, John A; Rao, P Nagesh; Randell, Scott H; Fishbein, Michael C; Burdick, Marie D; Strieter, Robert M

    2006-02-01

    Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases. PMID:16424223

  12. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  13. The buffer capacity of airway epithelial secretions

    PubMed Central

    Kim, Dusik; Liao, Jie; Hanrahan, John W.

    2014-01-01

    The pH of airway epithelial secretions influences bacterial killing and mucus properties and is reduced by acidic pollutants, gastric reflux, and respiratory diseases such as cystic fibrosis (CF). The effect of acute acid loads depends on buffer capacity, however the buffering of airway secretions has not been well characterized. In this work we develop a method for titrating micro-scale (30 μl) volumes and use it to study fluid secreted by the human airway epithelial cell line Calu-3, a widely used model for submucosal gland serous cells. Microtitration curves revealed that HCO−3 is the major buffer. Peak buffer capacity (β) increased from 17 to 28 mM/pH during forskolin stimulation, and was reduced by >50% in fluid secreted by cystic fibrosis transmembrane conductance regulator (CFTR)-deficient Calu-3 monolayers, confirming an important role of CFTR in HCO−3 secretion. Back-titration with NaOH revealed non-volatile buffer capacity due to proteins synthesized and released by the epithelial cells. Lysozyme and mucin concentrations were too low to buffer Calu-3 fluid significantly, however model titrations of porcine gastric mucins at concentrations near the sol-gel transition suggest that mucins may contribute to the buffer capacity of ASL in vivo. We conclude that CFTR-dependent HCO−3 secretion and epithelially-derived proteins are the predominant buffers in Calu-3 secretions. PMID:24917822

  14. Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels.

    PubMed

    Mukherjee, Bidisha; Bindhani, Banani; Saha, Hirak; Ray, Manas Ranjan

    2014-09-01

    To investigate whether biomass burning causes oxidative DNA damage and alters the expression of DNA base excision repair (BER) proteins in airway cells, sputum samples were collected from 80 premenopausal rural biomass-users and 70 age-matched control women who cooked with liquefied petroleum gas. Compared with control the airway cells of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine and lower percentages of BER proteins OGG1 and APE1 by immunocytochemical staining. Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase was depleted significantly among biomass-users. The concentrations of particulate matters were higher in biomass-using households which positively correlated with ROS generation and negatively with BER proteins expressions. ROS generation was positively correlated with 8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk for genotoxicity among rural women in their child-bearing age.

  15. Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels.

    PubMed

    Mukherjee, Bidisha; Bindhani, Banani; Saha, Hirak; Ray, Manas Ranjan

    2014-09-01

    To investigate whether biomass burning causes oxidative DNA damage and alters the expression of DNA base excision repair (BER) proteins in airway cells, sputum samples were collected from 80 premenopausal rural biomass-users and 70 age-matched control women who cooked with liquefied petroleum gas. Compared with control the airway cells of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine and lower percentages of BER proteins OGG1 and APE1 by immunocytochemical staining. Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase was depleted significantly among biomass-users. The concentrations of particulate matters were higher in biomass-using households which positively correlated with ROS generation and negatively with BER proteins expressions. ROS generation was positively correlated with 8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk for genotoxicity among rural women in their child-bearing age. PMID:25128766

  16. Epithelial Notch signaling regulates lung alveolar morphogenesis and airway epithelial integrity

    PubMed Central

    Tsao, Po-Nien; Matsuoka, Chisa; Wei, Shu-Chen; Sato, Atsuyasu; Sato, Susumu; Hasegawa, Koichi; Chen, Hung-kuan; Ling, Thai-Yen; Mori, Munemasa; Cardoso, Wellington V.; Morimoto, Mitsuru

    2016-01-01

    Abnormal enlargement of the alveolar spaces is a hallmark of conditions such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. Notch signaling is crucial for differentiation and regeneration and repair of the airway epithelium. However, how Notch influences the alveolar compartment and integrates this process with airway development remains little understood. Here we report a prominent role of Notch signaling in the epithelial–mesenchymal interactions that lead to alveolar formation in the developing lung. We found that alveolar type II cells are major sites of Notch2 activation and show by Notch2-specific epithelial deletion (Notch2cNull) a unique contribution of this receptor to alveologenesis. Epithelial Notch2 was required for type II cell induction of the PDGF-A ligand and subsequent paracrine activation of PDGF receptor-α signaling in alveolar myofibroblast progenitors. Moreover, Notch2 was crucial in maintaining the integrity of the epithelial and smooth muscle layers of the distal conducting airways. Our data suggest that epithelial Notch signaling regulates multiple aspects of postnatal development in the distal lung and may represent a potential target for intervention in pulmonary diseases. PMID:27364009

  17. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  18. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  19. Airway Epithelial Cell Integrity Protects from Cytotoxicity of Pseudomonas aeruginosa Quorum-Sensing Signals.

    PubMed

    Losa, Davide; Köhler, Thilo; Bacchetta, Marc; Saab, Joanna Bou; Frieden, Maud; van Delden, Christian; Chanson, Marc

    2015-08-01

    Cell-to-cell communication via gap junctions regulates airway epithelial cell homeostasis and maintains the epithelium host defense. Quorum-sensing molecules produced by Pseudomonas aeruginosa coordinate the expression of virulence factors by this respiratory pathogen. These bacterial signals may also incidentally modulate mammalian airway epithelial cell responses to the pathogen, a process called interkingdom signaling. We investigated the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). C12 degradation and its effects on cells were monitored in various airway epithelial cell models grown under nonpolarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. C12 rapidly altered epithelial integrity and decreased GJIC in nonpolarized airway epithelial cells, whereas other quorum-sensing molecules had no effect. The effects of C12 were dependent on [Ca(2+)]i and could be prevented by inhibitors of Src tyrosine family and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12 except when undergoing repair after wounding. In vivo during colonization of intubated patients, C12 did not accumulate, but it paralleled bacterial densities. In vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in nonpolarized cells, whereas PON2 expression was increased during epithelial polarization. The cytotoxicity of C12 on nonpolarized epithelial cells, combined with its impaired degradation allowing its accumulation, provides an additional pathogenic mechanism for P. aeruginosa infections.

  20. Epithelial repair mechanisms in the lung

    PubMed Central

    Crosby, Lynn M.

    2010-01-01

    The recovery of an intact epithelium following lung injury is critical for restoration of lung homeostasis. The initial processes following injury include an acute inflammatory response, recruitment of immune cells, and epithelial cell spreading and migration upon an autologously secreted provisional matrix. Injury causes the release of factors that contribute to repair mechanisms including members of the epidermal growth factor and fibroblast growth factor families (TGF-α, KGF, HGF), chemokines (MCP-1), interleukins (IL-1β, IL-2, IL-4, IL-13), and prostaglandins (PGE2), for example. These factors coordinate processes involving integrins, matrix materials (fibronectin, collagen, laminin), matrix metalloproteinases (MMP-1, MMP-7, MMP-9), focal adhesions, and cytoskeletal structures to promote cell spreading and migration. Several key signaling pathways are important in regulating these processes, including sonic hedgehog, Rho GTPases, MAP kinase pathways, STAT3, and Wnt. Changes in mechanical forces may also affect these pathways. Both localized and distal progenitor stem cells are recruited into the injured area, and proliferation and phenotypic differentiation of these cells leads to recovery of epithelial function. Persistent injury may contribute to the pathology of diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. For example, dysregulated repair processes involving TGF-β and epithelial-mesenchymal transition may lead to fibrosis. This review focuses on the processes of epithelial restitution, the localization and role of epithelial progenitor stem cells, the initiating factors involved in repair, and the signaling pathways involved in these processes. PMID:20363851

  1. Airway Epithelial Regulation of Allergic Sensitization in Asthma

    PubMed Central

    Poynter, Matthew E.

    2012-01-01

    While many of the contributing cell types and mediators of allergic asthma are known, less well understood are the factors that influence the development of allergic responses that lead to the development of allergic asthma. As the first airway cell type to respond to inhaled factors, the epithelium orchestrates downstream interactions between dendritic cells (DCs) and CD4+ T cells that quantitatively and qualitatively dictate the degree and type of the allergic asthma phenotype, making the epithelium of critical importance for the genesis of allergies that later manifest in allergic asthma. Amongst the molecular processes of critical importance in airway epithelium is the transcription factor, nuclear factor-kappaB (NF-κB). This review will focus primarily on the genesis of pulmonary allergies and the participation of airway epithelial NF-κB activation therein, using examples from our own work on nitrogen dioxide (NO2) exposure and genetic modulation of airway epithelial NF-κB activation. In addition, the mechanisms through which Serum Amyloid A (SAA), an NF-κB-regulated, epithelial-derived mediator, influences allergic sensitization and asthma severity will be presented. Knowledge of the molecular and cellular processes regulating allergic sensitization in the airways has the potential to provide powerful insight into the pathogenesis of allergy, as well as targets for the prevention and treatment of asthma. PMID:22579987

  2. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  3. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  4. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  5. Release of beryllium into artificial airway epithelial lining fluid.

    PubMed

    Stefaniak, Aleksandr B; Virji, M Abbas; Day, Gregory A

    2012-01-01

    Inhaled beryllium particles that deposit in the lung airway lining fluid may dissolve and interact with immune-competent cells resulting in sensitization. As such, solubilization of 17 beryllium-containing materials (ore, hydroxide, metal, oxide, alloys, and process intermediates) was investigated using artificial human airway epithelial lining fluid. The maximum beryllium release in 7 days was 11.78% (from a beryl ore melter dust), although release from most materials was < 1%. Calculated dissolution half-times ranged from 30 days (reduction furnace material) to 74,000 days (hydroxide). Despite rapid mechanical clearance, billions of beryllium ions may be released in the respiratory tract via dissolution in airway lining fluid. Beryllium-containing particles that deposit in the respiratory tract dissolve in artificial lung epithelial lining fluid, thereby providing ions for absorption in the lung and interaction with immune-competent cells in the respiratory tract.

  6. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  7. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  8. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    PubMed Central

    Bauer, Rebecca N.; Müller, Loretta; Brighton, Luisa E.; Duncan, Kelly E.

    2015-01-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell–Mac coculture model to investigate how epithelial cell–derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell–Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell–derived signals are important determinants of Mac immunophenotype and response to O3. PMID:25054807

  9. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    SciTech Connect

    Doupnik, C.A.; Leikauf, G.D. )

    1990-10-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with (3H)arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. (3H)arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein.

  10. Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation.

    PubMed

    de Vries, M; Heijink, I H; Gras, R; den Boef, L E; Reinders-Luinge, M; Pouwels, S D; Hylkema, M N; van der Toorn, M; Brouwer, U; van Oosterhout, A J M; Nawijn, M C

    2014-08-01

    Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC. PMID:24816488

  11. Impaired Capacity of Fibroblasts to Support Airway Epithelial Progenitors in Bronchiolitis Obliterans Syndrome

    PubMed Central

    Zhang, Su-Bei; Sun, Xin; Wu, Qi; Wu, Jun-Ping; Chen, Huai-Yong

    2016-01-01

    Background: Bronchiolitis obliterans syndrome (BOS) often develops in transplant patients and results in injury to the respiratory and terminal airway epithelium. Owing to its rising incidence, the pathogenesis of BOS is currently an area of intensive research. Studies have shown that injury to the respiratory epithelium results in dysregulation of epithelial repair. Airway epithelial regeneration is supported by stromal cells, including fibroblasts. This study aimed to investigate whether the supportive role of lung fibroblasts is altered in BOS. Methods: Suspensions of lung cells were prepared by enzyme digestion. Lung progenitor cells (LPCs) were separated by fluorescence-activated cell sorting. Lung fibroblasts from patients with BOS or healthy controls were mixed with sorted mouse LPCs to compare the colony-forming efficiency of LPCs by counting the number of colonies with a diameter of ≥50 μm in each culture. Statistical analyses were performed using the SPSS 17.0 software (SPSS Inc., USA). The paired Student's t-test was used to test for statistical significance. Results: LPCs were isolated with the surface phenotype of CD31- CD34- CD45- EpCAM+ Sca-1+. The colony-forming efficiency of LPCs was significantly reduced when co-cultured with fibroblasts isolated from patients with BOS. The addition of SB431542 increased the colony-forming efficiency of LPCs to 1.8%; however, it was still significantly less than that in co-culture with healthy control fibroblasts (P < 0.05). Conclusion: The epithelial-supportive capacity of fibroblasts is impaired in the development of BOS and suggest that inefficient repair of airway epithelium could contribute to persistent airway inflammation in BOS. PMID:27569228

  12. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  13. Restoring airway epithelial barrier dysfunction: a new therapeutic challenge in allergic airway disease.

    PubMed

    Steelant, B; Seys, S F; Boeckxstaens, G; Akdis, C A; Ceuppens, J L; Hellings, P W

    2016-09-01

    An intact functional mucosal barrier is considered to be crucial for the maintenance of airway homeostasis as it protects the host immune system from exposure to allergens and noxious environmental triggers. Recent data provided evidence for the contribution of barrier dysfunction to the development of inflammatory diseases in the airways, skin and gut. A defective barrier has been documented in chronic rhinosinusitis, allergic rhinitis, asthma, atopic dermatitis and inflammatory bowel diseases. However, it remains to be elucidated to what extent primary (genetic) versus secondary (inflammatory) mechanisms drive barrier dysfunction. The precise pathogenesis of barrier dysfunction in patients with chronic mucosal inflammation and its implications on tissue inflammation and systemic absorption of exogenous particles are only partly understood. Since epithelial barrier defects are linked with chronicity and severity of airway inflammation, restoring the barrier integrity may become a useful approach in the treatment of allergic diseases. We here provide a state-of-the-art review on epithelial barrier dysfunction in upper and lower airways as well as in the intestine and the skin and on how barrier dysfunction can be restored from a therapeutic perspective.

  14. Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

    SciTech Connect

    Skowron-zwarg, Marie; Boland, Sonja; Caruso, Nathalie; Coraux, Christelle; Marano, Francelyne; Tournier, Frederic . E-mail: f-tournier@paris7.jussieu.fr

    2007-07-15

    Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.

  15. Effects of ozone on airway epithelial permeability and ion transport

    SciTech Connect

    Bromberg, P.A.; Ranga, V.; Stutts, M.J. )

    1991-12-01

    Ozone is a highly reactive form of oxygen produced in the atmosphere by photochemical reactions involving substrates emitted from automobile engines. Outdoor air concentrations as high as 0.4 parts per million (ppm) occur. The respiratory tract extracts about 90% of inhaled ozone. From the chemical reactivity of ozone, it is expected to attack organic molecules located on or near the respiratory surfaces. The airways are covered with a cohesive layer of epithelial cells that forms the boundary between the external environment and the respiratory tissues. One important role of this epithelial layer is its barrier function. Airborne particles that deposit (and dissolve) in the airway surface liquid are not readily absorbed, and soluble tissue components are excluded from the surface liquid. The epithelium also controls the volume and composition of the surface liquid. One important process in this regard is the absorption and secretion of ions and water. We have studied the effects of inhalation of ozone on the barrier function (permeability to dissolved molecules) and the ion transport activity of epithelium using both in vivo and in vitro techniques. All our experiments were performed with male Hartley strain guinea pigs. Conscious, unrestrained animals were exposed to a concentration of ozone of 1 ppm for three hours in controlled environmental chambers in the Health Effects Research Laboratory, U.S. Environmental Protection Agency (EPA), Research Triangle Park, NC. Such exposures caused a marked increase in the rate of appearance in blood of various water-soluble compounds instilled onto the surface of the trachea, indicating increased permeability of the airway epithelium.

  16. RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

    PubMed Central

    Liesman, Rachael M.; Buchholz, Ursula J.; Luongo, Cindy L.; Yang, Lijuan; Proia, Alan D.; DeVincenzo, John P.; Collins, Peter L.; Pickles, Raymond J.

    2014-01-01

    Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease. PMID:24713657

  17. Effects of ozone on airway epithelial permeability and ion transport.

    PubMed

    Bromberg, P A; Ranga, V; Stutts, M J

    1991-12-01

    Ozone is a highly reactive form of oxygen produced in the atmosphere by photochemical reactions involving substrates emitted from automobile engines. Outdoor air concentrations as high as 0.4 parts per million (ppm) occur. The respiratory tract extracts about 90% of inhaled ozone. From the chemical reactivity of ozone, it is expected to attack organic molecules located on or near the respiratory surfaces. The airways are covered with a cohesive layer of epithelial cells that forms the boundary between the external environment and the respiratory tissues. One important role of this epithelial layer is its barrier function. Airborne particles that deposit (and dissolve) in the airway surface liquid are not readily absorbed, and soluble tissue components are excluded from the surface liquid. The epithelium also controls the volume and composition of the surface liquid. One important process in this regard is the absorption and secretion of ions and water. We have studied the effects of inhalation of ozone on the barrier function (permeability to dissolved molecules) and the ion transport activity of epithelium using both in vivo and in vitro techniques. All our experiments were performed with male Hartley strain guinea pigs. Conscious, unrestrained animals were exposed to a concentration of ozone of 1 ppm for three hours in controlled environmental chambers in the Health Effects Research Laboratory, U.S. Environmental Protection Agency (EPA), Research Triangle Park, NC. Such exposures caused a marked increase in the rate of appearance in blood of various water-soluble compounds instilled onto the surface of the trachea, indicating increased permeability of the airway epithelium. This interpretation was supported by electron microscopy, which showed that the tracer molecule horseradish peroxidase was present in the intercellular spaces of tracheal epithelium from ozone-exposed, but not air-exposed (control), animals. However, when the tracheas were excised after ozone

  18. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  19. Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling

    PubMed Central

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Sur, Sanjiv; Hegde, Muralidhar L.; Tian, Bing; Saavedra-Molina, Alfredo; Brasier, Allan R.; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER byproduct 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, Z3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling. PMID:26187872

  20. Airway Epithelial NF-κB Activation Promotes Mycoplasma pneumoniae Clearance in Mice

    PubMed Central

    Jiang, Di; Nelson, Mark L.; Gally, Fabienne; Smith, Sean; Wu, Qun; Minor, Maisha; Case, Stephanie; Thaikoottathil, Jyoti; Chu, Hong Wei

    2012-01-01

    Background/Objective Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression. Methodology/Main Results Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-CAIKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg−) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice. Conclusion By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression. PMID:23285237

  1. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  2. Basolateral Cl channels in primary airway epithelial cultures.

    PubMed

    Fischer, Horst; Illek, Beate; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2007-06-01

    Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.

  3. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    PubMed Central

    2009-01-01

    Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide. PMID:19650888

  4. Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

    PubMed Central

    Thevenot, Paul T.; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D.; Chustz, Regina E.; Mahne, Sarah; Kelley, Matthew A.; Hebert, Valeria Y.; Dellinger, Barry; Dugas, Tammy R.; DeMayo, Francesco J.

    2013-01-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  5. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    EPA Science Inventory

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  6. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  7. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice.

    PubMed

    Mall, Marcus; Grubb, Barbara R; Harkema, Jack R; O'Neal, Wanda K; Boucher, Richard C

    2004-05-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective epithelial cAMP-dependent Cl(-) secretion and increased airway Na(+) absorption. The mechanistic links between these altered ion transport processes and the pathogenesis of cystic fibrosis lung disease, however, are unclear. To test the hypothesis that accelerated Na(+) transport alone can produce cystic fibrosis-like lung disease, we generated mice with airway-specific overexpression of epithelial Na(+) channels (ENaC). Here we show that increased airway Na(+) absorption in vivo caused airway surface liquid (ASL) volume depletion, increased mucus concentration, delayed mucus transport and mucus adhesion to airway surfaces. Defective mucus transport caused a severe spontaneous lung disease sharing features with cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. We conclude that increasing airway Na(+) absorption initiates cystic fibrosis-like lung disease and produces a model for the study of the pathogenesis and therapy of this disease. PMID:15077107

  8. Plexins function in epithelial repair in both Drosophila and zebrafish

    PubMed Central

    Yoo, Sa Kan; Pascoe, Heath G.; Pereira, Telmo; Kondo, Shu; Jacinto, Antonio; Zhang, Xuewu; Hariharan, Iswar K.

    2016-01-01

    In most multicellular organisms, homeostasis is contingent upon maintaining epithelial integrity. When unanticipated insults breach epithelial barriers, dormant programmes of tissue repair are immediately activated. However, many of the mechanisms that repair damaged epithelia remain poorly characterized. Here we describe a role for Plexin A (PlexA), a protein with particularly well-characterized roles in axonal pathfinding, in the healing of damaged epithelia in Drosophila. Semaphorins, which are PlexA ligands, also regulate tissue repair. We show that Drosophila PlexA has GAP activity for the Rap1 GTPase, which is known to regulate the stability of adherens junctions. Our observations suggest that the inhibition of Rap1 activity by PlexA in damaged Drosophila epithelia allows epithelial remodelling, thus facilitating wound repair. We also demonstrate a role for Plexin A1, a zebrafish orthologue of Drosophila PlexA, in epithelial repair in zebrafish tail fins. Thus, plexins function in epithelial wound healing in diverse taxa. PMID:27452696

  9. Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells.

    PubMed

    Murai, Hiroki; Okazaki, Shintaro; Hayashi, Hisako; Kawakita, Akiko; Hosoki, Koa; Yasutomi, Motoko; Sur, Sanjiv; Ohshima, Yusei

    2015-09-01

    Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells.

  10. NADPH oxidase-dependent acid production in airway epithelial cells.

    PubMed

    Schwarzer, Christian; Machen, Terry E; Illek, Beate; Fischer, Horst

    2004-08-27

    The purpose of this study was to determine the role of NADPH oxidase in H(+) secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zinc-blockable plasma membrane H(+) conductance. However, the rate of H(+) secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H(+) secretion was blocked by mucosal ZnCl(2) and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22(phox)) and found Duox1, -2, and p22(phox) to be highly expressed in hTE, as well as the intracellular subunits p40(phox), p47(phox), and p67(phox). In contrast, Calu-3 lacked transcripts for Duox1, p40(phox), and p47(phox). Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na(+)/H(+) exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H(+) production and H(+) secretion, but not in H(+) conduction.

  11. Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

    PubMed Central

    Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-01-01

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

  12. Regenerative potential of human airway stem cells in lung epithelial engineering.

    PubMed

    Gilpin, Sarah E; Charest, Jonathan M; Ren, Xi; Tapias, Luis F; Wu, Tong; Evangelista-Leite, Daniele; Mathisen, Douglas J; Ott, Harald C

    2016-11-01

    Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair. PMID:27622532

  13. Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium.

    PubMed

    Rennolds, Jessica; Malireddy, Smitha; Hassan, Fatemat; Tridandapani, Susheela; Parinandi, Narasimham; Boyaka, Prosper N; Cormet-Boyaka, Estelle

    2012-01-01

    Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-κB dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation.

  14. Rho family GTPase functions in Drosophila epithelial wound repair.

    PubMed

    Verboon, Jeffrey M; Parkhurst, Susan M

    2015-01-01

    Epithelial repair in the Drosophila embryo is achieved through 2 dynamic cytoskeletal machineries: a contractile actomyosin cable and actin-based cellular protrusions. Rho family small GTPases (Rho, Rac, and Cdc42) are cytoskeletal regulators that control both of these wound repair mechanisms. Cdc42 is necessary for cellular protrusions and, when absent, wounds are slow to repair and never completely close. Rac proteins accumulate at specific regions in the wound leading edge cells and Rac-deficient embryos exhibit slower repair kinetics. Mutants for both Rho1 and its effector Rok impair the ability of wounds to close by disrupting the leading-edge actin cable. Our studies highlight the importance of these proteins in wound repair and identify a downstream effector of Rho1 signaling in this process.

  15. Microbial Patterns Signaling via Toll-Like Receptors 2 and 5 Contribute to Epithelial Repair, Growth and Survival

    PubMed Central

    Shaykhiev, Renat; Behr, Jürgen; Bals, Robert

    2008-01-01

    Epithelial cells (ECs) continuously interact with microorganisms and detect their presence via different pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs). Ligation of epithelial TLRs by pathogens is usually associated with the induction of pro-inflammatory mediators and antimicrobial factors. In this study, using human airway ECs as a model, we found that detection of microbial patterns via epithelial TLRs directly regulates tissue homeostasis. Staphylococcus aureus (S. aureus) and microbial patterns signaling via TLR2 and TLR5 induce a set of non-immune epithelial responses including cell migration, wound repair, proliferation, and survival of primary and cancerous ECs. Using small interfering RNA (siRNA) gene targeting, receptor-tyrosine kinase microarray and inhibition studies, we determined that TLR and the epidermal growth factor receptor (EGFR) mediate the stimulating effect of microbial patterns on epithelial repair. Microbial patterns signaling via Toll-like receptors 2 and 5 contribute to epithelial repair, growth and survival. This effect is independent of hematopoietic and other cells as well as inflammatory cytokines suggesting that epithelia are able to regulate their integrity in an autonomous non-inflammatory manner by sensing microbes directly via TLRs. PMID:18167552

  16. Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows

    PubMed Central

    Higuita-Castro, Natalia; Mihai, Cosmin; Hansford, Derek J.

    2014-01-01

    Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation. PMID:25213636

  17. Endogenous and exogenous stem cells: a role in lung repair and use in airway tissue engineering and transplantation

    PubMed Central

    2010-01-01

    Rapid repair of the denuded alveolar surface after injury is a key to survival. The respiratory tract contains several sources of endogenous adult stem cells residing within the basal layer of the upper airways, within or near pulmonary neuroendocrine cell rests, at the bronchoalveolar junction, and within the alveolar epithelial surface, which contribute to the repair of the airway wall. Bone marrow-derived adult mesenchymal stem cells circulating in blood are also involved in tracheal regeneration. However, an organism is frequently incapable of repairing serious damage and defects of the respiratory tract resulting from acute trauma, lung cancers, and chronic pulmonary and airway diseases. Therefore, replacement of the tracheal tissue should be urgently considered. The shortage of donor trachea remains a major obstacle in tracheal transplantation. However, implementation of tissue engineering and stem cell therapy-based approaches helps to successfully solve this problem. To date, huge progress has been achieved in tracheal bioengineering. Several sources of stem cells have been used for transplantation and airway reconstitution in animal models with experimentally induced tracheal defects. Most tracheal tissue engineering approaches use biodegradable three-dimensional scaffolds, which are important for neotracheal formation by promoting cell attachment, cell redifferentiation, and production of the extracellular matrix. The advances in tracheal bioengineering recently resulted in successful transplantation of the world's first bioengineered trachea. Current trends in tracheal transplantation include the use of autologous cells, development of bioactive cell-free scaffolds capable of supporting activation and differentiation of host stem cells on the site of injury, with a future perspective of using human native sites as micro-niche for potentiation of the human body's site-specific response by sequential adding, boosting, permissive, and recruitment impulses

  18. Nrf2 reduces allergic asthma in mice through enhanced airway epithelial cytoprotective function.

    PubMed

    Sussan, Thomas E; Gajghate, Sachin; Chatterjee, Samit; Mandke, Pooja; McCormick, Sarah; Sudini, Kuladeep; Kumar, Sarvesh; Breysse, Patrick N; Diette, Gregory B; Sidhaye, Venkataramana K; Biswal, Shyam

    2015-07-01

    Asthma development and pathogenesis are influenced by the interactions of airway epithelial cells and innate and adaptive immune cells in response to allergens. Oxidative stress is an important mediator of asthmatic phenotypes in these cell types. Nuclear erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that is the key regulator of the response to oxidative and environmental stress. We previously demonstrated that Nrf2-deficient mice have heightened susceptibility to asthma, including elevated oxidative stress, inflammation, mucus, and airway hyperresponsiveness (AHR) (Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, Yamamoto M, Kensler TW, Tuder RM, Georas SN, Biswal S. J Exp Med 202: 47-59, 2005). Here we dissected the role of Nrf2 in lung epithelial cells and tested whether genetic or pharmacological activation of Nrf2 reduces allergic asthma in mice. Cell-specific activation of Nrf2 in club cells of the airway epithelium significantly reduced allergen-induced AHR, inflammation, mucus, Th2 cytokine secretion, oxidative stress, and airway leakiness and increased airway levels of tight junction proteins zonula occludens-1 and E-cadherin. In isolated airway epithelial cells, Nrf2 enhanced epithelial barrier function and increased localization of zonula occludens-1 to the cell surface. Pharmacological activation of Nrf2 by 2-trifluoromethyl-2'-methoxychalone during the allergen challenge was sufficient to reduce allergic inflammation and AHR. New therapeutic options are needed for asthma, and this study demonstrates that activation of Nrf2 in lung epithelial cells is a novel potential therapeutic target to reduce asthma susceptibility.

  19. Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance

    PubMed Central

    Tosoni, Kendra; Cassidy, Diane; Kerr, Barry; Land, Stephen C.; Mehta, Anil

    2016-01-01

    Background Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial ‘set-point’ may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the ‘reference range of normality’ remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport. Methodology/Principal Findings Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm2; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport. Conclusions/Significance Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium

  20. Sex differences in the development of airway epithelial tolerance to naphthalene

    PubMed Central

    Sutherland, K. M.; Edwards, P. C.; Combs, T. J.

    2012-01-01

    Exposure to air pollution has been linked to pulmonary diseases. Naphthalene (NA), an abundant polycyclic aromatic hydrocarbon in tobacco smoke and urban air, is a model toxicant for air pollution effects in the lung. Repeated exposures to NA in male mice result in tolerance, defined as the emergence of a resistant cell phenotype after prior exposure. Tolerance has not been studied in females. Females have sex differences in airway epithelial responses and in the prevalence of certain airway diseases. Male and female mice were exposed to a tolerance-inducing regimen of NA, and lungs were examined by airway level to characterize the cellular changes associated with repeated NA exposure and to assess the expression of genes and proteins involved in NA bioactivation and detoxification. The airway epithelium in treated males resembled that of controls. Females in the tolerant state were characterized by dense populations of ciliated cells in midlevel, distal, and bifurcating airways and a lower abundance of Clara cells at all airway levels. Cytotoxicity following a secondary challenge dose was also greater in females than males. Furthermore, females had decreased gene/protein expression of CYP2F2, a P-450 that metabolizes NA to a toxic epoxide, and glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis, than NA-tolerant males at all airway levels examined. We conclude that, while females develop tolerance, sex differences exist in the tolerant state by airway level, and females remain more susceptible than males to repeated exposures to NA. PMID:22003090

  1. Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium

    SciTech Connect

    Rennolds, Jessica; Malireddy, Smitha; Hassan, Fatemat; Tridandapani, Susheela; Parinandi, Narasimham; Boyaka, Prosper N.; Cormet-Boyaka, Estelle

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cadmium induces secretion of IL-6 and IL-8 by two distinct pathways. Black-Right-Pointing-Pointer Cadmium increases NAPDH oxidase activity leading to Erk activation and IL-8 secretion. Black-Right-Pointing-Pointer Curcumin prevents cadmium-induced secretion of both IL-6 and IL-8 by airway cells. Black-Right-Pointing-Pointer Curcumin could be use to suppress lung inflammation due to cadmium inhalation. -- Abstract: Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-{kappa}B dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation.

  2. SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    Exposure of humans to PM results in increased mortality and morbidity. Recent toxicology studies have shown a number of pathophysiological pulmonary and car...

  3. TLR-2 IS INVOLVED IN AIRWAY EPITHELIAL CELL RESPONE TO AIR POLLUTION PARTICLES

    EPA Science Inventory

    Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of a number of oxidant stress response genes. Components of ambient air PM responsible for stim...

  4. CULTURE CONDITIONS AFFECT HUMAN AIRWAY EPITHELIAL CELL RESPONSE TO DIESEL PARTICLE EXPOSURE IN VITRO

    EPA Science Inventory

    Diesel exhaust particles (DEP) are a ubiquitous ambient air contaminant that may contribute to the health effects of particulate matter inhalation. In vitro studies have shown that DEP exposure induces pro-inflammatory proteins in human airway epithelial cells (HAEC) with varying...

  5. SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES
    Y.M. Kim, A.G. Lenz, R. Silbajoris, I. Jaspers and J.M. Samet. Department of Environmental Sciences and Engineering and Center for Environmental Medicine, University of North Carolina, ...

  6. [Allergens-induced sensitization alters airway epithelial adhesion molecules expression in mice].

    PubMed

    Zeng, Dan; Tan, Mei-Ling; Xiang, Yang; Qin, Xiao-Qun; Zhu, Li-Ming; Dai, Ai-Guo

    2015-12-25

    To explore the relationship between the epithelial adhesion molecules and immune responses of airway epithelium, we observed the expression of integrin β4 and intercellular adhesion molecule-1 (ICAM-1) in the mice airway epithelium after sensitization with allergens. BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) or house dust mite (HDM) and then developed airway hyper-responsiveness as determined by barometric whole-body plethysmography. Both OVA and HDM sensitization led to increases of the number of peripheral leukocytes as well as inflammatory cells infiltration in lungs. OVA sensitized mice showed more severe inflammatory cells infiltration than HDM sensitized mice. Immunohistochemistry analysis of mice lung tissues revealed that sensitization with both allergens also led to a decrease of integrin β4 expression and an increase of ICAM-1 expression in airway epithelia. OVA sensitized mice showed a more significant increase of ICAM-1 expression compared with HDM sensitized mice. siRNA mediated silencing of integrin β4 gene in 16HBE cells resulted in an up-regulation of ICAM-1 expression. Our results indicate a possible role of airway epithelial adhesion molecules in allergen-induced airway immune responses. PMID:26701635

  7. Store-operated Ca2+ channels in airway epithelial cell function and implications for asthma

    PubMed Central

    Samanta, Krishna; Parekh, Anant B.

    2016-01-01

    The epithelial cells of the lung are at the interface of a host and its environment and are therefore directly exposed to the inhaled air-borne particles. Rather than serving as a simple physical barrier, airway epithelia detect allergens and other irritants and then help organize the subsequent immune response through release of a plethora of secreted signals. Many of these signals are generated in response to opening of store-operated Ca2+ channels in the plasma membrane. In this review, we describe the properties of airway store-operated channels and their role in regulating airway epithelial cell function. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377718

  8. TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

    PubMed

    Suzuki, Shingo; Sargent, R Geoffrey; Illek, Beate; Fischer, Horst; Esmaeili-Shandiz, Alaleh; Yezzi, Michael J; Lee, Albert; Yang, Yanu; Kim, Soya; Renz, Peter; Qi, Zhongxia; Yu, Jingwei; Muench, Marcus O; Beyer, Ashley I; Guimarães, Alessander O; Ye, Lin; Chang, Judy; Fine, Eli J; Cradick, Thomas J; Bao, Gang; Rahdar, Meghdad; Porteus, Matthew H; Shuto, Tsuyoshi; Kai, Hirofumi; Kan, Yuet W; Gruenert, Dieter C

    2016-01-01

    Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways. PMID:26730810

  9. Airway epithelial NF-κB activation promotes the ability to overcome inhalational antigen tolerance

    PubMed Central

    Ather, Jennifer L.; Foley, Kathryn L.; Suratt, Benjamin T.; Boyson, Jonathan E.; Poynter, Matthew E.

    2015-01-01

    Background Inhalational antigen tolerance typically protects against the development of allergic airway disease but may be overcome to induce allergic sensitization preceding the development of asthma. Objectives We examined in vivo whether pre-existing inhalational antigen tolerance could be overcome by activation of the transcription factor NF-κB in conducting airway epithelial cells, and used a combination of in vivo and in vitro approaches to examine the mechanisms involved. Methods Wildtype and transgenic mice capable of expressing constitutively active IκB kinase β (CAIKKβ) in airway epithelium were tolerized to inhaled ovalbumin. Twenty-eight days later, the transgene was transiently expressed and mice were exposed to inhaled OVA on day 30 in an attempt to overcome inhalational tolerance. Results Following ovalbumin challenge on days 40-42, CAIKKβ mice in which the transgene had been activated exhibited characteristic features of allergic airway disease, including airway eosinophilia and methacholine hyperresponsiveness. Increases in the CD103+ and CD11bHI lung dendritic cell populations were present in CAIKKβ mice on day 31. Bronchoalveolar lavage from mice expressing CAIKKβ mice induced CD4+ T cells to secrete TH2 and TH17 cytokines, an effect that required IL-4 and IL-1 signaling, respectively. CAIKKβ mice on Dox demonstrated increased numbers of innate lymphoid type 2 cells (ILC2) in the lung, which also exhibited elevated mRNA expression of the TH2-polarizing cytokine IL-4. Finally, airway epithelial NF-kB activation induced allergic sensitization in CAIKKβ mice on Dox that required IL-4 and IL-1-signaling in vivo. Conclusions Our studies demonstrate that soluble mediators generated in response to airway epithelial NF-κB activation orchestrate the breaking of inhalational tolerance and allergic antigen sensitization through the effects of soluble mediators, including IL-1 and IL-4, on pulmonary dendritic cells as well as innate lymphoid and CD

  10. Epithelial barrier function: at the frontline of asthma immunology and allergic airway inflammation

    PubMed Central

    Georas, Steve N.; Rezaee, Fariba

    2014-01-01

    Airway epithelial cells form a barrier to the outside world, and are at the frontline of mucosal immunity. Epithelial apical junctional complexes are multi-protein subunits that promote cell-cell adhesion and barrier integrity. Recent studies in the skin and GI tract suggest that disruption of cell-cell junctions is required to initiate epithelial immune responses, but how this applies to mucosal immunity in the lung is not clear. Increasing evidence indicates that defective epithelial barrier function is a feature of airway inflammation in asthma. One challenge in this area is that barrier function and junctional integrity are difficult to study in the intact lung, but innovative approaches should provide new knowledge in this area in the near future. In this article, we review the structure and function of epithelial apical junctional complexes, emphasizing how regulation of the epithelial barrier impacts innate and adaptive immunity. We discuss why defective epithelial barrier function may be linked to Th2 polarization in asthma, and propose a rheostat model of barrier dysfunction that implicates the size of inhaled allergen particles as an important factor influencing adaptive immunity. PMID:25085341

  11. miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production.

    PubMed

    Oglesby, Irene K; Vencken, Sebastian F; Agrawal, Raman; Gaughan, Kevin; Molloy, Kevin; Higgins, Gerard; McNally, Paul; McElvaney, Noel G; Mall, Marcus A; Greene, Catherine M

    2015-11-01

    Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.

  12. Trehalose-Mediated Autophagy Impairs the Anti-Viral Function of Human Primary Airway Epithelial Cells

    PubMed Central

    Wu, Qun; Jiang, Di; Huang, Chunjian; van Dyk, Linda F.; Li, Liwu; Chu, Hong Wei

    2015-01-01

    Human rhinovirus (HRV) is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5) effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I) and IFN-β promoter stimulator 1 (IPS-1), two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations. PMID:25879848

  13. RACK1, a PKC Targeting Protein, Is Exclusively Localized to Basal Airway Epithelial Cells

    PubMed Central

    Slager, Rebecca E.; DeVasure, Jane M.; Pavlik, Jaqueline A.; Sisson, Joseph H.; Wyatt, Todd A.

    2008-01-01

    The novel isoform of protein kinase C (PKC), PKCɛ, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCɛ signaling in the airways and the potential role of the PKCɛ-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, β-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCɛ signaling in the basal airway cell may involve RACK1; however, PKCɛ regulation in ciliated cells uses RACK1-independent pathways. (J Histochem Cytochem 56:7–14, 2008) PMID:17875659

  14. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2010-03-01

    Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients. PMID:19947928

  15. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2010-01-27

    Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.

  16. Klotho expression is reduced in COPD airway epithelial cells: effects on inflammation and oxidant injury.

    PubMed

    Gao, Wei; Yuan, Cheng; Zhang, Jingying; Li, Lingling; Yu, Like; Wiegman, Coen H; Barnes, Peter J; Adcock, Ian M; Huang, Mao; Yao, Xin

    2015-12-01

    COPD (chronic obstructive pulmonary disease) is associated with sustained inflammation, excessive injury, and accelerated lung aging. Human Klotho (KL) is an anti-aging protein that protects cells against inflammation and damage. In the present study, we quantified KL expression in the lungs of COPD patients and in an ozone-induced mouse model of COPD, and investigated the mechanisms that control KL expression and function in the airways. KL distribution and levels in human and mouse airways were measured by immunohistochemistry and Western blotting. The effect of CSE (cigarette smoke extract) on KL expression was detected in human bronchial epithelial cells. Moreover, the effect of KL on CSE-mediated inflammation and hydrogen peroxide-induced cellular injury/apoptosis was determined using siRNAs. KL expression was decreased in the lungs of smokers and further reduced in patients with COPD. Similarly, 6 weeks of exposure to ozone decreased KL levels in airway epithelial cells. CSE and TNFα (tumour necrosis factor α) decreased KL expression and release from airway epithelial cells, which was associated with enhanced pro-inflammatory cytokine expression. Moreover, KL depletion increased cell sensitivity to cigarette smoke-induced inflammation and oxidative stress-induced cell damage. These effects involved the NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase) and Nrf2 (nuclear factor erythroid 2-related factor 2) pathways. Reduced KL expression in COPD airway epithelial cells was associated with increased oxidative stress, inflammation and apoptosis. These data provide new insights into the mechanisms associated with the accelerated lung aging in COPD development. PMID:26201096

  17. Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing

    PubMed Central

    Lendeckel, Derik; Eymann, Christine; Emicke, Philipp; Daeschlein, Georg; Darm, Katrin; O'Neil, Serena; Beule, Achim G.; von Woedtke, Thomas; Völker, Uwe; Weltmann, Klaus-Dieter; Jünger, Michael; Hosemann, Werner; Scharf, Christian

    2015-01-01

    Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine. PMID:26539504

  18. Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

    PubMed Central

    Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

  19. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  20. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO UTAH VALLEY PARTICULATE MATTER

    EPA Science Inventory

    Exposure to ambient particulate matter (PM) in the Utah Valley (UV) has previously been associated with a variety of adverse health effects. To investigate intracellular signaling mechanisms for pulmonary responses to UV PM inhalation, human primary airway epithelial cells (NHBE)...

  1. Continuous mucociliary transport by primary human airway epithelial cells in vitro

    PubMed Central

    Sears, Patrick R.; Yin, Wei-Ning

    2015-01-01

    Mucociliary clearance (MCC) is an important innate defense mechanism that continuously removes inhaled pathogens and particulates from the airways. Normal MCC is essential for maintaining a healthy respiratory system, and impaired MCC is a feature of many airway diseases, including both genetic (cystic fibrosis, primary ciliary dyskinesia) and acquired (chronic obstructive pulmonary disease, bronchiectasis) disorders. Research into the fundamental processes controlling MCC, therefore, has direct clinical application, but has been limited in part due to the difficulty of studying this complex multicomponent system in vitro. In this study, we have characterized a novel method that allows human airway epithelial cells to differentiate into a mucociliary epithelium that transports mucus in a continuous circular track. The mucociliary transport device allows the measurement and manipulation of all features of mucociliary transport in a controlled in vitro system. In this initial study, the effect of ciliary beat frequency and mucus concentration on the speed of mucociliary transport was investigated. PMID:25979076

  2. Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol

    PubMed Central

    Erle, David J.

    2016-01-01

    Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

  3. Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol.

    PubMed

    Khosravi, Ali Reza; Erle, David J

    2016-01-01

    Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

  4. Cathepsin B contributes to Na+ hyperabsorption in cystic fibrosis airway epithelial cultures.

    PubMed

    Tan, Chong Da; Hobbs, Carey; Sameni, Mansoureh; Sloane, Bonnie F; Stutts, M Jackson; Tarran, Robert

    2014-12-01

    In cystic fibrosis (CF) lung disease, the absence of functional CF transmembrane conductance regulator results in Cl(-)/HCO3 (-) hyposecretion and triggers Na(+) hyperabsorption through the epithelial Na(+) channel (ENaC), which contribute to reduced airway surface liquid (ASL) pH and volume. Prostasin, a membrane-anchored serine protease with trypsin-like substrate specificity has previously been shown to activate ENaC in CF airways. However, prostasin is typically inactive below pH 7.0, suggesting that it may be less relevant in acidic CF airways. Cathepsin B (CTSB) is present in both normal and CF epithelia and is secreted into ASL, but little is known about its function in the airways. We hypothesized that the acidic ASL seen in CF airways may stimulate CTSB to activate ENaC, contributing to Na(+) hyperabsorption and depletion of CF ASL volume. In Xenopus laevis oocytes, CTSB triggered α- and γENaC cleavage and induced an increase in ENaC activity. In bronchial epithelia from both normal and CF donor lungs, CTSB localized to the apical membrane. In normal and CF human bronchial epithelial cultures, CTSB was detected at the apical plasma membrane and in the ASL. CTSB activity was significantly elevated in acidic ASL, which correlated with increased abundance of ENaC in the plasma membrane and a reduction in ASL volume. This acid/CTSB-dependent activation of ENaC was ameliorated with the cell impermeable, CTSB-selective inhibitor CA074, suggesting that CTSB inhibition may have therapeutic relevance. Taken together, our data suggest that CTSB is a pathophysiologically relevant protease that activates ENaC in CF airways. PMID:25260629

  5. IL13 activates autophagy to regulate secretion in airway epithelial cells.

    PubMed

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

  6. IL13 activates autophagy to regulate secretion in airway epithelial cells

    PubMed Central

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    ABSTRACT Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent. PMID:26062017

  7. Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

    PubMed Central

    Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

    2016-01-01

    Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

  8. DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

    PubMed Central

    Habibovic, Aida; Hristova, Milena; Heppner, David E.; Danyal, Karamatullah; Ather, Jennifer L.; Janssen-Heininger, Yvonne M.W.; Irvin, Charles G.; Poynter, Matthew E.; Lundblad, Lennart K.; Dixon, Anne E.; Geiszt, Miklos

    2016-01-01

    Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management. PMID:27812543

  9. Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

    PubMed

    Statt, Sarah; Ruan, Jhen-Wei; Hung, Li-Yin; Chang, Ching-Yun; Huang, Chih-Ting; Lim, Jae Hyang; Li, Jian-Dong; Wu, Reen; Kao, Cheng-Yuan

    2015-11-01

    Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

  10. A mechanism of airway injury in an epithelial model of mucociliary clearance

    PubMed Central

    O'Brien, Darryl W; Morris, Melanie I; Ding, Jie; Zayas, J Gustavo; Tai, Shusheng; King, Malcolm

    2004-01-01

    We studied the action of sodium metabisulphite on mucociliary transport in a frog palate epithelial injury model, hypothesizing that it may be useful for the study of mechanisms of airway injury. Sodium metabisulphite (MB) releases SO2 on contact with water. SO2 is a pollutant in automobile fumes and may play a role in the exacerbation of airway disease symptoms. We first investigated its effect on mucociliary clearance. MB 10-1 M, increased mucociliary clearance time (MCT) by 254.5 ± 57.3% of control values, (p < 0.001, n = 7). MB 10-4 and 10-2 M did not interfere with mucus clearance time compared to control values. In MB-treated frog palates, MCT did not return to control values after one hour (control, 97.3 ± 6.3% vs. MB, 140.9 ± 46.3%, p < 0.001, n = 7). Scanning EM images of epithelial tissue were morphometrically analyzed and showed a 25 ± 12% loss of ciliated cells in MB palates compared to controls with an intact ciliary blanket. Intact cells or groups of ciliated cells were found in scanning EM micrographs of mucus from MB-treated palates. This was associated with increased matrix metalloproteinase (MMP-9) activity in epithelial tissue and mucus. We suggest that the loss of ciliated cells as a result of MMP-9 activation prevented full recovery of MCT after MB 10-1 M. The mechanism of action may be on epithelial cell-cell or cell-matrix attachments leading to cell loss and a disruption of MCT. Further studies are warranted to determine whether this is an inflammatory mediated response or the result of a direct action on epithelial cells and what role this mechanism may play in the progression to chronic airway diseases with impaired mucociliary clearance. PMID:15357883

  11. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    PubMed Central

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

  12. Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

    PubMed Central

    Nita-Lazar, Mihai; Banerjee, Aditi; Feng, Chiguang; Amin, Mohammed N.; Frieman, Matthew B.; Chen, Wilbur H.; Cross, Alan S.; Wang, Lai-Xi; Vasta, Gerardo R.

    2015-01-01

    The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence motif, with critical regulatory roles in development and immune homeostasis. In this study, we examined the possibility that galectins expressed in the airway epithelial cells might play a significant role in viral or pneumococcal adhesion to airway epithelial cells. Our results in a mouse model for influenza and pneumococcal infection revealed that the murine lung expresses a diverse galectin repertoire, from which selected galectins, including galectin 1 (Gal1) and galectin 3 (Gal3), are released to the bronchoalveolar space. Further, the results showed that influenza and subsequent S. pneumoniae infections significantly alter the glycosylation patterns of the airway epithelial surface and modulate galectin expression. In vitro studies on the human airway epithelial cell line A549 were consistent with the observations made in the mouse model, and further revealed that both Gal1 and Gal3 bind strongly to IAV and S. pneumoniae, and that exposure of the cells to viral neuraminidase or

  13. Gene Transfer by Guanidinium-Cholesterol Cationic Lipids into Airway Epithelial Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Oudrhiri, Noufissa; Vigneron, Jean-Pierre; Peuchmaur, Michel; Leclerc, Tony; Lehn, Jean-Marie; Lehn, Pierre

    1997-03-01

    Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis (guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

  14. Inter-alpha-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding.

    PubMed

    Adair, Jennifer E; Stober, Vandy; Sobhany, Mack; Zhuo, Lisheng; Roberts, John D; Negishi, Masahiko; Kimata, Koji; Garantziotis, Stavros

    2009-06-19

    Pulmonary epithelial injury is central to the pathogenesis of many lung diseases, such as asthma, pulmonary fibrosis, and the acute respiratory distress syndrome. Regulated epithelial repair is crucial for lung homeostasis and prevents scar formation and inflammation that accompany dysregulated healing. The extracellular matrix (ECM) plays an important role in epithelial repair after injury. Vitronectin is a major ECM component that promotes epithelial repair. However, the factors that modify cell-vitronectin interactions after injury and help promote epithelial repair are not well studied. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protein. IaI heavy chains contain von Willebrand A domains that can bind the arginine-glycine-aspartate domain of vitronectin. We therefore hypothesized that IaI can bind vitronectin and promote vitronectin-induced epithelial repair after injury. We show that IaI binds vitronectin at the arginine-glycine-aspartate site, thereby promoting epithelial adhesion and migration in vitro. Furthermore, we show that IaI-deficient mice have a dysregulated response to epithelial injury in vivo, consisting of decreased proliferation and epithelial metaplasia. We conclude that IaI interacts not only with hyaluronan, as previously reported, but also other ECM components like vitronectin and is an important regulator of cellular repair after injury. PMID:19395377

  15. Feasibility of a 3D human airway epithelial model to study respiratory absorption.

    PubMed

    Reus, Astrid A; Maas, Wilfred J M; Jansen, Harm T; Constant, Samuel; Staal, Yvonne C M; van Triel, Jos J; Kuper, C Frieke

    2014-03-01

    The respiratory route is an important portal for human exposure to a large variety of substances. Consequently, there is an urgent need for realistic in vitro strategies for evaluation of the absorption of airborne substances with regard to safety and efficacy assessment. The present study investigated feasibility of a 3D human airway epithelial model to study respiratory absorption, in particular to differentiate between low and high absorption of substances. Bronchial epithelial models (MucilAir™), cultured at the air-liquid interface, were exposed to eight radiolabeled model substances via the apical epithelial surface. Absorption was evaluated by measuring radioactivity in the apical compartment, the epithelial cells and the basolateral culture medium. Antipyrine, caffeine, naproxen and propranolol were highly transported across the epithelial cell layer (>5%), whereas atenolol, mannitol, PEG-400 and insulin were limitedly transported (<5%). Results indicate that the 3D human airway epithelial model used in this study is able to differentiate between substances with low and high absorption. The intra-experimental reproducibility of the results was considered adequate based on an average coefficient of variation (CV) of 15%. The inter-experimental reproducibility of highly absorbed compounds was in a similar range (CV of 15%), but this value was considerably higher for those compounds that were limitedly absorbed. No statistical significant differences between different donors and experiments were observed. The present study provides a simple method transposable in any lab, which can be used to rank the absorption of chemicals and pharmaceuticals, and is ready for further validation with respect to reproducibility and capacity of the method to predict respiratory transport in humans.

  16. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation.

  17. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation. PMID:26276826

  18. Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling

    PubMed Central

    2014-01-01

    Background Eotaxin proteins are a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Since inflammation is often associated with an increased generation of reactive oxygen species (ROS), oxidative stress is a mechanistically imperative factor in asthma. Astragalin (kaempferol-3-O-glucoside) is a flavonoid with anti-inflammatory activity and newly found in persimmon leaves and green tea seeds. This study elucidated that astragalin inhibited endotoxin-induced oxidative stress leading to eosinophilia and epithelial apoptosis in airways. Methods Airway epithelial BEAS-2B cells were exposed to lipopolysaccharide (LPS) in the absence and presence of 1–20 μM astragalin. Western blot and immunocytochemical analyses were conducted to determine induction of target proteins. Cell and nuclear staining was also performed for ROS production and epithelial apoptosis. Results When airway epithelial cells were exposed to 2 μg/ml LPS, astragalin nontoxic at ≤20 μM suppressed cellular induction of Toll-like receptor 4 (TLR4) and ROS production enhanced by LPS. Both LPS and H2O2 induced epithelial eotaxin-1 expression, which was blocked by astragalin. LPS activated and induced PLCγ1, PKCβ2, and NADPH oxidase subunits of p22phox and p47phox in epithelial cells and such activation and induction were demoted by astragalin or TLR4 inhibition antagonizing eotaxin-1 induction. H2O2-upregulated phosphorylation of JNK and p38 MAPK was dampened by adding astragalin to epithelial cells, while this compound enhanced epithelial activation of Akt and ERK. H2O2 and LPS promoted epithelial apoptosis concomitant with nuclear condensation or caspase-3 activation, which was blunted by astragalin. Conclusions Astragalin ameliorated oxidative stress-associated epithelial eosinophilia and apoptosis through disturbing TLR4-PKCβ2-NADPH oxidase-responsive signaling. Therefore, astragalin may be a potent agent antagonizing endotoxin

  19. Physical characterization and profiling of airway epithelial derived exosomes using light scattering

    PubMed Central

    Kesimer, Mehmet; Gupta, Richa

    2015-01-01

    Exosomes and other extracellular vesicles have been gaining interest during the last decade due to their emerging role in biology and, disease pathogenesis and their biomarker potential. Almost all published research related to exosomes and other extracellular vesicles include some form of physical characterization. Therefore, these vesicles should be precisely profiled and characterized physically before studying their biological role as intercellular messengers, biomarkers or therapeutic tools. Using a combination of light scattering techniques, including dynamic light scattering (DLS) and multi-angle laser light scattering combined with size exclusion separation (SEC-MALLS), we physically characterized and compared distinct extracellular vesicles derived from the apical secretions of two different cultured airway epithelial cells. The results indicated that epithelial cells release vesicles with distinct physical properties and sizes. Human primary tracheobronchial cell culture (HTBE) derived vesicles have a hydrodynamic radius (Rh) of approximately 340 nm while their radius of gyration (Rg) is approximately 200 nm. Electron microscopy analysis, however, revealed that their spherical component is 40-100 nm in size, and they carry filamentous, entangled membrane mucins on their surface that increases their overall radius. The mucin decoration on the surface defines their size and charge as measured using light scattering techniques. Their surface properties mirror the properties of the cells from which they are derived. This may provide a unique tool for researchers to elucidate the unanswered questions in normal airway biology and innate and adaptive defense, including the remodeling of airways during inflammation, tumorigenesis and metastasis. PMID:25823850

  20. Physical characterization and profiling of airway epithelial derived exosomes using light scattering.

    PubMed

    Kesimer, Mehmet; Gupta, Richa

    2015-10-01

    Exosomes and other extracellular vesicles have been gaining interest during the last decade due to their emerging role in biology and, disease pathogenesis and their biomarker potential. Almost all published research related to exosomes and other extracellular vesicles include some form of physical characterization. Therefore, these vesicles should be precisely profiled and characterized physically before studying their biological role as intercellular messengers, biomarkers or therapeutic tools. Using a combination of light scattering techniques, including dynamic light scattering (DLS) and multi-angle laser light scattering combined with size exclusion separation (SEC-MALLS), we physically characterized and compared distinct extracellular vesicles derived from the apical secretions of two different cultured airway epithelial cells. The results indicated that epithelial cells release vesicles with distinct physical properties and sizes. Human primary tracheobronchial cell culture (HTBE) derived vesicles have a hydrodynamic radius (Rh) of approximately 340 nm while their radius of gyration (Rg) is approximately 200 nm. Electron microscopy analysis, however, revealed that their spherical component is 40-100 nm in size, and they carry filamentous, entangled membrane mucins on their surface that increases their overall radius. The mucin decoration on the surface defines their size and charge as measured using light scattering techniques. Their surface properties mirror the properties of the cells from which they are derived. This may provide a unique tool for researchers to elucidate the unanswered questions in normal airway biology and innate and adaptive defense, including the remodeling of airways during inflammation, tumorigenesis and metastasis. PMID:25823850

  1. Engineering Airway Epithelium

    PubMed Central

    Soleas, John P.; Paz, Ana; Marcus, Paula; McGuigan, Alison; Waddell, Thomas K.

    2012-01-01

    Airway epithelium is constantly presented with injurious signals, yet under healthy circumstances, the epithelium maintains its innate immune barrier and mucociliary elevator function. This suggests that airway epithelium has regenerative potential (I. R. Telford and C. F. Bridgman, 1990). In practice, however, airway regeneration is problematic because of slow turnover and dedifferentiation of epithelium thereby hindering regeneration and increasing time necessary for full maturation and function. Based on the anatomy and biology of the airway epithelium, a variety of tissue engineering tools available could be utilized to overcome the barriers currently seen in airway epithelial generation. This paper describes the structure, function, and repair mechanisms in native epithelium and highlights specific and manipulatable tissue engineering signals that could be of great use in the creation of artificial airway epithelium. PMID:22523471

  2. Soft TCPTP Agonism-Novel Target to Rescue Airway Epithelial Integrity by Exogenous Spermidine.

    PubMed

    Ghisalberti, Carlo A; Borzì, Rosa M; Cetrullo, Silvia; Flamigni, Flavio; Cairo, Gaetano

    2016-01-01

    A reparative approach of disrupted epithelium in obstructive airway diseases, namely asthma and chronic obstructive pulmonary disease (COPD), may afford protection and long-lasting results compared to conventional therapies, e.g., corticosteroids or immunosuppressant drugs. Here, we propose the polyamine spermidine as a novel therapeutic agent in airways diseases, based on a recently identified mode of action: T-cell protein tyrosine phosphatase (TCPTP) agonism. It may include and surpass single-inhibitors of stress and secondary growth factor pathway signaling, i.e., the new medicinal chemistry in lung diseases. Enhanced polyamine biosynthesis has been charged with aggravating prognosis by competing for L-arginine at detriment of nitric oxide (NO) synthesis with bronchoconstrictive effects. Although excess spermine, a higher polyamine, is harmful to airways physiology, spermidine can pivot the cell homeostasis during stress conditions by the activation of TCPTP. In fact, the dephosphorylating activity of TCPTP inhibits the signaling cascade that leads to the expression of genes involved in detachment and epithelial-to-mesenchymal transition (EMT), and increases the expression of adhesion and tight junction proteins, thereby enhancing the barrier functionality in inflammation-prone tissues. Moreover, a further beneficial effect of spermidine may derive from its ability to promote autophagy, possibly in a TCPTP-dependent way. Since doses of spermidine in the micromolar range are sufficient to activate TCPTP, low amounts of spermidine administered in sustained release modality may provide an optimal pharmacologic profile for the treatment of obstructive airway diseases. PMID:27375482

  3. Soft TCPTP Agonism—Novel Target to Rescue Airway Epithelial Integrity by Exogenous Spermidine

    PubMed Central

    Ghisalberti, Carlo A.; Borzì, Rosa M.; Cetrullo, Silvia; Flamigni, Flavio; Cairo, Gaetano

    2016-01-01

    A reparative approach of disrupted epithelium in obstructive airway diseases, namely asthma and chronic obstructive pulmonary disease (COPD), may afford protection and long-lasting results compared to conventional therapies, e.g., corticosteroids or immunosuppressant drugs. Here, we propose the polyamine spermidine as a novel therapeutic agent in airways diseases, based on a recently identified mode of action: T-cell protein tyrosine phosphatase (TCPTP) agonism. It may include and surpass single-inhibitors of stress and secondary growth factor pathway signaling, i.e., the new medicinal chemistry in lung diseases. Enhanced polyamine biosynthesis has been charged with aggravating prognosis by competing for L-arginine at detriment of nitric oxide (NO) synthesis with bronchoconstrictive effects. Although excess spermine, a higher polyamine, is harmful to airways physiology, spermidine can pivot the cell homeostasis during stress conditions by the activation of TCPTP. In fact, the dephosphorylating activity of TCPTP inhibits the signaling cascade that leads to the expression of genes involved in detachment and epithelial-to-mesenchymal transition (EMT), and increases the expression of adhesion and tight junction proteins, thereby enhancing the barrier functionality in inflammation-prone tissues. Moreover, a further beneficial effect of spermidine may derive from its ability to promote autophagy, possibly in a TCPTP-dependent way. Since doses of spermidine in the micromolar range are sufficient to activate TCPTP, low amounts of spermidine administered in sustained release modality may provide an optimal pharmacologic profile for the treatment of obstructive airway diseases. PMID:27375482

  4. Distal airway epithelial progenitor cells are radiosensitive to High-LET radiation

    PubMed Central

    McConnell, Alicia M.; Konda, Bindu; Kirsch, David G.; Stripp, Barry R.

    2016-01-01

    Exposure to high-linear energy transfer (LET) radiation occurs in a variety of situations, including charged particle radiotherapy, radiological accidents, and space travel. However, the extent of normal tissue injury in the lungs following high-LET radiation exposure is unknown. Here we show that exposure to high-LET radiation led to a prolonged loss of in vitro colony forming ability by airway epithelial progenitor cells. Furthermore, exposure to high-LET radiation induced clonal expansion of a subset of progenitor cells in the distal airway epithelium. Clonal expansion following high-LET radiation exposure was correlated with elevated progenitor cell apoptosis, persistent γ-H2AX foci, and defects in mitotic progression of distal airway progenitors. We discovered that the effects of high-LET radiation exposure on progenitor cells occur in a p53-dependent manner. These data show that high-LET radiation depletes the distal airway progenitor pool by inducing cell death and loss of progenitor function, leading to clonal expansion. Importantly, high-LET radiation induces greater long-term damage to normal lung tissue than the relative equivalent dose of low-LET γ-rays, which has implications in therapeutic development and risk assessment. PMID:27659946

  5. Intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells

    SciTech Connect

    Verhaeghe, Catherine; Tabruyn, Sebastien P.; Oury, Cecile; Bours, Vincent . E-mail: vbours@ulg.ac.be; Griffioen, Arjan W.

    2007-05-11

    Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications.

  6. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses.

    PubMed

    Eckerle, Isabella; Ehlen, Lukas; Kallies, René; Wollny, Robert; Corman, Victor M; Cottontail, Veronika M; Tschapka, Marco; Oppong, Samuel; Drosten, Christian; Müller, Marcel A

    2014-01-01

    Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells. PMID:24454736

  7. Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review

    PubMed Central

    Croft, Carys A.; Culibrk, Luka; Moore, Margo M.; Tebbutt, Scott J.

    2016-01-01

    Aspergillus fumigatus is an environmental filamentous fungus that also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell (AEC). The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with AECs is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and AECs, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by AECs, the immune response of AECs, as well as the role of fungal virulence factors, and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. fumigatus could point to novel targets for therapy and prophylaxis. PMID:27092126

  8. IL-1α mediates cellular cross-talk in the airway epithelial mesenchymal trophic unit

    PubMed Central

    Hill, Alison R.; Donaldson, Jessica E.; Blume, Cornelia; Smithers, Natalie; Tezera, Liku; Tariq, Kamran; Dennison, Patrick; Rupani, Hitasha; Edwards, Matthew J.; Howarth, Peter H.; Grainge, Christopher; Davies, Donna E.; Swindle, Emily J.

    2016-01-01

    ABSTRACT The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections. EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection. PMID:27583193

  9. IL-1α mediates cellular cross-talk in the airway epithelial mesenchymal trophic unit.

    PubMed

    Hill, Alison R; Donaldson, Jessica E; Blume, Cornelia; Smithers, Natalie; Tezera, Liku; Tariq, Kamran; Dennison, Patrick; Rupani, Hitasha; Edwards, Matthew J; Howarth, Peter H; Grainge, Christopher; Davies, Donna E; Swindle, Emily J

    2016-01-01

    The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesized that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections. EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1α which was unaffected by the presence of fibroblasts. Blockade of IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1α induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signaling within the EMTU to propagate inflammatory responses to viral infection. PMID:27583193

  10. Role of β-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

    PubMed

    Zemans, Rachel L; McClendon, Jazalle; Aschner, Yael; Briones, Natalie; Young, Scott K; Lau, Lester F; Kahn, Michael; Downey, Gregory P

    2013-03-15

    Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that β-catenin signaling promotes epithelial repair after inflammatory injury, but the β-catenin target genes that mediate this effect are unknown. Herein, we examined which β-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of β-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the β-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that β-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

  11. The innate immune function of airway epithelial cells in inflammatory lung disease

    PubMed Central

    Hiemstra, Pieter S.; McCray, Paul B.; Bals, Robert

    2016-01-01

    The airway epithelium is now considered central to the orchestration of pulmonary inflammatory and immune responses, and is also key to tissue remodelling. It acts as a first barrier in the defence against a wide range of inhaled challenges, and is critically involved in the regulation of both innate and adaptive immune responses to these challenges. Recent progress in our understanding of the developmental regulation of this tissue, the differentiation pathways, recognition of pathogens and antimicrobial responses is now exploited to help understand how epithelial cell function and dysfunction contributes to the pathogenesis of a variety of inflammatory lung diseases. In the review, advances in our knowledge of the biology of airway epithelium, as well as its role and (dys)function in asthma, COPD and cystic fibrosis, are discussed. PMID:25700381

  12. Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.

    PubMed

    Le Cras, Timothy D; Acciani, Thomas H; Mushaben, Elizabeth M; Kramer, Elizabeth L; Pastura, Patricia A; Hardie, William D; Korfhagen, Thomas R; Sivaprasad, Umasundari; Ericksen, Mark; Gibson, Aaron M; Holtzman, Michael J; Whitsett, Jeffrey A; Hershey, Gurjit K Khurana

    2011-03-01

    Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.

  13. Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

    PubMed

    Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

    2013-12-01

    Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.

  14. Targeting TSLP With shRNA Alleviates Airway Inflammation and Decreases Epithelial CCL17 in a Murine Model of Asthma

    PubMed Central

    Chen, Yi-Lien; Chiang, Bor-Luen

    2016-01-01

    Airway epithelium defends the invasion from microorganisms and regulates immune responses in allergic asthma. Thymic stromal lymphopoietin (TSLP) from inflamed epithelium promotes maturation of dendritic cells (DCs) to prime Th2 responses via CCL17, which induces chemotaxis of CD4+ T cells to mediate inflammation. However, few studies have investigated the regulation of epithelial CCL17. In this study, we used shRNA against TSLP to clarify the role of TSLP in the airway inflammation and whether TSLP affects the airway inflammation via epithelial CCL17. Specific shTSLP was delivered by lentivirus and selected by the knockdown efficiency. Allergic mice were intratracheally pretreated with the lentivirus and followed by intranasal ovalbumin (OVA) challenges. The sera antibody levels, airway inflammation, airway hyper-responsiveness (AHR), cytokine levels in bronchoalveolar lavage fluids, and CCL17 expressions in lungs were determined. In vivo, TSLP attenuation reduced the AHR, decreased the airway inflammation, inhibited the maturations of DCs, and suppressed the migration of T cells. Furthermore, the expression of CCL17 was particularly decreased in bronchial epithelium. In vitro, CCL17 induction was regulated by TSLP. In conclusion, TSLP might coordinate airway inflammation partially via CCL17-mediated responses and this study provides the vital utility of TSLP to develop the therapeutic approach in allergic airway inflammation. PMID:27138176

  15. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells

    PubMed Central

    Konishi, Satoshi; Gotoh, Shimpei; Tateishi, Kazuhiro; Yamamoto, Yuki; Korogi, Yohei; Nagasaki, Tadao; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Ito, Isao; Tsukita, Sachiko; Mishima, Michiaki

    2015-01-01

    Summary Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1+-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM+ VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a “9 + 2” microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine. PMID:26724905

  16. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells.

    PubMed

    Konishi, Satoshi; Gotoh, Shimpei; Tateishi, Kazuhiro; Yamamoto, Yuki; Korogi, Yohei; Nagasaki, Tadao; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Ito, Isao; Tsukita, Sachiko; Mishima, Michiaki

    2016-01-12

    Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1(+)-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM(+) VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a "9 + 2" microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine. PMID:26724905

  17. In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

    PubMed

    Broadbent, Lindsay; Villenave, Remi; Guo-Parke, Hong; Douglas, Isobel; Shields, Michael D; Power, Ultan F

    2016-01-01

    The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis. PMID:27464691

  18. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells.

    PubMed

    Konishi, Satoshi; Gotoh, Shimpei; Tateishi, Kazuhiro; Yamamoto, Yuki; Korogi, Yohei; Nagasaki, Tadao; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Ito, Isao; Tsukita, Sachiko; Mishima, Michiaki

    2016-01-12

    Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1(+)-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM(+) VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a "9 + 2" microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine.

  19. Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells☆

    PubMed Central

    Hristova, Milena; Veith, Carmen; Habibovic, Aida; Lam, Ying-Wai; Deng, Bin; Geiszt, Miklos; Janssen-Heininger, Yvonne M.W.; van der Vliet, Albert

    2014-01-01

    The NADPH oxidase homolog dual oxidase 1 (DUOX1) plays an important role in innate airway epithelial responses to infection or injury, but the precise molecular mechanisms are incompletely understood and the cellular redox-sensitive targets for DUOX1-derived H2O2 have not been identified. The aim of the present study was to survey the involvement of DUOX1 in cellular redox signaling by protein S-glutathionylation, a major mode of reversible redox signaling. Using human airway epithelial H292 cells and stable transfection with DUOX1-targeted shRNA as well as primary tracheal epithelial cells from either wild-type or DUOX1-deficient mice, DUOX1 was found to be critical in ATP-stimulated transient production of H2O2 and increased protein S-glutathionylation. Using cell pre-labeling with biotin-tagged GSH and analysis of avidin-purified proteins by global proteomics, 61 S-glutathionylated proteins were identified in ATP-stimulated cells compared to 19 in untreated cells. Based on a previously established role of DUOX1 in cell migration, various redox-sensitive proteins with established roles in cytoskeletal dynamics and/or cell migration were evaluated for S-glutathionylation, indicating a critical role for DUOX1 in ATP-stimulated S-glutathionylation of β-actin, peroxiredoxin 1, the non-receptor tyrosine kinase Src, and MAPK phosphatase 1. Overall, our studies demonstrate the importance of DUOX1 in epithelial redox signaling through reversible S-glutathionylation of a range of proteins, including proteins involved in cytoskeletal regulation and MAPK signaling pathways involved in cell migration. PMID:24624333

  20. LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.

    PubMed

    Luz, Simão; Cihil, Kristine M; Brautigan, David L; Amaral, Margarida D; Farinha, Carlos M; Swiatecka-Urban, Agnieszka

    2014-05-23

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.

  1. Proteinases release /sup 35/S-labeled macromolecules from cultured airway epithelial cells

    SciTech Connect

    Varsano, S.; Borson, D.B.; Gold, M.; Forsberg, S.; Basbaum, C.B.; Nadel, J.A.

    1986-03-05

    To determine whether proteinases release radiolabeled macromolecules from airway cells devoid of secretory granules, they studied canine cultured tracheal epithelial cells grown to confluency. At this time the cells are bound by tight junctions, maintain anion transport, have a well developed glycocalyx, but contain no secretory granules. They labeled the cells with /sup 35/SO/sub 4/ (50..mu..ci/ml/24h) then changed the medium every 20 min and measured nondialyzable /sup 35/S released into the medium. Two h later, the rate of spontaneous release of /sup 35/S-labeled-macromolecules was 5700 +/- 1600 CPM/20 min (mean +/- SD). At this time trypsin, thermolysin, pseudomonas elastase and alkaline proteinase, each released /sup 35/S-labeled-macromolecules, whereas aspergillus acid proteinase did not. In more detailed studies, trypsin released /sup 35/S in a concentration dependent fashion, with a threshold below 10 units/ml and a response to 1000 units/ml of 1092 +/- 173% (mean +/- SD; n=5 cultures) above pre-trypsin baseline. Sepharose CL4B chromatography of the radiolabeled materials released by trypsin showed a void volume fraction (MW greater than or equal to 10/sup 6/), and a second, included fraction (MW 2-3 x 10/sup 5/). These results indicate that cultured airway epithelial cells synthesize macromolecules and release them into the medium, and that proteinases increase the rate of macromolecule release markedly.

  2. Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells

    PubMed Central

    Jairaman, Amit; Maguire, Chelsea H.; Schleimer, Robert P.; Prakriya, Murali

    2016-01-01

    Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca2+ is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca2+ elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca2+ elevations in AECs through the activation of Ca2+ release-activated Ca2+ (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca2+ entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway. PMID:27604412

  3. Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells.

    PubMed

    Jairaman, Amit; Maguire, Chelsea H; Schleimer, Robert P; Prakriya, Murali

    2016-09-08

    Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca(2+) is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca(2+) elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca(2+) elevations in AECs through the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca(2+) entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway.

  4. Nitrite Modulates Bacterial Antibiotic Susceptibility and Biofilm Formation in Association with Airway Epithelial Cells

    PubMed Central

    Zemke, Anna C; Shiva, Sruti; Burn, Jane L.; Moskowitz, Samuel M.; Pilewski, Joseph M.; Gladwin, Mark T.; Bomberger, Jennifer M.

    2014-01-01

    Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, while the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects in other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain ‘poisons’, can be safely nebulized at high concentration in humans. PMID:25229185

  5. Rhinoviral infection and asthma: the detection and management of rhinoviruses by airway epithelial cells.

    PubMed

    Parker, L C; Stokes, C A; Sabroe, I

    2014-01-01

    Human rhinoviruses (HRV) have been linked to the development of childhood asthma and recurrent acute asthma exacerbations throughout life, and contribute considerably to the healthcare and economic burden of this disease. However, the ability of HRV infections to trigger exacerbations, and the link between allergic status and HRV responsiveness, remains incompletely understood. Whilst the receptors on human airway cells that detect and are utilized by most HRV group A and B, but not C serotypes are known, how endosomal pattern recognition receptors (PRRs) detect HRV replication products that are generated within the cytoplasm remains somewhat of an enigma. In this article, we explore a role for autophagy, a cellular homeostatic process that allows the cell to encapsulate its own cytosolic constituents, as the crucial mechanism controlling this process and regulating the innate immune response of airway epithelial cells to viral infection. We will also briefly describe some of the recent insights into the immune responses of the airway to HRV, focusing on neutrophilic inflammation that is a potentially unwanted feature of the acute response to viral infection, and the roles of IL-1 and Pellinos in the regulation of responses to HRV.

  6. Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells.

    PubMed

    Jairaman, Amit; Maguire, Chelsea H; Schleimer, Robert P; Prakriya, Murali

    2016-01-01

    Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca(2+) is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca(2+) elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca(2+) elevations in AECs through the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca(2+) entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway. PMID:27604412

  7. Junctional abnormalities in human airway epithelial cells expressing F508del CFTR

    PubMed Central

    Stauffer, Brandon; Moriarty, Hannah K.; Kim, Agnes H.; McCarty, Nael A.; Koval, Michael

    2015-01-01

    Cystic fibrosis (CF) has a profound impact on airway physiology. Accumulating evidence suggests that intercellular junctions are impaired in CF. We examined changes to CF transmembrane conductance regulator (CFTR) function, tight junctions, and gap junctions in NuLi-1 (CFTRwt/wt) and CuFi-5 (CFTRΔF508/ΔF508) cells. Cells were studied at air-liquid interface (ALI) and compared with primary human bronchial epithelial cells. On the basis of fluorescent lectin binding, the phenotype of the NuLi-1 and CuFi-5 cells at week 8 resembled that of serous, glycoprotein-rich airway cells. After week 7, CuFi-5 cells possessed 130% of the epithelial Na+ channel activity and 17% of the CFTR activity of NuLi-1 cells. In both cell types, expression levels of CFTR were comparable to those in primary airway epithelia. Transepithelial resistance of NuLi-1 and CuFi-5 cells stabilized during maturation in ALI culture, with significantly lower transepithelial resistance for CuFi-5 than NuLi-1 cells. We also found that F508del CFTR negatively affects gap junction function in the airway. NuLi-1 and CuFi-5 cells express the connexins Cx43 and Cx26. While both connexins were properly trafficked by NuLi-1 cells, Cx43 was mistrafficked by CuFi-5 cells. Cx43 trafficking was rescued in CuFi-5 cells treated with 4-phenylbutyric acid (4-PBA), as assessed by intracellular dye transfer. 4-PBA-treated CuFi-5 cells also exhibited an increase in forskolin-induced CFTR-mediated currents. The Cx43 trafficking defect was confirmed using IB3-1 cells and found to be corrected by 4-PBA treatment. These data support the use of NuLi-1 and CuFi-5 cells to examine the effects of F508del CFTR expression on tight junction and gap junction function in the context of serous human airway cells. PMID:26115671

  8. Pseudomonas aeruginosa pyocyanin modulates mucin glycosylation with sialyl-Lewisx to increase binding to airway epithelial cells

    PubMed Central

    Choi, Woosuk; Choe, Shawn; Miao, Jinfeng; Xu, Ying; Powell, Rebecca; Lin, Jingjun; Kuang, Zhizhou; Gaskins, H Rex; Lau, Gee W.

    2015-01-01

    Cystic fibrosis (CF) patients battle life-long pulmonary infections with the respiratory pathogen Pseudomonas aeruginosa (PA). An overabundance of mucus in CF airways provides a favorable niche for PA growth. When compared to that of non-CF individuals, mucus of CF airways is enriched in sialyl-Lewisx, a preferred binding receptor for PA. Notably, the levels of sialyl-Lewisx directly correlate with infection severity in CF patients. However, the mechanism by which PA causes increased sialylation remains uncharacterized. In this study, we examined the ability of PA virulence factors to modulate sialyl-Lewisx modification in airway mucins. We found pyocyanin (PCN) to be a potent inducer of sialyl-Lewisx in both mouse airways and in primary and immortalized CF and non-CF human airway epithelial cells. PCN increased the expression of C2/4GnT and ST3Gal-IV, two of the glycosyltransferases responsible for the stepwise biosynthesis of sialyl-Lewisx, through a TNF-α-mediated phosphoinositol-specific phospholipase C (PI-PLC) dependent pathway. Furthermore, PA bound more efficiently to airway epithelial cells pre-exposed to PCN through a flagellar cap-dependent manner. Importantly, antibodies against sialyl-Lewisx and anti-TNF-α attenuated PA binding. These results indicate that PCN secretes PCN to induce a favorable environment for chronic colonization of CF lungs by increasing the glycosylation of airway mucins with sialyl-Lewisx. PMID:26555707

  9. Evidence of K+ channel function in epithelial cell migration, proliferation, and repair

    PubMed Central

    Girault, Alban

    2013-01-01

    Efficient repair of epithelial tissue, which is frequently exposed to insults, is necessary to maintain its functional integrity. It is therefore necessary to better understand the biological and molecular determinants of tissue regeneration and to develop new strategies to promote epithelial repair. Interestingly, a growing body of evidence indicates that many members of the large and widely expressed family of K+ channels are involved in regulation of cell migration and proliferation, key processes of epithelial repair. First, we briefly summarize the complex mechanisms, including cell migration, proliferation, and differentiation, engaged after epithelial injury. We then present evidence implicating K+ channels in the regulation of these key repair processes. We also describe the mechanisms whereby K+ channels may control epithelial repair processes. In particular, changes in membrane potential, K+ concentration, cell volume, intracellular Ca2+, and signaling pathways following modulation of K+ channel activity, as well as physical interaction of K+ channels with the cytoskeleton or integrins are presented. Finally, we discuss the challenges to efficient, specific, and safe targeting of K+ channels for therapeutic applications to improve epithelial repair in vivo. PMID:24196531

  10. Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells.

    PubMed

    Zhao, Rui; Fallon, Timothy R; Saladi, Srinivas Vinod; Pardo-Saganta, Ana; Villoria, Jorge; Mou, Hongmei; Vinarsky, Vladimir; Gonzalez-Celeiro, Meryem; Nunna, Naveen; Hariri, Lida P; Camargo, Fernando; Ellisen, Leif W; Rajagopal, Jayaraj

    2014-07-28

    Our understanding of how stem cells are regulated to maintain appropriate tissue size and architecture is incomplete. We show that Yap (Yes-associated protein 1) is required for the actual maintenance of an adult mammalian stem cell. Without Yap, adult airway basal stem cells are lost through their unrestrained differentiation, resulting in the simplification of a pseudostratified epithelium into a columnar one. Conversely, Yap overexpression increases stem cell self-renewal and blocks terminal differentiation, resulting in epithelial hyperplasia and stratification. Yap overexpression in differentiated secretory cells causes them to partially reprogram and adopt a stem cell-like identity. In contrast, Yap knockdown prevents the dedifferentiation of secretory cells into stem cells. We then show that Yap functionally interacts with p63, the cardinal transcription factor associated with myriad epithelial basal stem cells. In aggregate, we show that Yap regulates all of the cardinal behaviors of airway epithelial stem cells and determines epithelial architecture.

  11. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

    PubMed Central

    Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee

    2015-01-01

    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases. PMID:26599511

  12. Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells

    PubMed Central

    Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina A; O'Neal, Wanda; Gabriel, Sherif; Abdullah, Lubna; Davis, C William; Boucher, Richard C; Lazarowski, Eduardo R

    2007-01-01

    The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca2+-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca2+-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca2+-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y2 receptor-stimulated increase of cytosolic Ca2+ concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL. PMID:17656429

  13. The Role of Lysophosphatidic Acid on Airway Epithelial Cell Denudation in a Murine Heterotopic Tracheal Transplant Model

    PubMed Central

    Tando, Yukiko; Ota, Chiharu; Yamada, Mitsuhiro; Kamata, Satoshi; Yamaya, Mutsuo; Kano, Kuniyuki; Okudaira, Shinichi; Aoki, Junken; Kubo, Hiroshi

    2015-01-01

    Background Chronic rejection is the major leading cause of morbidity and mortality after lung transplantation. Obliterative bronchiolitis (OB), a fibroproliferative disorder of the small airways, is the main manifestation of chronic lung allograft rejection. However, there is currently no treatment for the disease. We hypothesized that lysophosphatidic acid (LPA) participates in the progression of OB. The aim of this study was to reveal the involvement of LPA on the lesion of OB. Methods Ki16198, an antagonist specifically for LPA1 and LPA3, was daily administered into the heterotopic tracheal transplant model mice at the day of transplantation. At days 10 and 28, the allografts were isolated and evaluated histologically. The messenger RNA levels of LPAR in microdissected mouse airway regions were assessed to reveal localization of lysophosphatidic acid receptors. The human airway epithelial cell was used to evaluate the mechanism of LPA-induced suppression of cell adhesion to the extracellular matrix (ECM). Results The administration of Ki16198 attenuated airway epithelial cell loss in the allograft at day 10. Messenger RNAs of LPA1 and LPA3 were detected in the airway epithelial cells of the mice. Lysophosphatidic acid inhibited the attachment of human airway epithelial cells to the ECM and induced cell detachment from the ECM, which was mediated by LPA1 and Rho-kinase pathway. However, Ki16198 did not prevent obliteration of allograft at day 28. Conclusions The LPA signaling is involved in the status of epithelial cells by distinct contribution in 2 different phases of the OB lesion. This finding suggests a role of LPA in the pathogenesis of OB. PMID:27500235

  14. The deubiquitinating enzyme USP10 regulates the endocytic recycling of CFTR in airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Barnaby, Roxanna L; Stanton, Bruce A

    2010-01-01

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cyclic AMP-regulated chloride channel that plays an important role in regulating the volume of the lung airway surface liquid, and thereby mucociliary clearance and elimination of pathogens from the lung. In epithelial cells, cell surface CFTR abundance is determined in part by regulating both CFTR endocytosis from the apical plasma membrane and recycling back to the plasma membrane. We recently reported, using an activity-based chemical screen to identify active deubiquitinating enzymes (DUBs) in human airway epithelial cells, that Ubiquitin Specific Protease-10 (USP10) is located and active in the early endosomal compartment and regulates the deubiquitination of CFTR and thereby promotes its endocytic recycling. siRNA-mediated knockdown of USP10 increased the multi-ubiquitination and lysosomal degradation of CFTR and decreased the endocytic recycling and the half-life of CFTR in the apical membrane, as well as CFTR-mediated chloride secretion. Overexpression of wild-type USP10 reduced CFTR multi-ubiquitination and degradation, while overexpression of a dominant-negative USP10 promoted increased multi-ubiquitination and lysosomal degradation of CFTR. In the current study, we show localization and activity of USP10 in the early endosomal compartment of primary bronchial epithelial cells, as well as an interaction between CFTR and USP10 in this compartment. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes, thereby enhancing the endocytic recycling and cell surface expression of CFTR.

  15. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

    PubMed Central

    Berman, Reena; Jiang, Di; Wu, Qun; Chu, Hong Wei

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection. PMID:27354786

  16. Effect of Polarization on Airway Epithelial Conditioning of Monocyte-Derived Dendritic Cells.

    PubMed

    Papazian, Dick; Chhoden, Tashi; Arge, Maria; Vorup-Jensen, Thomas; Nielsen, Claus H; Lund, Kaare; Würtzen, Peter A; Hansen, Soren

    2015-09-01

    Airway epithelial cells (AECs) form polarized barriers that interact with inhaled allergens and are involved in immune homeostasis. We examined how monocyte-derived dendritic cells (MDDCs) are affected by contact with the airway epithelium. In traditional setups, bronchial epithelial cell lines were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs' immunoregulatory properties; thus, previous observations obtained using traditional setups should be considered with caution. Using the optimized setup, AEC conditioning of MDDCs led to increased expression of programmed death 1 ligand 1, immunoglobulin-like transcript 3, CD40, CD80, and CD23. This increased expression was accompanied by decreased secretion of monocyte chemotactic protein 1 and eotaxin and donor-variable effects on IL-12 and IL-10 secretion. Conditioning varied between maturation states and depended partly on direct contact between AECs and MDDCs. The setup allowed MDDCs on the basal side of the epithelium to sample allergens administered to the apical side. Allergen uptake depended on polarization and the nature of the allergen. AEC conditioning led to decreased birch allergen-specific proliferation of autologous T cells and a trend toward decreased secretion of the Th2-specific cytokines IL-5 and IL-13. In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens.

  17. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770

    PubMed Central

    Van Goor, Fredrick; Hadida, Sabine; Grootenhuis, Peter D. J.; Burton, Bill; Cao, Dong; Neuberger, Tim; Turnbull, Amanda; Singh, Ashvani; Joubran, John; Hazlewood, Anna; Zhou, Jinglan; McCartney, Jason; Arumugam, Vijayalaksmi; Decker, Caroline; Yang, Jennifer; Young, Chris; Olson, Eric R.; Wine, Jeffery J.; Frizzell, Raymond A.; Ashlock, Melissa; Negulescu, Paul

    2009-01-01

    Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl− secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na+ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway. PMID:19846789

  18. Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells.

    PubMed

    Rondanino, Christine; Poland, Paul A; Kinlough, Carol L; Li, Hui; Rbaibi, Youssef; Myerburg, Michael M; Al-bataineh, Mohammad M; Kashlan, Ossama B; Pastor-Soler, Nuria M; Hallows, Kenneth R; Weisz, Ora A; Apodaca, Gerard; Hughey, Rebecca P

    2011-09-01

    Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair. PMID:21677144

  19. Lipidome and Transcriptome Profiling of Pneumolysin Intoxication Identifies Networks Involved in Statin-Conferred Protection of Airway Epithelial Cells

    PubMed Central

    Statt, Sarah; Ruan, Jhen-Wei; Huang, Chih-Ting; Wu, Reen; Kao, Cheng-Yuan

    2015-01-01

    Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin. PMID:26023727

  20. Kidney injury molecule-1 (KIM-1) mediates renal epithelial cell repair via ERK MAPK signaling pathway

    PubMed Central

    Zhang, Zhiwei; Cai, Cindy X

    2016-01-01

    The expression of kidney injury molecule-1 (KIM-1), a very promising sensitive and specific urinary biomarker for acute renal injury, is markedly upregulated in injured and regenerating renal proximal tubular epithelial cells following ischemic or toxic insults, suggesting a possible role for this molecule in renal repair process. In the present study we report that expression of KIM-1 facilitates renal tubular epithelial cell repair by promoting cell migration and proliferation. KIM-1 expression also enhances ERK MAPK activation, and the modulatory effect of KIM-1 on cellular repair process is likely mediated via ERK MAPK signaling pathway. PMID:27084535

  1. Airway epithelial cell-derived insulin-like growth factor-1 triggers skewed CD8(+) T cell polarization.

    PubMed

    Zou, Jian-Yong; Huang, Shao-hong; Li, Yun; Chen, Hui-guo; Rong, Jian; Ye, Sheng

    2014-10-01

    Skewed CD8(+) T cell responses are important in airway inflammation. This study investigates the role of the airway epithelial cell-derived insulin-like growth factor 1 (IGF1) in contributing to CD8(+) T cell polarization. Expression of IGF1 in the airway epithelial cell line, RPMI2650 cells, was assessed by quantitative real time RT-PCR and Western blotting. The role of IGF1 in regulating CD8(+) T cell activation was observed by coculture of mite allergen-primed RPMI2650 cells and naïve CD8(+) T cells. CD8(+) T cell polarization was assessed by the carboxyfluorescein succinimidyl ester-dilution assay and the determination of cytotoxic cytokine levels in the culture medium. Exposure to mite allergen, Der p1, increased the expression of IGF1 by RPMI2650 cells. The epithelial cell-derived IGF1 prevented the activation-induced cell death by inducing the p53 gene hypermethylation. Mite allergen-primed RPMI2650 cells induced an antigen-specific CD8(+) T cell polarization. We conclude that mite allergens induce airway epithelial cell line, RPMI2650 cells, to produce IGF1; the latter contributes to antigen-specific CD8(+) T cell polarization. PMID:24844927

  2. INCREASED IL-8 AND IL-6 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    INCREASED IL-6 AND IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES.
    R Silbajoris1, A G Lenz2, I Jaspers3, J M Samet1. 1NHEERL, USEPA, RTP, NC, USA; 2GSF-Institute for Inhalation Biology, Neuherberg, Germany; 3 CEMLB, UNC-CH, Chapel Hill, ...

  3. ZN2+-INDUCED IL-8 EXPRESSION INVOLVES AP-1, JNK, AND ERK ACTIVITIES IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Exposure to zinc-laden particulate matter (PM) in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zin...

  4. ULTRAFINE CARBON PARTICLES INDUCE IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS THROUGH A POST-TRANSCRIPTIONAL MECHANISM

    EPA Science Inventory

    Ultrafine carbon particles induce IL-8 expression in human airway
    epithelial cells through a post-transcritpional mechanism
    Epidemiological studies suggest that ultrafine particles contribute to
    particulate matter (PM) - induced adverse health effects. IL-8 is an
    i...

  5. IN VITRO EFFECTS OF PARTICULATE MATTER ON AIRWAY EPITHELIAL CELLS ISOLATED FROM CONCENTRATED AIR PARTICLES-EXPOSED SPONTANEOUS HYPERTENSIVE RATS

    EPA Science Inventory

    In vitro effects of particulate matter on airway epithelial cells isolated from concentrated air particles-exposed spontaneous hypertensive rats

    Ines Pagan, Urmila Kodavanti, Paul Evansky, Daniel L Costa and Janice A Dye. U.S. Environmental Protection Agency, ORD, National...

  6. Trichostatin A Inhibits Epithelial Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium

    PubMed Central

    Shin, Jae-Min; Lee, Heung-Man

    2016-01-01

    Background and Objectives Tissue remodeling is believed to cause recalcitrant chronic rhinosinusitis (CRS). Epithelial-mesenchymal transition (EMT) is a novel clinical therapeutic target in many chronic airway diseases related with tissue remodeling. The aim of this study was to investigate the effect of trichostatin A (TSA) on transforming growth factor (TGF)-β1-induced EMT in airway epithelium and nasal tissue. Materials and Methods A549 cells, primary nasal epithelial cells (PNECs), or inferior nasal turbinate organ culture were exposed to TSA prior to stimulation with TGF-β1. Expression levels of E-cadherin, vimentin, fibronectin, α-smooth muscle actin (SMA), histone deacetylase 2 (HDAC2), and HDAC4 were determined by western blotting and/or immunofluorescent staining. Hyperacetylation of histone H2 and H4 by TSA was measured by western blotting. After siHDAC transfection, the effects of HDAC2 and HDAC4 silencing on expression of E-cadherin, vimentin, fibronectin, α-SMA, HDAC2, and HDAC4 in TGF-β1-induced A549 were determined by RT-PCR and/or western blotting. We assessed the change in migration capacity of A549 cells by using cell migration assay and transwell invasion assay. Results TGF-β1 altered mRNA and protein expression levels of EMT markers including E-cadherin, vimentin, fibronectin, α-SMA, slug, and snail in A549 cells. Inhibition and silencing of HDAC2 and HDAC4 by TSA and siRNA enhanced TGF-β1-induced EMT in A549 cells. TSA blocked the effect of TGF-β1 on the migratory ability of A549 cells. In experiments using PNECs and inferior turbinate organ cultures, TSA suppressed expression of EMT markers induced by TGF-β1. Conclusions We showed that EMT is induced by TGF-β1 in airway epithelial cells and nasal tissue via activation of HDAC2 and HDAC4, and that inhibition of HDAC2 and HDAC4 by TSA reduces TGF-β1-induced EMT. This observation indicates that histone deacetylase inhibitors such as TSA could be potential candidates for treatment of

  7. Parallel activities and interactions between antimicrobial peptides and complement in host defense at the airway epithelial surface.

    PubMed

    Hiemstra, Pieter S

    2015-11-01

    Antimicrobial peptides and complement components contribute to host defense as well as inflammation and tissue injury in the respiratory tract. The airway epithelial surface is the main site of action of these immune effectors, and airway epithelial cells contribute markedly to their local production. Whereas both antimicrobial peptides and complement display overlapping functions, it is increasingly clear that both effector mechanisms also interact. Furthermore, excessive or uncontrolled release of antimicrobial peptides as well as complement activation may contribute to inflammatory lung diseases. Therefore, further knowledge of interactions between these systems may provide more insight into the pathogenesis of a range of lung diseases. In this review, recent findings on the functions, collaborations and other interactions between antimicrobial peptides and complement are discussed with a specific focus on the airway epithelium.

  8. Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells.

    PubMed

    Berger, John P; Simet, Samantha M; DeVasure, Jane M; Boten, Jessica A; Sweeter, Jenea M; Kharbanda, Kusum K; Sisson, Joseph H; Wyatt, Todd A

    2014-08-01

    Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3-7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3 to 7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.

  9. Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway.

    PubMed

    Xue, Di; Ma, Yan; Li, Min; Li, Yanan; Luo, Haixia; Liu, Xiaoming; Wang, Yujiong

    2015-01-15

    Mycoplasma ovipneumoniae (M. ovipneumoniae) is a bacterium that specifically infects sheep and goat and causes ovine infectious pleuropneumonia. In an effort to understand the pathogen-host interaction between the M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory response using a primary air-liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (Ovis aries). The ALI culture of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. Exposure of ALI cultures to M. ovipneumoniae led to increased expression of Toll-like receptors (TLRs), and components of the myeloid differentiation factor 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, infection with M. ovipneumoniae failed to induce the expression of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of the MyD88-independent signaling pathway. These results suggest that the MyD88-dependent TLR pathway may play a crucial role in sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating pathogen-host interactions between M. ovipneumoniae and airway epithelial cells.

  10. Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

    SciTech Connect

    Bao, X.; Sinha, M. |; Liu, T.; Hong, C.; Luxon, B.A. |; Garofalo, R.P. ||; Casola, A. ||

    2008-04-25

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

  11. Chromium(VI) stimulates Fyn to initiate innate immune gene induction in human airway epithelial cells

    PubMed Central

    Nemec, Antonia A.; Zubritsky, Lindsey M.; Barchowsky, Aaron

    2009-01-01

    Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes and we hypothesized this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation. PMID:19994902

  12. Aggregates of mutant CFTR fragments in airway epithelial cells of CF lungs: new pathologic observations.

    PubMed

    Du, Kai; Karp, Philip H; Ackerley, Cameron; Zabner, Joseph; Keshavjee, Shaf; Cutz, Ernest; Yeger, Herman

    2015-03-01

    Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl(-) channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary "conformational protein-associated" diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.

  13. Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

    PubMed Central

    Spiess, Page C.; Morin, Dexter; Williams, Chase R.; Buckpitt, Alan R.

    2010-01-01

    Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, β-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA. PMID:19843705

  14. Ex Vivo and In Vivo Lentivirus-Mediated Transduction of Airway Epithelial Progenitor Cells.

    PubMed

    Leoni, Giulia; Wasowicz, Marguerite Y; Chan, Mario; Meng, Cuixiang; Farley, Raymond; Brody, Steven L; Inoue, Makoto; Hasegawa, Mamoru; Alton, Eric W F W; Griesenbach, Uta

    2015-01-01

    A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration. PMID:26471068

  15. Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells.

    PubMed

    Shaykhiev, Renat; Crystal, Ronald G

    2014-12-01

    The airway epithelium is the primary site of the earliest pathologic changes induced by smoking, contributing to the development of chronic obstructive pulmonary disease (COPD). The normal human airway epithelium is composed of several major cell types, including differentiated ciliated and secretory cells, intermediate undifferentiated cells, and basal cells (BC). BC contain the stem/progenitor cell population responsible for maintenance of the normally differentiated airway epithelium. Although inflammatory and immune processes play a significant role in the pathogenesis of COPD, the earliest lesions include hyperplasia of the BC population, suggesting that the disease may start with this cell type. Apart from BC hyperplasia, smoking induces a number of COPD-relevant airway epithelial remodeling phenotypes that are likely initiated in the BC population, including mucous cell hyperplasia, squamous cell metaplasia, epithelial-mesenchymal transition, altered ciliated and nonmucous secretory cell differentiation, and suppression of junctional barrier integrity. Significant progress has been recently made in understanding the biology of human airway BC, including gene expression features, stem/progenitor, and other functions, including interaction with other airway cell types. Accumulating evidence suggests that human airway BC function as both sensors and cellular sources of various cytokines and growth factors relevant to smoking-associated airway injury, as well as the origin of various molecular and histological phenotypes relevant to the pathogenesis of COPD. In the context of these considerations, we suggest that early BC-specific smoking-induced molecular changes are critical to the pathogenesis of COPD, and these represent a candidate target for novel therapeutic approaches to prevent COPD progression in susceptible individuals.

  16. Effects of corexit oil dispersants and the WAF of dispersed oil on DNA damage and repair in cultured human bronchial airway cells, BEAS-2B

    PubMed Central

    Major, Danielle; Derbes, Rebecca S.; Wang, He; Roy-Engel, Astrid M.

    2016-01-01

    Large quantities of dispersants were used as a method to disperse the roughly 210 million gallons of spilled crude oil that consumed the Gulf of Mexico. Little is known if the oil-dispersant and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to Corexit dispersants EC9500 and EC9527, Water Accommodated Fraction (WAF) -crude, WAF-9500 + Oil, and WAF-9527 + Oil. Cellular cytotoxicity to WAF-dispersed oil samples was observed at concentrations greater than 1000 ppm with over 70% of observed cellular death. At low concentration exposures (100 and 300 ppm) DNA damage was evidenced by the detection of single strand breaks (SSBs) and double strand breaks (DSBs) as measured by alkaline and neutral comet assay analyses. Immunoblot analyses of the phosphorylated histone H2A.X (ɣ-H2A.X) and tumor suppressor p53 protein confirmed activation of the DNA damage response due to the exposure-induced DNA breaks. Although, many xenobiotics interfere with DNA repair pathways, in vitro evaluation of the nucleotide excision repair (NER) and DSB repair pathways appear to be unaffected by the oil-dispersant mixtures tested. Overall, this study supports that oil-dispersant mixtures induce genotoxic effects in culture. PMID:27563691

  17. Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

    PubMed Central

    MacPherson, Heather; Keir, Pamela A; Edwards, Carol J; Webb, Sheila; Dorin, Julia R

    2006-01-01

    Background Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells. Methods Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations. Results The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0 – 1.6% with whole marrow and 0.6 – 1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any

  18. Type I Interferons link viral infection to enhanced epithelial turnover and repair

    PubMed Central

    Sun, Lulu; Miyoshi, Hiroyuki; Origanti, Sofia; Nice, Timothy J.; Barger, Alexandra C.; Manieri, Nicholas A.; Fogel, Leslie A.; French, Anthony R.; Piwnica-Worms, David; Piwnica-Worms, Helen; Virgin, Herbert W.; Lenschow, Deborah J.; Stappenbeck, Thaddeus S.

    2014-01-01

    Summary The host immune system functions constantly to maintain chronic commensal and pathogenic organisms in check. The consequences of these immune responses on host physiology are as yet unexplored, and may have long-term implications in health and disease. We show that chronic viral infection increased epithelial turnover in multiple tissues, and the antiviral cytokines Type I interferons (IFNs) mediates this response. Using a murine model with persistently elevated Type I IFNs in the absence of exogenous viral infection, the Irgm1-/- mouse, we demonstrate that Type I IFNs act through non-epithelial cells, including macrophages, to promote increased epithelial turnover and wound repair. Downstream of Type I IFN signaling, the highly related IFN-stimulated genes Apolipoprotein L9a and b activate epithelial proliferation through ERK activation. Our findings demonstrate that the host immune response to chronic viral infection has systemic effects on epithelial turnover through a myeloid-epithelial circuit. PMID:25482432

  19. Oxidative stress in Nipah virus-infected human small airway epithelial cells

    PubMed Central

    Escaffre, Olivier; Halliday, Hailey; Borisevich, Viktoriya; Casola, Antonella

    2015-01-01

    Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development. PMID:26297489

  20. Linking optics and mechanics in an in vivo model of airway fibrosis and epithelial injury

    NASA Astrophysics Data System (ADS)

    Raub, Christopher B.; Mahon, Sari; Narula, Navneet; Tromberg, Bruce J.; Brenner, Matthew; George, Steven C.

    2010-01-01

    Chronic mucosal and submucosal injury can lead to persistent inflammation and tissue remodeling. We hypothesized that microstructural and mechanical properties of the airway wall could be derived from multiphoton images. New Zealand White rabbits were intubated, and the tracheal epithelium gently denuded every other day for five days (three injuries). Three days following the last injury, the tracheas were excised for multiphoton imaging, mechanical compression testing, and histological analysis. Multiphoton imaging and histology confirm epithelial denudation, mucosal ulceration, subepithelial thickening, collagen deposition, immune cell infiltration, and a disrupted elastin network. Elastase removes the elastin network and relaxes the collagen network. Purified collagenase removes epithelium with subtle subepithelial changes. Young's modulus [(E) measured in kiloPascal] was significantly elevated for the scrape injured (9.0+/-3.2) trachea, and both collagenase (2.6+/-0.4) and elastase (0.8+/-0.3) treatment significantly reduced E relative to control (4.1+/-0.7). E correlates strongly with second harmonic generation (SHG) signal depth decay for enzyme-treated and control tracheas (R2=0.77), but not with scrape-injured tracheas. We conclude that E of subepithelial connective tissue increases on repeated epithelial wounding, due in part to changes in elastin and collagen microstructure and concentration. SHG depth decay is sensitive to changes in extracellular matrix content and correlates with bulk Young's modulus.

  1. Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

    PubMed Central

    2012-01-01

    Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells. PMID:22694362

  2. Particulate matter (PM₁₀) induces metalloprotease activity and invasion in airway epithelial cells.

    PubMed

    Morales-Bárcenas, Rocío; Chirino, Yolanda I; Sánchez-Pérez, Yesennia; Osornio-Vargas, Álvaro Román; Melendez-Zajgla, Jorge; Rosas, Irma; García-Cuellar, Claudia María

    2015-09-17

    Airborne particulate matter with an aerodynamic diameter ≤ 10 μm (PM10) is a risk factor for the development of lung diseases and cancer. The aim of this work was to identify alterations in airway epithelial (A549) cells induced by PM10 that could explain how subtoxic exposure (10 μg/cm(2)) promotes a more aggressive in vitro phenotype. Our results showed that cells exposed to PM10 from an industrial zone (IZ) and an urban commercial zone (CZ) induced an increase in protease activity and invasiveness; however, the cell mechanism is different, as only PM10 from CZ up-regulated the activity of metalloproteases MMP-2 and MMP-9 and disrupted E-cadherin/β-catenin expression after 48 h of exposure. These in vitro findings are relevant in terms of the mechanism action of PM10 in lung epithelial cells, which could be helpful in understanding the pathogenesis of some human illness associated with highly polluted cities.

  3. Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells.

    PubMed

    Schwarzer, Christian; Fischer, Horst; Machen, Terry E

    2016-01-01

    Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10-80 fold increase, termed "swarming"), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds. PMID:27031335

  4. Role of neutrophilic inflammation in ozone-induced epithelial alterations in the nasal airways of rats

    NASA Astrophysics Data System (ADS)

    Cho, Hye Youn

    Ozone is a principal oxidant air pollutant in photochemical smog. Epithelial cells lining the centriacinar region of lung and the proximal aspects of nasal passage are primary target sites for ozone-induced injury in laboratory animals. Acute exposure of rats to high ambient concentrations of ozone (e.g., 0.5 ppm) results in neutrophilic inflammation, epithelial hyperplasia and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) lining the proximal nasal airways. The principal purpose of the present study was to investigate the role of pre-metaplastic cellular responses, especially neutrophilic inflammation, in the pathogenesis of ozone-induced MCM in rat NTE. For this purpose, three specific hypotheses-based whole-animal inhalation studies were conducted. Male F344/N rats were exposed in whole-body inhalation chambers to 0 (filtered air) or 0.5 ppm ozone for 1-3 days (8 h/day). Histochemical, immunochemical, molecular and morphometric techniques were used to investigate the ozone-induced cellular and molecular events in the NTE. Two in vitro studies were also conducted to examine the effects of ozone-inducible cytokines (i.e., tumor necrosis factor-alpha; TNF- a, and interleukin-6; IL-6) on mucin gene (rMuc-5AC) expression. Ozone induced a rapid increase of rMuc-5AC mRNA in nasal tissues within hours after the start of exposure. It preceded the appearance of MCM, and persisted with MCM. Ozone-induced neutrophilic inflammation accompanied the mucin gene upregulation, but was resolved when MCM first appeared in the NTE. Antibody-mediated depletion of circulating neutrophils attenuated ozone-induced MCM, although it did not affect the ozone-induced epithelial hyperplasia and mucin mRNA upregulation. In another study, it was found that preexisting neutrophilic rhinitis induced by endotoxin augmented the ozone-induced MCM. However, pre-existing rhinitis did not alter the severity of ozone-induced epithelial hyperplasia and mucin gene upregulation

  5. Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model.

    PubMed

    Lever, Amanda R; Park, Hyoungshin; Mulhern, Thomas J; Jackson, George R; Comolli, James C; Borenstein, Jeffrey T; Hayden, Patrick J; Prantil-Baun, Rachelle

    2015-04-01

    Respiratory viruses invade the upper airway of the lung, triggering a potent immune response that often exacerbates preexisting conditions such as asthma and COPD. Poly(I:C) is a synthetic analog of viral dsRNA that induces the characteristic inflammatory response associated with viral infection, such as loss of epithelial integrity, and increased production of mucus and inflammatory cytokines. Here, we explore the mechanistic responses to poly(I:C) in a well-defined primary normal human bronchial epithelial (NHBE) model that recapitulates in vivo functions and responses. We developed functional and quantifiable methods to evaluate the physiology of our model in both healthy and inflamed states. Through gene and protein expression, we validated the differentiation state and population of essential cell subtypes (i.e., ciliated, goblet, club, and basal cells) as compared to the human lung. Assays for total mucus production, cytokine secretion, and barrier function were used to evaluate in vitro physiology and response to viral insult. Cells were treated apically with poly(I:C) and evaluated 48 h after induction. Results revealed a dose-dependent increase in goblet cell differentiation, as well as, an increase in mucus production relative to controls. There was also a dose-dependent increase in secretion of IL-6, IL-8, TNF-α, and RANTES. Epithelial barrier function, as measured by TEER, was maintained at 1501 ± 355 Ω*cm² postdifferentiation, but dropped significantly when challenged with poly(I:C). This study provides first steps toward a well-characterized model with defined functional methods for understanding dsRNA stimulated inflammatory responses in a physiologically relevant manner.

  6. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.

    PubMed

    Luissint, Anny-Claude; Parkos, Charles A; Nusrat, Asma

    2016-10-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  7. Selective regulation of MAP kinases and Chemokine expression after ligation of ICAM-1 on human airway epithelial cells

    PubMed Central

    Krunkosky, Thomas M; Jarrett, Carla L

    2006-01-01

    Background Intercellular adhesion molecule 1 (ICAM-1) is an immunoglobulin-like cell adhesion molecule expressed on the surface of multiple cell types, including airway epithelial cells. It has been documented that cross-linking ICAM-1 on the surface of leukocytes results in changes in cellular function through outside-inside signaling; however, the effect of cross-linking ICAM-1 on the surface of airway epithelial cells is currently unknown. The objective of this study was to investigate whether or not cross-linking ICAM-1 on the surface of airway epithelial cells phosphorylated MAP kinases or stimulated chemokine expression and secretion. Methods The human lung adenocarcinoma (A549) cells and primary cultures of normal human bronchial epithelial (NHBE) cells were used in these studies. To increase ICAM-1 surface expression, cultures were stimulated with TNFα to enhance ICAM-1 surface expression. Following ICAM-1 upregulation, ICAM-1 was ligated with a murine anti-human ICAM-1 antibody and subsequently cross-linked with a secondary antibody (anti-mouse IgG(ab')2) in the presence or absence of the MAP kinase inhibitors. Following treatments, cultures were assessed for MAPK activation and chemokine gene expression and secretion. Control cultures were treated with murine IgG1 antibody or murine IgG1 antibody and anti-mouse IgG(ab')2 to illustrate specificity. Data were analyzed for significance using a one-way analysis of variance (ANOVA) with Bonferroni post-test correction for multiple comparisons, and relative gene expression was analyzed using the 2-ΔΔCT method. Results ICAM-1 cross-linking selectively phosphorylated both ERK and JNK MAP kinases as detected by western blot analysis. In addition, cross-linking resulted in differential regulation of chemokine expression. Specifically, IL-8 mRNA and protein secretion was not altered by ICAM-1 cross-linking, in contrast, RANTES mRNA and protein secretion was induced in both epithelial cultures. These events were

  8. Roflumilast combined with adenosine increases mucosal hydration in human airway epithelial cultures after cigarette smoke exposure

    PubMed Central

    Tyrrell, Jean; Qian, Xiaozhong; Freire, Jose

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality worldwide. Recent studies have shown that cigarette smoke (CS) induces cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, which leads to airway-surface liquid (ASL) dehydration. This in turn contributes to the mucus dehydration and impaired mucociliary clearance that are seen in the chronic bronchitis form of COPD. Roflumilast is a phosphodiesterase 4 inhibitor that may improve lung function and reduce the frequency of exacerbations in patients with COPD. Although roflumilast can affect cAMP metabolism, little is known about the downstream pharmacological effects in the airways. We hypothesized that roflumilast would increase ASL rehydration in human bronchial epithelial cultures (HBECs) after chronic CS exposure. cAMP production was measured by Förster resonance energy transfer in HEK293T cells and by ELISA in HBECs. ASL height was measured by xz-confocal microscopy after air exposure or following HBEC exposure to freshly produced CS. Roflumilast had little effect on cAMP or ASL height when applied on its own; however, roflumilast significantly potentiated adenosine-induced increases in cAMP and ASL height in CS-exposed HBECs. Roflumilast increased the rate of ASL height recovery in cultures after CS exposure compared with controls. In contrast, the β2-adrenergic receptor agonists isoproterenol and salmeterol failed to increase ASL height after CS exposure. Our data suggest that roflumilast can increase ASL hydration in CS-exposed HBECs, which is predicted to be beneficial for the treatment of mucus dehydration/mucus stasis in patients with COPD chronic bronchitis. PMID:25795727

  9. REGULATION OF CYTOKINE PRODUCTION IN HUMAN ALVEOLAR MACHROPHAGES AND AIRWAY EPITHELIAL CELLS IN RESPONSE TO AMBIENT AIR POLLUTION PARTICLES: FURTHER MECHANISTIC STUDIES

    EPA Science Inventory

    In order to better understand how ambient air particulate matter (PM) affect lung health, the two main airway cell types likely to interact with inhaled particles, alveolar macrophages (AM) and airway epithelial cells have been exposed to particles in vitro and followed for endp...

  10. Human Airway Epithelial Cell Responses to Single Walled Carbon Nanotube Exposure: Nanorope-Residual Body Formation

    SciTech Connect

    Panessa-Warren, Barbara J.; Warren, John B.; Kisslinger, Kim; Crosson, Kenya; Maye, Mathew M.

    2012-11-01

    This investigation examines the 'first contact responses' of in vitro human epithelial airway cells exposed to unrefined single walled carbon nanotubes (SWCNTs) [containing metal catalyst, carbon black, amorphous carbon, graphitic shells, and SWCNTs], and refined acid/peroxide cleaned and cut SWCNTs at low and high dose exposures (0.16 ug/L and 1.60 ug/L) for 2, 3 and 3.5 hours. FTIR, X-ray compositional analysis, morphological TEM analysis and UV-Vis were used to physicochemically characterize the SWCNTs in this study. Following SWCNT exposure to human lung NCI-H292 epithelial monolayers, the airway cells were prepared for light microscopy vital staining, or fixed in glutaraldehyde for SEM/TEM imaging to determine SWCNT binding, uptake, intracellular processing and organellar/SWCNT fate within the exposure period. At 2 hr exposures to both unrefined Carbolex, and refined SWCNTs (at both high and low doses), there were no increases in lung cell necrosis compared to controls. However high dose, 3 hr exposures to unrefined Carbolex material produced severe cell damage (apical and basal plasma membrane holes, decreased mitochondria, numerous intracellular vesicles containing nanomaterial and membrane fragments) and increased cell necrosis. The refined SWCNTs exposed for 3 hr at low dose produced no increase in cell death, although high dose exposure produced significant cell death. By TEM, Acid/peroxide cleaned SWCNT 3 hr exposures at high and low doses, revealed SWCNTs attachment to cell surface mucin, and SWCNT uptake into the cells during membrane recycling. Membranes and SWCNTs were seen within cytoplasmic lamellar body-type vesicles, where vesicular contents were bio-degraded, eventually forming long SWCNT-nanoropes, which were subsequently released into the cytoplasm as clusters of attached nanoropes, as the vesicle membranes fragmented. These Nanorope-Residual Bodies did not cause damage to the surrounding organelles or cytoplasm, and seemed very stabile in the

  11. Cystic fibrosis transmembrane conductance regulator trafficking modulates the barrier function of airway epithelial cell monolayers.

    PubMed

    LeSimple, Pierre; Liao, Jie; Robert, Renaud; Gruenert, Dieter C; Hanrahan, John W

    2010-04-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane glycoprotein which functions as an anion channel and influences diverse cellular processes. We studied its role in the development of epithelial tightness by expressing wild-type (WT-CFTR) or mutant (Delta F508-CFTR) CFTR in human airway epithelial cell monolayers cultured at the air-liquid interface. Green fluorescent protein (GFP)-tagged WT or Delta F508 constructs were expressed in the CF bronchial cell line CFBE41o(-) using adenoviruses, and the results were compared with those obtained using CFBE41o(-) lines stably complemented with wild-type or mutant CFTR. As predicted, GFP-Delta WT-CFTR reached the apical membrane whereas GFP-F508-CFTR was only detected intracellularly. Although CFTR expression would be expected to reduce transepithelial resistance (TER), expressing GFP-CFTR significantly increased the TER of CFBE41o(-) monolayers whilst GFP-Delta F508-CFTR had no effect. Similar results were obtained with cell lines stably overexpressing Delta F508-CFTR or WT-CFTR. Preincubating Delta F508-CFTR monolayers at 29 degrees C reduced mannitol permeability and restored TER, and the effect on TER was reversible during temperature oscillations. Expression of GFP-Delta F508-CFTR or GFP-WT-CFTR in a cell line already containing endogenous WT-CFTR (Calu-3) did not alter TER. The CFTR- and temperature-dependence of TER were not affected by the CFTR inhibitor CFTR(inh)172 or low-chloride medium; therefore the effect of CFTR on barrier function was unrelated to its ion channel activity. Modulation of TER was blunted but not eliminated by genistein, implying the involvement of tyrosine phosphorylation and other mechanisms. Modulation of CFTR trafficking was correlated with an increase in tight junction depth. The results suggest that CFTR trafficking is required for the normal organisation and function of tight junctions. A reduction in barrier function caused by endoplasmic reticulum

  12. Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys

    PubMed Central

    Ballinger, Carol A.; Plopper, Charles G.; McDonald, Ruth J.; Bartolucci, Alfred A.; Postlethwait, Edward M.; Harkema, Jack R.

    2011-01-01

    Children chronically exposed to high levels of ozone (O3), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O3 [0.5 parts per million (ppm), 8 h/day; “1-cycle”] or filtered air (FA) or 11 biweekly cycles of O3 (FA days 1–9; 0.5 ppm, 8 h/day on days 10–14; “11-cycle”). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH2), and uric acid (UA) concentration. Eleven-cycle O3 induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O3 also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments. PMID:21131400

  13. Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles

    SciTech Connect

    Tal, Tamara L.; Simmons, Steven O.; Silbajoris, Robert; Dailey, Lisa; Cho, Seung-Hyun; Ramabhadran, Ram; Linak, William; Reed, William; Bromberg, Philip A.; Samet, James M.

    2010-02-15

    Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

  14. Arylhydrocarbon receptor (AhR) activation in airway epithelial cells induces MUC5AC via reactive oxygen species (ROS) production.

    PubMed

    Chiba, Takahito; Uchi, Hiroshi; Tsuji, Gaku; Gondo, Hisaki; Moroi, Yoichi; Furue, Masutaka

    2011-02-01

    The dioxins and dioxin-like compounds in cigarette smoke regulate various immunological responses via the arylhydrocarbon receptor (AhR). These environmental toxicants are known to cause bronchitis, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Recent studies have demonstrated that AhR activation upregulates the expression of mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) in the airway epithelial cell line. However, the mechanism for the production of mucin has not been clarified. In this study, we investigated the role and pathway of AhR in airway epithelial cells by using selective agonists and antagonists. After stimulation with or without benzopyrene (B[a]P), an AhR agonist, MUC5AC expression was measured by real-time RT-PCR. The mechanism of AhR-induced MUC5AC expression in airway epithelial cells was studied in terms of the production of cytokine and reactive oxygen species (ROS). Treatment with B[a]P increased ROS generation in NCI-H₂₉₂ cells. Furthermore, B[a]P-induced MUC5AC upregulation and mucin production were inhibited by AhR siRNA or the use of an antioxidative agent. These results suggest that the AhR-induced increase of mucin production is partially mediated by ROS generation. An antioxidant therapy approach may help to cure AhR-induced mucus hypersecretory diseases. PMID:20709182

  15. Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia.

    PubMed

    Ribeiro, Carla M Pedrosa; Paradiso, Anthony M; Carew, Mark A; Shears, Stephen B; Boucher, Richard C

    2005-03-18

    In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether

  16. Innate Immune Responses after Airway Epithelial Stimulation with Mycobacterium bovis Bacille-Calmette Guérin

    PubMed Central

    Tenland, Erik; Håkansson, Gisela; Alaridah, Nader; Lutay, Nataliya; Rönnholm, Anna; Hallgren, Oskar; Westergren-Thorsson, Gunilla; Godaly, Gabriela

    2016-01-01

    Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung. PMID:27723804

  17. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

    Plikus, Maksim V.; Gay, Denise L.; Treffeisen, Elsa; Wang, Anne; Supapannachart, Rarinthip June; Cotsarelis, George

    2012-01-01

    Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis hasa mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new inter-follicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover cellular and signaling basis of this remarkable adult wound regeneration phenomenon. PMID:23085626

  18. Primary Paediatric Bronchial Airway Epithelial Cell in Vitro Responses to Environmental Exposures

    PubMed Central

    McInnes, Neil; Davidson, Matthew; Scaife, Alison; Miller, David; Spiteri, Daniella; Engelhardt, Tom; Semple, Sean; Devereux, Graham; Walsh, Garry; Turner, Steve

    2016-01-01

    The bronchial airway epithelial cell (BAEC) is the site for initial encounters between inhaled environmental factors and the lower respiratory system. Our hypothesis was that release of pro inflammatory interleukins (IL)-6 and IL-8 from primary BAEC cultured from children will be increased after in vitro exposure to common environmental factors. Primary BAEC were obtained from children undergoing clinically indicated routine general anaesthetic procedures. Cells were exposed to three different concentrations of lipopolysaccharide (LPS) or house dust mite allergen (HDM) or particulates extracted from side stream cigarette smoke (SSCS). BAEC were obtained from 24 children (mean age 7.0 years) and exposed to stimuli. Compared with the negative control, there was an increase in IL-6 and IL-8 release after exposure to HDM (p ≤ 0.001 for both comparisons). There was reduced IL-6 after higher compared to lower SSCS exposure (p = 0.023). There was no change in BAEC release of IL-6 or IL-8 after LPS exposure. BAEC from children are able to recognise and respond in vitro with enhanced pro inflammatory mediator secretion to some inhaled exposures. PMID:27023576

  19. Effect of guaifenesin on mucin production, rheology, and mucociliary transport in differentiated human airway epithelial cells.

    PubMed

    Seagrave, JeanClare; Albrecht, Helmut; Park, Yong Sung; Rubin, Bruce; Solomon, Gail; Kim, K Chul

    2011-12-01

    Guaifenesin is widely used to alleviate symptoms of excessive mucus accumulation in the respiratory tract. However, its mechanism of action is poorly understood. The authors hypothesized that guaifenesin improves mucociliary clearance in humans by reducing mucin release, by decreasing mucus viscoelasticity, and by increasing mucociliary transport. To test these hypotheses, human differentiated airway epithelial cells, cultured at an air-liquid interface, were treated with clinically relevant concentrations of guaifenesin by addition to the basolateral medium. To evaluate the effect on mucin secretion, the authors used an anzyme-linked immunosorbent assay (ELISA) to measure the amounts of MUC5AC protein in apical surface fluid and cell lysates. To measure mucociliary transportability, additional cultures were treated for 1 or 6 hours with guaifenesin, and the movement of cell debris was measured from video data. Further, the authors measured mucus dynamic viscoelasticity using a micro cone and plate rheometer with nondestructive creep transformation. Guaifenesin suppressed mucin production in a dose-dependent manner at clinically relevant concentrations. The reduced mucin production was associated with increased mucociliary transport and decreased viscoelasticity of the mucus. Viability of the cultures was not significantly affected. These results suggest that guaifenesin could improve mucociliary clearance in humans by reducing the release and/or production of mucins, thereby altering mucus rheology. PMID:22044398

  20. Intracellular calcium mobilization and phospholipid degradation in sphingosylphosphorylcholine-stimulated human airway epithelial cells.

    PubMed Central

    Orlati, S; Porcelli, A M; Hrelia, S; Lorenzini, A; Rugolo, M

    1998-01-01

    Extracellular sphingosylphosphorylcholine (SPC) caused a remarkable elevation in the intracellular Ca2+ concentration ([Ca2+]i) in immortalized human airway epithelial cells (CFNP9o-). An increase in total inositol phosphates formation was determined; however, the dose responses for [Ca2+]i elevation and inositol phosphates production were slightly different and, furthermore, PMA and pertussis toxin almost completely inhibited [Ca2+]i mobilization by SPC, whereas inositol phosphates production was only partially reduced. The possible direct interaction of SPC with Ca2+ channels of intracellular stores was determined by experiments with permeabilized cells, where SPC failed to evoke Ca2+ release, whereas lysophosphatidic acid was shown to be effective. The level of phosphatidic acid was increased by SPC only in the presence of AACOCF3, a specific inhibitor of phospholipase A2 (PLA2) and blocked by both pertussis toxin and R59022, an inhibitor of diacylglycerol kinase. R59022 enhanced diacylglycerol production by SPC and also significantly reduced [Ca2+]i mobilization. Only polyunsaturated diacylglycerol and phosphatidic acid were generated by SPC. Lastly, SPC caused stimulation of arachidonic acid release, indicating the involvement of PLA2. Taken together, these data suggest that, after SPC stimulation, phospholipase C-derived diacylglycerol is phosphorylated by a diacylglycerol kinase to phosphatidic acid, which is further hydrolysed by PLA2 activity to arachidonic and lysophosphatidic acids. We propose that lysophosphatidic acid might be the intracellular messenger able to release Ca2+ from internal stores. PMID:9729473

  1. Establishment and transformation of telomerase-immortalized human small airway epithelial cells by heavy ions

    NASA Astrophysics Data System (ADS)

    Zhao, Y. L.; Piao, C. Q.; Hei, T. K.

    Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of 1 GeV/nucleon of 56Fe ions accelerated at the Brookhaven National Laboratory. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

  2. Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

    SciTech Connect

    Ulanova, Marina . E-mail: marina.ulanova@normed.ca; Marcet-Palacios, Marcelo; Munoz, Samira; Asfaha, Samuel; Kim, Moo-Kyung; Schreiber, Alan D.; Befus, A. Dean

    2006-12-15

    We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in {beta}1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via {beta}1 integrins, suggesting that TNF and {beta}1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-{kappa}B. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.

  3. Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3.

    PubMed

    Yamamoto, Keisuke; Yamamoto, Soh; Ogasawara, Noriko; Takano, Kenichi; Shiraishi, Tsukasa; Sato, Toyotaka; Miyata, Ryo; Kakuki, Takuya; Kamekura, Ryuta; Kojima, Takashi; Tsutsumi, Hiroyuki; Himi, Tetsuo; Yokota, Shin-Ichi

    2016-09-01

    Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-β and -λ production. Furthermore, IFN-β promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role. PMID:27468646

  4. Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Barchowsky, Aaron

    2009-01-01

    Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

  5. Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

    PubMed

    Hendrick, Siobhán M; Mroz, Magdalena S; Greene, Catherine M; Keely, Stephen J; Harvey, Brian J

    2014-09-01

    Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 μM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid.

  6. Identification of Nrf2-dependent airway epithelial adaptive response to proinflammatory oxidant-hypochlorous acid challenge by transcription profiling.

    PubMed

    Zhu, Lingxiang; Pi, Jingbo; Wachi, Shinichiro; Andersen, Melvin E; Wu, Reen; Chen, Yin

    2008-03-01

    In inflammatory diseases of the airway, a high level (estimated to be as high as 8 mM) of HOCl can be generated through a reaction catalyzed by the leukocyte granule enzyme myeloperoxidase (MPO). HOCl, a potent oxidative agent, causes extensive tissue injury through its reaction with various cellular substances, including thiols, nucleotides, and amines. In addition to its physiological source, HOCl can also be generated by chlorine gas inhalation from an accident or a potential terrorist attack. Despite the important role of HOCl-induced airway epithelial injury, the underlying molecular mechanism is largely unknown. In the present study, we found that HOCl induced dose-dependent toxicity in airway epithelial cells. By transcription profiling using GeneChip, we identified a battery of HOCl-inducible antioxidant genes, all of which have been reported previously to be regulated by nuclear factor erythroid-related factor 2 (Nrf2), a transcription factor that is critical to the lung antioxidant response. Consistent with this finding, Nrf2 was found to be activated time and dose dependently by HOCl. Although the epidermal growth factor receptor-MAPK pathway was also highly activated by HOCl, it was not involved in Nrf2 activation and Nrf2-dependent gene expression. Instead, HOCl-induced cellular oxidative stress appeared to lead directly to Nrf2 activation. To further understand the functional significance of Nrf2 activation, small interference RNA was used to knock down Nrf2 level by targeting Nrf2 or enhance nuclear accumulation of Nrf2 by targeting its endogenous inhibitor Keap1. By both methods, we conclude that Nrf2 directly protects airway epithelial cells from HOCl-induced toxicity.

  7. Effects of air pollution-related heavy metals on the viability and inflammatory responses of human airway epithelial cells.

    PubMed

    Honda, Akiko; Tsuji, Kenshi; Matsuda, Yugo; Hayashi, Tomohiro; Fukushima, Wataru; Sawahara, Takahiro; Kudo, Hitomi; Murayama, Rumiko; Takano, Hirohisa

    2015-01-01

    Various metals produced from human activity are ubiquitously detected in ambient air. The metals may lead to induction and/or exacerbation of respiratory diseases, but the significant metals and factors contributing to such diseases have not been identified. To compare the effects of each metal and different oxidation states of metals on human airway, we examined the viability and production of interleukin (IL)-6 and IL-8 using BEAS-2B cell line, derived from human airway epithelial cells. Airway epithelial cells were exposed to Mn(2+), V(4+), V(5+), Cr(3+), Cr(6+), Zn(2+), Ni(2+), and Pb(2+) at a concentration of 0.5, 5, 50, or 500 μmol/L for 24 hours. Mn and V decreased the cell viability in a concentration-dependent manner, and V(5+) tended to have a greater effect than V(4+). The Cr decreased the cell viability, and (Cr(+6)) at concentrations of 50 and 500 μmol/L was more toxic than (Cr(+3)). Zn at a concentration of 500 μmol/L greatly decreased the cell viability, whereas Ni at the same concentration increased it. Pb produced fewer changes. Mn and Ni at a concentration of 500 μmol/L induced the significant production of IL-6 and IL-8. However, most of the metals including (V(+4), V(+5)), (Cr(+3), Cr(+6)), Zn, and Pb inhibited the production of both IL-6 and IL-8. The present results indicate that various heavy metals have different effects on toxicity and the proinflammatory responses of airway epithelial cells, and those influences also depend on the oxidation states of the metals.

  8. Linking polymorphic p53 response elements with gene expression in airway epithelial cells of smokers and cancer risk.

    PubMed

    Wang, Xuting; Pittman, Gary S; Bandele, Omari J; Bischof, Jason J; Liu, Gang; Brothers, John F; Spira, Avrum; Bell, Douglas A

    2014-12-01

    Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA-damaging agents, creating a field of molecular injury in the airway and altering gene expression. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression, and this suggests that single-nucleotide polymorphisms (SNPs) in functional p53 binding sites (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with gene expression and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis expression quantitative trait loci (eQTL) analysis, assessing associations between SNP genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further test and validate these genotype-expression associations, we searched published eQTL studies from independent populations and determined that 53% (39/74) of the bronchial epithelial eQTLs were observed in at least one of other studies. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein-DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies or candidate gene studies. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers.

  9. Rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells

    PubMed Central

    Chattoraj, Sangbrita S.; Ganesan, Shyamala; Jones, Andrew M.; Helm, Jennifer M; Comstock, Adam T; Bright-Thomas, Rowland; LiPuma, John J.; Hershenson, Marc B.; Sajjan, Umadevi S.

    2011-01-01

    Background Intermittent viral exacerbations in cystic fibrosis (CF) patients with chronic P. aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are associated with majority of viral-related exacerbations in CF and required prolonged intravenous antibiotic treatment. These observations imply that acute RV infection may increase lower respiratory symptoms by increasing planktonic bacterial load. However, the underlying mechanisms are not known. Methods Primary CF airway epithelial cells differentiated into mucociliary phenotype were infected with mucoid PA (MPA) followed by RV and examined for bacterial density, biofilm mass, levels of chemokines and hydrogen peroxide (H2O2). Requirement of dual oxidase 2 in RV-induced generation of H2O2 in CF cells was assessed by using gene-specific siRNA. Results Super infection with RV increased chemokine responses in CF mucociliary-differentiated airway epithelial cells with pre-existing MPA infection in the form of biofilm. This was associated with the presence of planktonic bacteria at both the apical and basolateral epithelial cell surfaces. Further, RV-induced generation of H2O2 via dual oxidase 2, a component of NADPH oxidase in CF cells was sufficient for dispersal of planktonic bacteria from biofilm. Inhibition of NADPH oxidase reduced bacterial transmigration across mucociliary-differentiated CF cells and IL-8 response in MPA and RV-infected cells. Conclusion We show that acute infection with RV liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterpart stimulates increased chemokine responses in CF airway epithelial cells, which in turn may contribute to pathogenesis of CF exacerbations. PMID:21289024

  10. Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways

    PubMed Central

    Deng, Xuefeng; Yan, Ziying; Cheng, Fang; Engelhardt, John F.; Qiu, Jianming

    2016-01-01

    Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase–related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways. PMID:26765330

  11. Systematic Analysis of Multiwalled Carbon Nanotube-Induced Cellular Signaling and Gene Expression in Human Small Airway Epithelial Cells

    PubMed Central

    Snyder-Talkington, Brandi N.

    2013-01-01

    Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment. PMID:23377615

  12. Genetic Variation in Genes Encoding Airway Epithelial Potassium Channels Is Associated with Chronic Rhinosinusitis in a Pediatric Population

    PubMed Central

    Mentch, Frank; Grant, Struan F. A.; Desrosiers, Martin; Hakonarson, Hakon; Toskala, Elina

    2014-01-01

    Background Apical potassium channels regulate ion transport in airway epithelial cells and influence air surface liquid (ASL) hydration and mucociliary clearance (MCC). We sought to identify whether genetic variation within genes encoding airway potassium channels is associated with chronic rhinosinusitis (CRS). Methods Single nucleotide polymorphism (SNP) genotypes for selected potassium channels were derived from data generated on the Illumnia HumanHap550 BeadChip or Illumina Human610-Quad BeadChip for 828 unrelated individuals diagnosed with CRS and 5,083 unrelated healthy controls from the Children's Hospital of Philadelphia (CHOP). Statistical analysis was performed with set-based tests using PLINK, and corrected for multiple testing. Results Set-based case control analysis revealed the gene KCNMA1 was associated with CRS in our Caucasian subset of the cohort (598 CRS cases and 3,489 controls; p = 0.022, based on 10,000 permutations). In addition there was borderline evidence that the gene KCNQ5 (p = 0.0704) was associated with the trait in our African American subset of the cohort (230 CRS cases and 1,594 controls). In addition to the top significant SNPs rs2917454 and rs6907229, imputation analysis uncovered additional genetic variants in KCNMA1 and in KCNQ5 that were associated with CRS. Conclusions We have implicated two airway epithelial potassium channels as novel susceptibility loci in contributing to the pathogenesis of CRS. PMID:24595210

  13. Immunohistochemical characteristics of epithelial cell rests of Malassez during cementum repair.

    PubMed

    Hasegawa, Naohiko; Kawaguchi, Hiroyuki; Ogawa, Tetsuji; Uchida, Takashi; Kurihara, Hidemi

    2003-02-01

    To clarify the roles of epithelial cell rests of Malassez (ECRM) during periodontal repair, experimental root resorption was induced in rats and then the ECRM that existed in periodontal ligament during cementum repair was investigated using morphological and immunohistochemical approaches. At day 7, after mechanical injury, root resorption was observed and ECRM were present adjacent to the site of resorption lacunae. They were observed in periodontal ligament adjacent to site of the resorption lacunae. These ECRM were immunoreactive for bone morphogenetic protein-2. During the stage of early cementum repair, the ECRM were immunoreactive for osteopontin and ameloblastin. They strongly reacted to proliferating cell nuclear antigen. In uninjured control sections, ECRM located in the periodontal ligament adjacent to cementum were not immunoreactive for any antibodies. These findings suggested that ECRM may be related to cementum repair by activating their potential to secrete matrix proteins which have been expressed in tooth development. PMID:12558937

  14. Epithelial expression of profibrotic mediators in a model of allergen-induced airway remodeling.

    PubMed

    Kelly, Margaret M; Leigh, Richard; Bonniaud, Philippe; Ellis, Russ; Wattie, Jennifer; Smith, Mary Jo; Martin, Gail; Panju, Mohammed; Inman, Mark D; Gauldie, Jack

    2005-02-01

    Airway remodeling, including subepithelial fibrosis, is a characteristic feature of asthma and likely contributes to the pathogenesis of airway hyperresponsiveness. We examined expression of genes related to airway wall fibrosis in a model of chronic allergen-induced airway dysfunction using laser capture microdissection and quantitative real-time PCR. BALB/c mice were sensitized and subjected to chronic ovalbumin exposure over a 12-wk period, after which they were rested and then harvested 2 and 8 wk after the last exposure. Chronic allergen-exposed mice had significantly increased indices of airway remodeling and airway hyperreactivity at all time points, although no difference in expression of fibrosis-related genes was found when mRNA extracted from whole lung was examined. In contrast, fibrosis-related gene expression was significantly upregulated in mRNA obtained from microdissected bronchial wall at 2 wk after chronic allergen exposure. In addition, when bronchial wall epithelium and smooth muscle were separately microdissected, gene expression of transforming growth factor-beta1 and plasminogen activating inhibitor-1 were significantly upregulated only in the airway epithelium. These data suggest that transforming growth factor-beta1 and other profibrotic mediators produced by airway wall, and specifically, airway epithelium, play an important role in the pathophysiology of airway remodeling.

  15. Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1.

    PubMed

    Martino, Mary E B; Olsen, John C; Fulcher, Nanette B; Wolfgang, Matthew C; O'Neal, Wanda K; Ribeiro, Carla M P

    2009-05-29

    Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses. PMID:19321437

  16. Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways.

    PubMed

    Gianotti, Ambra; Ferrera, Loretta; Philp, Amber R; Caci, Emanuela; Zegarra-Moran, Olga; Galietta, Luis J V; Flores, Carlos A

    2016-06-15

    Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice. PMID:27063443

  17. Human metapneumovirus infection induces significant changes in small noncoding RNA expression in airway epithelial cells.

    PubMed

    Deng, Junfang; Ptashkin, Ryan N; Wang, Qingrong; Liu, Guangliang; Zhang, Guanping; Lee, Inhan; Lee, Yong Sun; Bao, Xiaoyong

    2014-05-20

    Small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), virus-derived sncRNAs, and more recently identified tRNA-derived RNA fragments, are critical to posttranscriptional control of genes. Upon viral infection, host cells alter their sncRNA expression as a defense mechanism, while viruses can circumvent host defenses and promote their own propagation by affecting host cellular sncRNA expression or by expressing viral sncRNAs. Therefore, characterizing sncRNA profiles in response to viral infection is an important tool for understanding host-virus interaction, and for antiviral strategy development. Human metapneumovirus (hMPV), a recently identified pathogen, is a major cause of lower respiratory tract infections in infants and children. To investigate whether sncRNAs play a role in hMPV infection, we analyzed the changes in sncRNA profiles of airway epithelial cells in response to hMPV infection using ultrahigh-throughput sequencing. Of the cloned sncRNAs, miRNA was dominant in A549 cells, with the percentage of miRNA increasing in a time-dependent manner after the infection. In addition, several hMPV-derived sncRNAs and corresponding ribonucleases for their biogenesis were identified. hMPV M2-2 protein was revealed to be a key viral protein regulating miRNA expression. In summary, this study revealed several novel aspects of hMPV-mediated sncRNA expression, providing a new perspective on hMPV-host interactions.

  18. Nickel Mobilizes Intracellular Zinc to Induce Metallothionein in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Leikauf, George D.; Pitt, Bruce R.; Wasserloos, Karla J.; Barchowsky, Aaron

    2009-01-01

    We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release. PMID:19097988

  19. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation.

  20. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. PMID:25883089

  1. Inhibition of endotoxin-induced airway epithelial cell injury by a novel family of pyrrol derivates.

    PubMed

    Cabrera-Benítez, Nuria E; Pérez-Roth, Eduardo; Ramos-Nuez, Ángela; Sologuren, Ithaisa; Padrón, José M; Slutsky, Arthur S; Villar, Jesús

    2016-06-01

    Inflammation and apoptosis are crucial mechanisms for the development of the acute respiratory distress syndrome (ARDS). Currently, there is no specific pharmacological therapy for ARDS. We have evaluated the ability of a new family of 1,2,3,5-tetrasubstituted pyrrol compounds for attenuating lipopolysaccharide (LPS)-induced inflammation and apoptosis in an in vitro LPS-induced airway epithelial cell injury model based on the first steps of the development of sepsis-induced ARDS. Human alveolar A549 and human bronchial BEAS-2B cells were exposed to LPS, either alone or in combination with the pyrrol derivatives. Rhein and emodin, two representative compounds with proven activity against the effects of LPS, were used as reference compounds. The pyrrol compound that was termed DTA0118 had the strongest inhibitory activity and was selected as the lead compound to further explore its properties. Exposure to LPS caused an intense inflammatory response and apoptosis in both A549 and BEAS-2B cells. DTA0118 treatment downregulated Toll-like receptor-4 expression and upregulated nuclear factor-κB inhibitor-α expression in cells exposed to LPS. These anti-inflammatory effects were accompanied by a significantly lower secretion of interleukin-6 (IL-6), IL-8, and IL-1β. The observed antiapoptotic effect of DTA0118 was associated with the upregulation of antiapoptotic Bcl-2 and downregulation of proapoptotic Bax and active caspase-3 protein levels. Our findings demonstrate the potent anti-inflammatory and antiapoptotic properties of the pyrrol DTA0118 compound and suggest that it could be considered as a potential drug therapy for the acute phase of sepsis and septic ARDS. Further investigations are needed to examine and validate these mechanisms and effects in a clinically relevant animal model of sepsis and sepsis-induced ARDS. PMID:26999659

  2. An NF-κB-independent and Erk1/2-dependent mechanism controls CXCL8/IL-8 responses of airway epithelial cells to cadmium.

    PubMed

    Cormet-Boyaka, Estelle; Jolivette, Kalyn; Bonnegarde-Bernard, Astrid; Rennolds, Jessica; Hassan, Fatemat; Mehta, Payal; Tridandapani, Susheela; Webster-Marketon, Jeanette; Boyaka, Prosper N

    2012-02-01

    Airway epithelial cells in the lung are the first line of defense against pathogens and environmental pollutants. Inhalation of the environmental pollutant cadmium has been linked to the development of lung cancer and chronic obstructive pulmonary disease, which are diseases characterized by chronic inflammation. To address the role of airway epithelial cells in cadmium-induced lung inflammation, we investigated how cadmium regulates secretion of interleukin 8 (IL-8) by airway epithelial cells. We show that exposure of human airway epithelial cells to subtoxic doses of cadmium in vitro promotes a characteristic inflammatory cytokine response consisting of IL-8, but not IL-1β or tumor necrosis factor-alpha. We also found that intranasal delivery of cadmium increases lung levels of the murine IL-8 homologs macrophage inflammatory protein-2 and keracinocyte-derived chemokine and results in an influx of Gr1+ cells into the lung. We determined that inhibition of the nuclear factor-κB (NF-κB) pathway had no effect on cadmium-induced IL-8 secretion by human airway epithelial cells, suggesting that IL-8 production was mediated through an NF-κB-independent pathway. Mitogen-activated protein kinases (MAPKs) are often involved in proinflammatory signaling. Cadmium could activate the main MAPKs (i.e., p38, JNK, and Erk1/2) in human airway epithelial cells. However, only pharmacological inhibition of Erk1/2 pathway or knockdown of the expression of Erk1 and Erk2 using small interfering RNAs suppressed secretion of IL-8 induced by cadmium. Our findings identify cadmium as a potent activator of the proinflammatory cytokine IL-8 in lung epithelial cells and reveal for the first time the role of an NF-κB-independent but Erk1/2-dependent pathway in cadmium-induced lung inflammation. PMID:22094458

  3. Responses of well-differentiated nasal epithelial cells exposed to particles: Role of the epithelium in airway inflammation

    SciTech Connect

    Auger, Floriane; Gendron, Marie-Claude; Chamot, Christophe; Marano, Francelyne; Dazy, Anne-Catherine . E-mail: dazy@paris7.jussieu.fr

    2006-09-15

    Numerous epidemiological studies support the contention that ambient air pollution particles can adversely affect human health. To explain the acute inflammatory process in airways exposed to particles, a number of in vitro studies have been performed on cells grown submerged on plastic and poorly differentiated, and on cell lines, the physiology of which is somewhat different from that of well-differentiated cells. In order to obtain results using a model system in which epithelial cells are similar to those of the human airway in vivo, apical membranes of well-differentiated human nasal epithelial (HNE) cells cultured in an air-liquid interface (ALI) were exposed for 24 h to diesel exhaust particles (DEP) and Paris urban air particles (PM{sub 2.5}). DEP and PM{sub 2.5} (10-80 {mu}g/cm{sup 2}) stimulated both IL-8 and amphiregulin (ligand of EGFR) secretion exclusively towards the basal compartment. In contrast, there was no IL-1{beta} secretion and only weak non-reproducible secretion of TNF-{alpha}. IL-6 and GM-CSF were consistently stimulated towards the apical compartment and only when cells were exposed to PM{sub 2.5}. ICAM-1 protein expression on cell surfaces remained low after particle exposure, although it increased after TNF-{alpha} treatment. Internalization of particles, which is believed to initiate oxidative stress and proinflammatory cytokine expression, was restricted to small nanoparticles ({<=} 40 nm). Production of reactive oxygen species (ROS) was detected, and DEP were more efficient than PM{sub 2.5}. Collectively, our results suggest that airway epithelial cells exposed to particles augment the local inflammatory response in the lung but cannot alone initiate a systemic inflammatory response.

  4. In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.

    PubMed

    Anderson, Gregory G; Moreau-Marquis, Sophie; Stanton, Bruce A; O'Toole, George A

    2008-04-01

    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.

  5. Role of mucus in the repair of gastric epithelial damage in the rat. Inhibition of epithelial recovery by mucolytic agents.

    PubMed

    Wallace, J L; Whittle, B J

    1986-09-01

    A role for mucus in providing a microenvironment over sites of gastric damage, which is conducive to reepithelialization, has been proposed. We tested this hypothesis by examining the effects of disruption of such mucus on the recovery of epithelial integrity after damage induced by 50% ethanol. Exposure of an ex vivo chambered gastric mucosa to topically applied 50% ethanol resulted in copious release of mucus, cellular debris, and plasma, which formed a continuous cap over the mucosal surface. Ethanol-induced gastric damage was accompanied by extensive surface epithelial cell damage and a marked decrease in transmucosal potential difference. During the 30 min after ethanol was removed from the chamber, the epithelium became reestablished and the potential difference gradually recovered to 94% of the level before ethanol treatment. However, if the mucolytic agents N-acetylcysteine (5%) or pepsin (0.5%) were added to the bathing solutions, the "mucoid cap" disintegrated and the recovery of potential difference was significantly retarded (recovering to only 51% and 52% of levels before ethanol treatment). Histologic evaluation confirmed that mucosae treated with either agent had significantly less (p less than 0.005) intact epithelium at the end of the experiment. Removal of the mucoid cap with forceps caused a similar inhibition of the repair of the epithelium and the recovery of potential difference. Both mechanical and chemical (N-acetylcysteine) disruption of the mucoid cap resulted in a significant increase in the mucosal leakage of albumin and hemoglobin, supporting previous histologic evidence that the mucoid cap traps blood components over the damaged mucosa. These studies support the hypothesis that mucus released in response to topical application of an irritant plays an important role in the repair of epithelial damage through the process of restitution.

  6. Baicalin Inhibits Lipopolysaccharide-Induced Inflammation Through Signaling NF-κB Pathway in HBE16 Airway Epithelial Cells.

    PubMed

    Dong, Shou-jin; Zhong, Yun-qing; Lu, Wen-ting; Li, Guan-hong; Jiang, Hong-li; Mao, Bing

    2015-08-01

    Baicalin, a flavonoid monomer derived from Scutellaria baicalensis called Huangqin in mandarin, is the main active ingredient contributing to S. baicalensis' efficacy. It is known in China that baicalin has potential therapeutic effects on inflammatory diseases. However, its anti-inflammatory mechanism has still not been fully interpreted. We aim to investigate the anti-inflammatory effect of baicalin on lipopolysaccharide (LPS)-induced inflammation in HBE16 airway epithelial cells and also to explore the underlying signaling mechanisms. The anti-inflammatory action of baicalin was evaluated in human airway epithelial cells HBE16 treated with LPS. Airway epithelial cells HBE16 were pretreated with a range of concentrations of baicalin for 30 min and then stimulated with 10 μg/ml LPS. The secretions of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) in cell culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expressions of IL-6, IL-8, and TNF-α were tested by quantitative real-time polymerase chain reaction (real-time RT-PCR). Furthermore, Western blotting was used to determine whether the signaling pathway NF-κB was involved in the anti-inflammatory action of baicalin. The inflammatory cell model was successfully built with 10 μg/ml LPS for 24 h in our in vitro experiments. Both the secretions and the mRNA expressions of IL-6, IL-8, and TNF-α were significantly inhibited by baicalin. Moreover, the expression levels of phospho-IKKα/β and phospho-NF-κB p65 were downregulated, and the phospho-IκB-α level was upregulated by baicalin. These findings suggest that the anti-inflammatory properties of baicalin may be resulted from the inhibition of IL-6, IL-8, and TNF-α expression via preventing signaling NF-κB pathway in HBE16 airway epithelial cells. In addition, this study provides evidence to understand the therapeutic effects of baicalin on inflammatory diseases in

  7. Airway epithelial NF-kappaB activation modulates asbestos-induced inflammation and mucin production in vivo.

    PubMed

    Haegens, Astrid; Barrett, Trisha F; Gell, Joanna; Shukla, Arti; Macpherson, Maximilian; Vacek, Pamela; Poynter, Matthew E; Butnor, Kelly J; Janssen-Heininger, Yvonne M; Steele, Chad; Mossman, Brooke T

    2007-02-01

    To investigate the role of bronchiolar epithelial NF-kappaB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IkappaBalpha mutant (IkappaBalphasr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IkappaBalphasr Tg(+) and Tg(-) mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1beta were increased (p < or = 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p < or = 0.05) in Tg(+) vs Tg(-) mice. Asbestos also caused increases in IL-4, MIP-1beta, and MCP-1 in BALF that were more elevated (p < or = 0.05) in Tg(+) mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p < or = 0.05) in Tg(+) mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p < or = 0.05) in Tg(+) vs Tg(-) mice. Our findings demonstrate that airway epithelial NF-kappaB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.

  8. NOD-Like Receptors in Intestinal Homeostasis and Epithelial Tissue Repair

    PubMed Central

    Parlato, Marianna; Yeretssian, Garabet

    2014-01-01

    The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease. PMID:24886810

  9. The Effects of Gas Humidification with High-Flow Nasal Cannula on Cultured Human Airway Epithelial Cells

    PubMed Central

    Chidekel, Aaron; Zhu, Yan; Wang, Jordan; Mosko, John J.; Rodriguez, Elena; Shaffer, Thomas H.

    2012-01-01

    Humidification of inspired gas is important for patients receiving respiratory support. High-flow nasal cannula (HFNC) effectively provides temperature and humidity-controlled gas to the airway. We hypothesized that various levels of gas humidification would have differential effects on airway epithelial monolayers. Calu-3 monolayers were placed in environmental chambers at 37°C with relative humidity (RH) < 20% (dry), 69% (noninterventional comparator), and >90% (HFNC) for 4 and 8 hours with 10 L/min of room air. At 4 and 8 hours, cell viability and transepithelial resistance measurements were performed, apical surface fluid was collected and assayed for indices of cell inflammation and function, and cells were harvested for histology (n = 6/condition). Transepithelial resistance and cell viability decreased over time (P < 0.001) between HFNC and dry groups (P < 0.001). Total protein secretion increased at 8 hours in the dry group (P < 0.001). Secretion of interleukin (IL)-6 and IL-8 in the dry group was greater than the other groups at 8 hours (P < 0.001). Histological analysis showed increasing injury over time for the dry group. These data demonstrate that exposure to low humidity results in reduced epithelial cell function and increased inflammation. PMID:22988501

  10. Anti-Inflammatory Effects of Levalbuterol-Induced 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Airway Epithelial Cells

    PubMed Central

    Randall, Matthew J.; Kostin, Shannon F.; Burgess, Edward J.; Hoyt, Laura R.; Ather, Jennifer L.; Lundblad, Lennart K.; Poynter, Matthew E.

    2015-01-01

    Airway epithelial NF-κB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting β2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-κB activity. Since β2-agonists can induce expression of 11β-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-κB activation induced by the β-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC) with (R)-albuterol (levalbuterol), but not with (S)- or a mixture of (R + S)- (racemic) albuterol, augmented mRNA expression of 11β-HSD1. MTCC were stably transfected with luciferase (luc) reporter constructs under transcriptional regulation by NF-κB (NF-κB/luc) or glucocorticoid response element (GRE/luc) consensus motifs. Stimulation of NF-κB/luc MTCC with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) induced luc activity, which was inhibited by pretreatment with (R)-, but not (S)- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R)-, but not with (S)- or racemic albuterol, augmented 11-keto corticosteroid (cortisone) induced luc activity, which was diminished by the 11β-HSD inhibitor glycyrrhetinic acid (18β-GA), indicating that there was a conversion of inactive 11-keto to active 11-hydroxy corticosteroids. LPS- and TNFα-induced NF-κB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R)-albuterol, an effect that was inhibited by 18β-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R)-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine

  11. High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway

    PubMed Central

    HUANG, WUFENG; ZHAO, HAIJIN; DONG, HANGMING; WU, YUE; YAO, LIHONG; ZOU, FEI; CAI, SHAOXI

    2016-01-01

    Recent studies have indicated that high-mobility group box 1 protein (HMGB1) and the receptor for advanced glycation end-products (RAGE) contribute to the pathogenesis of asthma. However, whether the activation of the HMGB1/RAGE axis mediates airway epithelial barrier dysfunction remains unknown. Thus, the aim of this study was to examine the effects of HMGB1 and its synergistic action with interleukin (IL)-1β on airway epithelial barrier properties. We evaluated the effects of recombinant human HMGB1 alone or in combination with IL-1β on ionic and macromolecular barrier permeability, by culturing air-liquid interface 16HBE cells with HMGB1 to mimic the differentiated epithelium. Western blot analysis and immunofluorescence staining were utilized to examine the level and structure of major junction proteins, namely E-cadherin, β-catenin, occludin and claudin-1. Furthermore, we examined the effects of RAGE neutralizing antibodies and mitogen-activated protein kinase (MAPK) inhibitors on epithelial barrier properties in order to elucidate the mechanisms involved. HMGB1 increased FITC-dextran permeability, but suppressed epithelial resistance in a dose-and time-dependent manner. HMGB1-mediated barrier hyperpermeability was accompanied by a disruption of cell-cell contacts, the selective downregulation of occludin and claudin-1, and the redistribution of E-cadherin and β-catenin. HMGB1 in synergy with IL-1β induced a similar, but greater barrier hyperpermeability and induced the disruption of junction proteins. Furthermore, HMGB1 elicited the activation of the RAGE/extracellular signal-related kinase (ERK)1/2 signaling pathway, which correlated with barrier dysfunction in the 16HBE cells. Anti-RAGE antibody and the ERK1/2 inhibitor, U0126, attenuated the HMGB1-mediated changes in barrier permeability, restored the expression levels of occludin and claudin-1 and pevented the redistribution of E-cadherin and β-catenin. Taken together, the findings of our study

  12. Anti-inflammatory effects of long-chain n-3 PUFA in rhinovirus-infected cultured airway epithelial cells.

    PubMed

    Saedisomeolia, Ahmad; Wood, Lisa G; Garg, Manohar L; Gibson, Peter G; Wark, Peter A B

    2009-02-01

    Long-chain n-3 PUFA (LCn-3PUFA) including DHA and EPA, are known to decrease inflammation by inhibiting arachidonic acid (AA) metabolism to eicosanoids, decreasing the production of pro-inflammatory cytokines and reducing immune cell function. The aim of this study was to determine if EPA and DHA reduced the release of inflammatory mediators from airway epithelial cells infected with rhinovirus (RV). Airway epithelial cells (Calu-3) were incubated with EPA, DHA and AA for 24 h, followed by rhinovirus infection for 48 h. IL-6, IL-8 and interferon-gamma-induced protein-10 (IP-10) released by cells were measured using ELISA. Viral replication was measured by serial titration assays. The fatty acid content of cells was analysed using GC. Cellular viability was determined by visual inspection of cells and lactate dehydrogenase release. DHA (400 microm) resulted in a significant 16% reduction in IL-6 release after RV-43 infection, 29% reduction in IL-6 release after RV-1B infection, 28% reduction in IP-10 release after RV-43 infection and 23 % reduction in IP-10 release after RV-1B infection. Cellular DHA content negatively correlated with IL-6 and IP-10 release. None of the fatty acids significantly modified rhinovirus replication. DHA supplementation resulted in increased cellular content of DHA at the cost of AA, which may explain the decreased inflammatory response of cells. EPA and AA did not change the release of inflammatory biomarkers significantly. It is concluded that DHA has a potential role in suppressing RV-induced airway inflammation.

  13. Restoration of Chloride Efflux by Azithromycin in Airway Epithelial Cells of Cystic Fibrosis Patients▿

    PubMed Central

    Saint-Criq, Vinciane; Rebeyrol, Carine; Ruffin, Manon; Roque, Telma; Guillot, Loïc; Jacquot, Jacky; Clement, Annick; Tabary, Olivier

    2011-01-01

    Azithromycin (AZM) has shown promising anti-inflammatory properties in chronic obstructive pulmonary diseases, and clinical studies have presented an improvement in the respiratory condition of cystic fibrosis (CF) patients. The aim of this study was to investigate, in human airway cells, the mechanism by which AZM has beneficial effects in CF. We demonstrated that AZM did not have any anti-inflammatory effect on CF airway cells but restored Cl− efflux. PMID:21220528

  14. Gap junctional coupling is essential for epithelial repair in the avian cochlea.

    PubMed

    Jagger, Daniel J; Nickel, Regina; Forge, Andrew

    2014-11-26

    The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration.

  15. Gap junctional coupling is essential for epithelial repair in the avian cochlea.

    PubMed

    Jagger, Daniel J; Nickel, Regina; Forge, Andrew

    2014-11-26

    The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration. PMID:25429127

  16. Nicotine suppresses inflammatory factors in HBE16 airway epithelial cells after exposure to cigarette smoke extract and lipopolysaccharide.

    PubMed

    Li, Qi; Zhou, Xiangdong; Kolosov, Victor P; Perelman, Juliy M

    2010-12-01

    Cigarette smoke is a major cause of chronic inflammatory pulmonary disease, leading to inflammation, mucin (MUC) production, tissue damage, and remodeling. It is also well known that the major addictive component of cigarette smoke is nicotine. This study focused on the role of nicotine in the development of inflammatory pulmonary disease induced by cigarette smoke. HBE16 human airway epithelial cells were treated with serial dilutions of cigarette smoke chloroform extract (CE), lipopolysaccharide (LPS), and nicotine. The release of MUC5AC, tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-6 protein were assayed by enzyme-linked immunosorbent assay. The MUC5AC protein also was observed by immunofluorescence. The expression of MUC5AC, TNF-α, IL-8, and IL-6 mRNA were detected by real-time polymerase chain reaction. We found that the mRNA of the proinflammatory mediators TNF-α, IL-8, and IL-6, as well as MUC5AC was highly expressed after CE and LPS stimulation. Nicotine did not cause an excessive expression of TNF-α, IL-8, and IL-6, nor did it affect protein production from the MUC5AC gene. Nicotine not only failed to stimulate production of TNF-α, IL-8, and IL-6, but its presence was shown to suppress the activation resulting from exposure to CE and LPS (P < 0.05). Preincubation with nicotine also would reduce the level of MUC5AC protein in culture supernatants of CE- and LPS-treated cells. However, mRNA expression of MUC5AC showed no significant change in nicotine-treated cells when compared with normal control cells. This distinctive pattern implies that nicotine may have potential to suppress airway inflammation and maintain the mucus over retention in airway secretory cells to some extent, thus forming a balance between mucus hyperproduction and hypersecretion in airways exposed to smoking and LPS. PMID:21078494

  17. S-nitrosothiols regulate cell-surface pH buffering by airway epithelial cells during the human immune response to rhinovirus.

    PubMed

    Carraro, Silvia; Doherty, Joseph; Zaman, Khalequz; Gainov, Iain; Turner, Ronald; Vaughan, John; Hunt, John F; Márquez, Javier; Gaston, Benjamin

    2006-05-01

    Human rhinovirus infection is a common trigger for asthma exacerbations. Asthma exacerbations and rhinovirus infections are both associated with markedly decreased pH and ammonium levels in exhaled breath condensates. This observation is thought to be related, in part, to decreased activity of airway epithelial glutaminase. We studied whether direct rhinovirus infection and/or the host immune response to the infection decreased airway epithelial cell surface pH in vitro. Interferon-gamma and tumor necrosis factor-alpha, but not direct rhinovirus infection, decreased pH, an effect partly associated with decreased ammonium concentrations. This effect was 1) prevented by nitric oxide synthase inhibition; 2) independent of cyclic GMP; 3) associated with an increase in endogenous airway epithelial cell S-nitrosothiol concentration; 4) mimicked by the exogenous S-nitrosothiol, S-nitroso-N-acetyl cysteine; and 5) independent of glutaminase expression and activity. We then confirmed that decreased epithelial pH inhibits human rhinovirus replication in airway epithelial cells. These data suggest that a nitric oxide synthase-dependent host response to viral infection mediated by S-nitrosothiols, rather than direct infection itself, plays a role in decreased airway surface pH during human rhinovirus infection. This host immune response may serve to protect the lower airways from direct infection in the normal host. In patients with asthma, however, this fall in pH could be associated with the increased mucus production, augmented inflammatory cell degranulation, bronchoconstriction, and cough characteristic of an asthma exacerbation. PMID:16603595

  18. Vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells

    PubMed Central

    Rogel, Micah R.; Soni, Pritin N.; Troken, James R.; Sitikov, Albert; Trejo, Humberto E.; Ridge, Karen M.

    2011-01-01

    The physiological and pathophysiological implications of the expression of vimentin, a type III intermediate filament protein, in alveolar epithelial cells (AECs) are unknown. We provide data demonstrating that vimentin is regulated by TGFβ1, a major cytokine released in response to acute lung injury and that vimentin is required for wound repair and remodeling of the alveolar epithelium. Quantitative real-time PCR shows a 16-fold induction of vimentin mRNA in TGFβ1-treated transformed AECs. Luciferase assays identify a Smad-binding element in the 5′ promoter of vimentin responsible for TGFβ1-induced transcription. Notably, TGFβ1 induces vimentin protein expression in AECs, which is associated with a 2.5-fold increase in cell motility, resulting in increased rates of migration and wound closure. These effects are independent of cell proliferation. TGFβ1-mediated vimentin protein expression, cell migration, and wound closure are prevented by a pharmacological inhibitor of the Smad pathway and by expression of Ad-shRNA against vimentin. Conversely, overexpression of mEmerald-vimentin is sufficient for increased cell-migration and wound-closure rates. These results demonstrate that vimentin is required and sufficient for increased wound repair in an in vitro model of lung injury.—Rogel, M. R., Soni, P. N., Troken, J. R., Sitikov, A., Trejo, H. E., Ridge, K. M. Vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells. PMID:21803859

  19. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    SciTech Connect

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F.

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  20. Hyperosmolar solution effects in guinea pig airways. IV. Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity.

    PubMed

    Johnston, Richard A; Van Scott, Michael R; Kommineni, Choudari; Millecchia, Lyndell L; Dortch-Carnes, Juanita; Fedan, Jeffrey S

    2004-01-01

    We investigated the in vivo and in vitro effects of lipopolysaccharide (LPS) treatment (4 mg/kg i.p.) on guinea pig airway smooth muscle reactivity and epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Hyperosmolar challenge of the epithelium releases epithelium-derived relaxing factor (EpDRF). Using a two-chamber, whole body plethysmograph 18 h post-treatment, animals treated with LPS were hyporeactive to inhaled MCh aerosol. This could involve an increase in the release and/or actions of EpDRF, because LPS treatment enhanced EpDRF-induced smooth muscle relaxation in vitro in the isolated perfused trachea apparatus. In isolated perfused tracheas the basal transepithelial potential difference (Vt) was increased after LPS treatment. The increase in Vt was inhibited by amiloride and indomethacin. Concentration-response curves for changes in Vt in response to serosally and mucosally applied MCh were biphasic (hyperpolarization, <3 x 10(-7)M; depolarization, >3 x 10(-7)M); MCh was more potent when applied serosally. The hyperpolarization response to MCh, but not the depolarization response, was potentiated after LPS treatment. In both treatment groups, mucosally applied hyperosmolar solution (using added NaCl) depolarized the epithelium; this response was greater in tracheas from LPS-treated animals. The results of this study indicate that airway hyporeactivity in vivo after LPS treatment is accompanied by an increase in the release and/or actions of EpDRF in vitro. These changes may involve LPS-induced bioelectric alterations in the epithelium. PMID:14566002

  1. Usefulness of intraoperative bronchoscopy during surgical repair of a congenital cardiac anomaly with possible airway obstruction: three cases report.

    PubMed

    Oh, JongEun; Kim, Jung-Won; Shin, Won-Jung; Gwak, Mijeung; Park, Pyung Hwan

    2016-02-01

    Compression of the airway is relatively common in pediatric patients, although it is often an unrecognized complication of congenital cardiac and aortic arch anomalies. Aortopexy has been established as a surgical treatment for tracheobronchial obstruction associated with vascular anomaly, aortic arch anomaly, esophageal atresia, and tracheoesophageal fistula. The tissue-to-tissue arch repair technique could result in severe airway complication such as compression of the left main bronchus which was not a problem before the correction. We report three cases of corrective open heart surgery monitored by intraoperative bronchoscopy performed during prebypass, and performed immediately before weaning from bypass, to evaluate tracheobronchial obstruction caused by congenital, complex cardiac anomalies in the operating room. PMID:26885306

  2. Usefulness of intraoperative bronchoscopy during surgical repair of a congenital cardiac anomaly with possible airway obstruction: three cases report

    PubMed Central

    Oh, JongEun; Kim, Jung-won; Shin, Won-Jung; Park, Pyung Hwan

    2016-01-01

    Compression of the airway is relatively common in pediatric patients, although it is often an unrecognized complication of congenital cardiac and aortic arch anomalies. Aortopexy has been established as a surgical treatment for tracheobronchial obstruction associated with vascular anomaly, aortic arch anomaly, esophageal atresia, and tracheoesophageal fistula. The tissue-to-tissue arch repair technique could result in severe airway complication such as compression of the left main bronchus which was not a problem before the correction. We report three cases of corrective open heart surgery monitored by intraoperative bronchoscopy performed during prebypass, and performed immediately before weaning from bypass, to evaluate tracheobronchial obstruction caused by congenital, complex cardiac anomalies in the operating room. PMID:26885306

  3. Airway Epithelial Cells are the Site of Expression of a Mammalian Antimicrobial Peptide Gene

    NASA Astrophysics Data System (ADS)

    Diamond, Gill; Jones, Douglas E.; Bevins, Charles L.

    1993-05-01

    We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5'exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.

  4. Growth of airway epithelial cells at an air-liquid interface changes both the response to particle exposure and iron homeostasis.

    EPA Science Inventory

    RATIONALE: We tested the hypothesis that 1) relative to submerged cells, airway epithelial cells grown at an air-liquid interface and allowed to differentiate would have an altered response to particle exposure and 2) that these differences would be associated with indices of iro...

  5. Growth of airway epithelial cells at an air-liquid interface changes both the response to particle exposure and iron homeostasis

    EPA Science Inventory

    We tested the hypothesis that 1) relative to submerged cells, airway epithelial cells grown at an air-liquid interface and allowed to differentiate would have an altered response to particle exposure and 2) that these differences would be associated with indices of iron homeostas...

  6. MATRIX METALLOPROTEINS (MMP)-MEDIATED PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZINC (ZN)

    EPA Science Inventory

    Matrix Metalloproteinase (MMP)-Mediated Phosphorylation of The Epidermal Growth Factor Receptor (EGFR) in Human Airway Epithelial Cells (HAEC) Exposed to Zinc (Zn)
    Weidong Wu, James M. Samet, Robert Silbajoris, Lisa A. Dailey, Lee M. Graves, and Philip A. Bromberg
    Center fo...

  7. IL-1β stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a.

    PubMed

    Raymond, Meera; Marchbank, Tania; Moyer, Mary P; Playford, Raymond J; Sanderson, Ian R; Kruidenier, Laurens

    2012-12-01

    Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/β-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1β-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1β-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1β-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1β stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1β-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1β-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound

  8. ANESTHETIC MANAGEMENT OF FEMORAL FRACTURE REPAIR IN A PATIENT WITH CERVICAL MYELOPATHY, AUTONOMIC DYSFUNCTION, AND DIFFICULT AIRWAY.

    PubMed

    Vellore, Ajay R; Robards, Christopher B; Clendenen, Steven R

    2016-02-01

    Spinal stenosis is a potentially serious condition that can lead to myelopathies and autonomic instability, both of which, as a result, may complicate anesthetic management. Additionally, neuraxial anesthesia appears to increase the risk of worsened neurological outcomes in this population. A 56-year-old female with spinal stenosis, autonomic dysfunction, and known difficult airway who required anesthesia for repair of a femur fracture is presented. After pre-operative arterial line and femoral block placement, an ultrasound guided subarachnoid block was safely placed. This supports the notion that in the appropriate setting, a safe, successful neuraxial blockade can be performed when a general anesthetic may be fraught with more risk. PMID:27382821

  9. PROINFLAMMATORY OXIDANT HYPOCHLOROUS ACID (HOCL) INDUCES DUAL SIGNALING PATHWAYS IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    In the airway of inflammatory diseases such as bacterial infection, cystic fibrosis and COPD, high level of HOCL (local concentration of up to 5mM) can be generated through a reaction catalyzed by leukocyte granule enzyme- Myeloperoxidase (MPO). HOCL is a very potent oxidative ag...

  10. The effects of an epithelial barrier protective cationic aerosol on allergen-induced airway inflammation in asthma: a randomized, placebo-controlled clinical trial.

    PubMed

    Nair, P; Denis, S; Cancelliere, L; Radford, K; Efthimiadis, A; Rosano, M; Hanrahan, J

    2014-09-01

    Inhaled cationic airway lining modulator (iCALM) is a cationic aerosol therapy comprised of 1.29% calcium chloride dissolved in 0.9% isotonic saline that enhances the biophysical barrier function of the airway lining fluid and primes the host defense response. It's ability to attenuate bronchitis caused by inhaled particles was investigated using an allergen-inhalation model in a proof-of-concept study. In a randomized, double-blind, placebo-controlled cross-over trial of 6 mild atopic steroid-naïve asthmatic subjects, 3 doses of iCALM were well tolerated and they attenuated allergen-induced increase in sputum eosinophils, and levels of IL-5, MCP-1 and eotaxin. This study provides an opportunity to investigate the role of enhancing epithelial barrier to decrease airway inflammation provoked by inhaled particles in a variety of airway diseases.

  11. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ -1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non

  12. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

    PubMed

    Baumstark-Khan, C; Heilmann, J; Rink, H

    2003-01-01

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of

  13. Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck

    PubMed Central

    O'hara, Kimberley A.; Vaghjiani, Rasilaben J.; Nemec, Antonia A.; Klei, Linda R.; Barchowsky, Aaron

    2006-01-01

    Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein–DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 μM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3α and STAT3β. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression. PMID:17078813

  14. Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck.

    PubMed

    O'Hara, Kimberley A; Vaghjiani, Rasilaben J; Nemec, Antonia A; Klei, Linda R; Barchowsky, Aaron

    2007-03-01

    Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein-DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 microM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3alpha and STAT3beta. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression.

  15. Wood Smoke Enhances Cigarette Smoke–Induced Inflammation by Inducing the Aryl Hydrocarbon Receptor Repressor in Airway Epithelial Cells

    PubMed Central

    Awji, Elias G.; Chand, Hitendra; Bruse, Shannon; Smith, Kevin R.; Colby, Jennifer K.; Mebratu, Yohannes; Levy, Bruce D.

    2015-01-01

    Our previous studies showed that cigarette smokers who are exposed to wood smoke (WS) are at an increased risk for chronic bronchitis and reduced lung function. The present study was undertaken to determine the mechanisms for WS-induced adverse effects. We studied the effect of WS exposure using four cohorts of mice. C57Bl/6 mice were exposed for 4 or 12 weeks to filtered air, to 10 mg/m3 WS for 2 h/d, to 250 mg/m3 cigarette smoke (CS) for 6 h/d, or to CS followed by WS (CW). Inflammation was absent in the filtered air and WS groups, but enhanced by twofold in the bronchoalveolar lavage of the CW compared with CS group as measured by neutrophil numbers and levels of the neutrophil chemoattractant, keratinocyte-derived chemokine. The levels of the anti-inflammatory lipoxin, lipoxin A4, were reduced by threefold along with cyclo-oxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in airway epithelial cells and PGE2 levels in the bronchoalveolar lavage of CW compared with CS mice. We replicated, in primary human airway epithelial cells, the changes observed in mice. Immunoprecipitations showed that WS blocked the interaction of aryl hydrocarbon receptor (AHR) with AHR nuclear transporter to reduce expression of COX-2 and mPGES-1 by increasing expression of AHR repressor (AHRR). Collectively, these studies show that exposure to low concentrations of WS enhanced CS-induced inflammation by inducing AHRR expression to suppress AHR, COX-2, and mPGES-1 expression, and levels of PGE2 and lipoxin A4. Therefore, AHRR is a potential therapeutic target for WS-associated exacerbations of CS-induced inflammation. PMID:25137396

  16. The effect of ambroxol on chloride transport, CFTR and ENaC in cystic fibrosis airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Hussain, Rashida; Strid, Hilja; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2013-11-01

    Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl(-) efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) influx via the epithelial Na(+) channel (ENaC). In cells expressing wt-CFTR, ambroxol increased the Cl(-) conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl(-) transport was measured by Cl(-) efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl(-) efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α-ENaC, and of the CFTR protein. No significant difference was observed in β-ENaC after exposure to ambroxol, whereas mRNA expression of γ-ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca(2+) concentration. Upregulation of CFTR and enhanced Cl(-) efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients.

  17. The effect of ambroxol on chloride transport, CFTR and ENaC in cystic fibrosis airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Hussain, Rashida; Strid, Hilja; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2013-11-01

    Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl(-) efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) influx via the epithelial Na(+) channel (ENaC). In cells expressing wt-CFTR, ambroxol increased the Cl(-) conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl(-) transport was measured by Cl(-) efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl(-) efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α-ENaC, and of the CFTR protein. No significant difference was observed in β-ENaC after exposure to ambroxol, whereas mRNA expression of γ-ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca(2+) concentration. Upregulation of CFTR and enhanced Cl(-) efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients. PMID:23765701

  18. Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery.

    PubMed

    Valencia-Gattas, Monica; Conner, Gregory E; Fregien, Nevis L

    2016-01-01

    Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases. PMID:27532261

  19. Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Coutermarsh, Bonita A; Barnaby, Roxanna L; Stanton, Bruce A

    2012-05-18

    Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

  20. Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery

    PubMed Central

    Valencia-Gattas, Monica; Conner, Gregory E.; Fregien, Nevis L.

    2016-01-01

    Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases. PMID:27532261

  1. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

    SciTech Connect

    Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon; Heo, Won Il; Kim, Kyung Won; Kim, Kyu Earn; Sohn, Myung Hyun

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally

  2. Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells.

    PubMed

    Rubenstein, Ronald C; Lockwood, Shannon R; Lide, Ellen; Bauer, Rebecca; Suaud, Laurence; Grumbach, Yael

    2011-01-01

    The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o⁻ model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o⁻ parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o⁻ cells that stably express wild-type (wt) CFTR. CFBE41o⁻ wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o⁻ wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may

  3. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    PubMed Central

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β−NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  4. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis.

    PubMed

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β-NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  5. Reduced expression of Tis7/IFRD1 protein in murine and human cystic fibrosis airway epithelial cell models homozygous for the F508del-CFTR mutation.

    PubMed

    Blanchard, Elise; Marie, Solenne; Riffault, Laure; Bonora, Monique; Tabary, Olivier; Clement, Annick; Jacquot, Jacky

    2011-08-01

    12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o(-) cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o(-) cells compared to normal bronchial epithelial cells 16HBE14o(-). Surprisingly, messenger RNA level of IFRD1 in CFBE41o(-) cells was found elevated. Treating CFBE41o(-) cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.

  6. Intranasal Immunization Strategy To Impede Pilin-Mediated Binding of Pseudomonas aeruginosa to Airway Epithelial Cells

    PubMed Central

    Hsieh, Jennifer C.; Tham, Doris M.; Feng, Weijun; Huang, Fan; Embaie, Selamawit; Liu, Keyi; Dean, Deborah; Hertle, Ralf; FitzGerald, David J.; Mrsny, Randall J.

    2005-01-01

    Prevention of pulmonary Pseudomonas aeruginosa infections represents a critical unmet medical need for cystic fibrosis (CF) patients. We have examined the tenet that a mucosal immunization approach can reduce interactions of a piliated form of this opportunistic pathogen with respiratory epithelial cells. Vaccinations were performed using ntPEpilinPAK, a protein chimera composed of a nontoxic form of P. aeruginosa exotoxin A (ntPE), where the C-terminal loop amino acid sequence of the PAK strain pilin protein was inserted in place of the ntPE Ib domain. Intranasal (i.n.) immunization of BALB/c mice with ntPEpilinPAK generated both serum and saliva immune responses. A series of in vitro studies showed that diluted samples of saliva obtained from immunized mice reduced pilin-dependent P. aeruginosa binding to polarized human tracheal epithelial cells, protected human pulmonary epithelial cells from cytotoxic actions associated with bacterial challenge, and reduced exotoxin A toxicity. Overall, i.n. administration of ntPEpilinPAK induced mucosal and systemic immune responses that may be beneficial for blocking early stage adhesion and/or infection events of epithelial cell-P. aeruginosa interactions at oropharyngeal surfaces. PMID:16239575

  7. 1α,25-dihydroxyvitamin D₃ counteracts the effects of cigarette smoke in airway epithelial cells.

    PubMed

    Zhang, Ruhui; Zhao, Haijin; Dong, Hangming; Zou, Fei; Cai, Shaoxi

    2015-06-01

    Cigarette smoke extracts (CSE) alter calpain-1 expression via ERK signaling pathway in bronchial epithelial cells. 1α,25-dihydroxyvitamin D3 (1,25D3) inhibits cigarette smoke-induced epithelial barrier disruption. This study was aimed to explore whether the 1,25D3 counteracted the CSE effects in a human bronchial epithelial cell line (16HBE). In particular, transepithelial electrical resistance (TER) and permeability, expression and distribution of E-cadherin and β-catenin, calpain-1 expression, and ERK phosphorylation were assessed in the CSE-stimulated 16HBE cells. The CSE induced the ERK phosphorylation, improved the calpain-1 expression, increased the distribution anomalies and the cleaving of E-cadherin and β-catenin, and resulted in the TER reduction and the permeability increase. The 1,25D3 reduced these pathological changes. The 1,25D3 mediated effects were associated with a reduced ERK phosphorylation. In conclusion, the present study provides compelling evidences that the 1,25D3 may be considered a possible valid therapeutic option in controlling the cigarette smoke-induced epithelial barrier disruption.

  8. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO METALS

    EPA Science Inventory

    We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms respons...

  9. Efficient intratracheal delivery of airway epithelial cells in mice and pigs

    PubMed Central

    Gui, Liqiong; Qian, Hong; Rocco, Kevin A.; Grecu, Loreta

    2014-01-01

    Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases. PMID:25416381

  10. Efficient intratracheal delivery of airway epithelial cells in mice and pigs.

    PubMed

    Gui, Liqiong; Qian, Hong; Rocco, Kevin A; Grecu, Loreta; Niklason, Laura E

    2015-01-15

    Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.

  11. Effects of ozone on airway epithelial permeability and ion transport. Research report, Aug 83-Jan 91

    SciTech Connect

    Bromberg, P.; Ranga, V.; Stutts, M.J.

    1991-01-01

    The authors studied the effects of ozone inhalation on the permeability to dissolved molecules and the ion transport activity of airway epithelium using in vivo and in vitro techniques. Conscious unrestrained guinea pigs were exposed to 1 part per million (ppm) ozone for three hours in controlled environmental chambers. The rate of appearance in blood of various water-soluble compounds instilled onto the surface of the trachea increased, indicating increased permeability of the airway epithelium. The interpretation was supported by the observation of horseradish peroxidase in the intercellular spaces of the tracheal epithelium from ozone-exposed, but not air-exposed (control), animals. However, when the tracheas were excised after ozone exposure and mounted in a tissue bath before measurement of permeability, no increase was found. When the authors exposed animals to 1 ppm for three hours daily, the increased permeability observed in vivo was no longer demonstrable after the fourth exposure, suggesting that 'adaptation' had occurred. Ozone exposure caused a sharp increase in active ion transport by tracheal epithelium. This is probably due to increased absorption of sodium ion because it was inhibited by 30 micromolar amiloride. The increased transport was observed in excised tissues and was present in tracheas removed from animals up to three days after a single exposure.

  12. A Human Espophageal Epithelial Cell Model for Study of Radiation Induced Cancer and DNA Damage Repair

    NASA Technical Reports Server (NTRS)

    Huff, Janice L.; Patel, Zarana S.; Hada, Megumi; Cucinotta, Francis A.

    2008-01-01

    For cancer risk assessment in astronauts and for countermeasure development, it is essential to understand the molecular mechanisms of radiation carcinogenesis and how these mechanisms are influenced by exposure to the types of radiation found in space. We are developing an in vitro model system for the study of radiation-induced initiation and progression of esophageal carcinoma, a type of cancer found to have a significant enhancement in incidence in the survivors of the atomic bomb detonations in Japan. Here we present the results of our preliminary characterization of both normal and hTERT immortalized esophageal epithelial cells grown in 2-dimensional culture. We analyzed DNA repair capacity by measuring the kinetics of formation and loss of - H2AX foci following radiation exposure. Additionally, we analyzed induction of chromosomal aberrations using 3-color fluorescence in situ hybridization (FISH). Data were generated using both low LET (gamma rays) and high LET ions (1000 MeV/nucleon iron).

  13. Negative impact of DEP exposure on human airway epithelial cell adhesion, stiffness, and repair.

    PubMed

    Doornaert, Blandine; Leblond, Valerie; Galiacy, Stephane; Gras, Gabriel; Planus, Emmanuelle; Laurent, Valerie; Isabey, Daniel; Lafuma, Chantal

    2003-01-01

    Epidemiological and experimental studies suggest that diesel exhaust particles (DEPs) may be associated with increased respiratory mortality and morbidity. Several recent studies have also shown that DEPs increase the production of inflammatory cytokines by human bronchial epithelium (HBE) cells in vitro. The present study investigates the effects of DEPs on the interaction of l-HBE cells (16HBE14o-) with the cell and matrix microenvironment based on evaluation of integrin-type cell/matrix ligand expression, cytoskeleton (CSK) stiffness, and matrix remodeling via matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 expression. The results showed that DEP exposure induced: 1) a net dose-dependent decrease in CSK stiffness through actin fibers, 2) a concomitant specific reduction of both alpha(3)- and beta(1)-integrin subunits extensively expressed on the HBE cell surface, 3) a decrease in the level of CD44, which is a major HBE cell-cell and HBE cell-matrix adhesion molecule; and 4) an isolated decrease in MMP-1 expression without any change in tissue inhibitor of matrix metalloproteinase (TIMP)-1 or TIMP-2 tissue inhibitors. Restrictive modulation of cell-matrix interaction, cell-cell connection, CSK stiffness, and fibrillary collagen remodeling results in a decreased wound closure capacity and an increased deadhesion capacity. In conclusion, on the basis of these results, we can propose that, in addition to their ability to increase the production of inflammatory cytokines, DEPs could also alter the links between actin CSK and the extracellular matrix, suggesting that they might facilitate HBE cell detachment in vivo. PMID:12471014

  14. Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells.

    PubMed

    Popova, Taissia G; Turell, Michael J; Espina, Virginia; Kehn-Hall, Kylene; Kidd, Jessica; Narayanan, Aarthi; Liotta, Lance; Petricoin, Emanuel F; Kashanchi, Fatah; Bailey, Charles; Popov, Serguei G

    2010-01-01

    Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK) and downstream transcriptional factors [STAT1 (Y701), ATF2 (T69/71), MSK1 (S360) and CREB (S133)]. NF-κB phosphorylation was also increased. Activation of p53 (S15, S46) correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473), along with phosphorylation of FOX 01/03 (T24/31) which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication. PMID:21072193

  15. A murine model of airway fibrosis induced by repeated naphthalene exposure.

    PubMed

    Aoshiba, Kazutetsu; Tsuji, Takao; Itoh, Masayuki; Semba, Seitaro; Yamaguchi, Kazuhiro; Nakamura, Hiroyuki; Watanabe, Hidehiro

    2014-07-01

    The airway epithelium serves as a biological barrier essential for host defense against inhaled pollutants. While chronic epithelial injury, commonly associated with chronic obstructive pulmonary disease and bronchiolitis obliterans syndrome, often results in airway fibrosis, limited animal models of airway fibrosis have been established. Club cells (Clara cells) in the small airways represent an important population of epithelial progenitor cells and also the principal site of localization of the cytochrome P-450 monooxygenase system, which metabolically activates xenobiotic chemicals such as naphthalene by converting them to toxic epoxide intermediates. We hypothesized that repeated exposure to naphthalene may cause prolonged loss of club cells, triggering aberrant local epithelial repair mechanisms that lead to peribronchial fibrosis. We administered intraperitoneal injections of naphthalene to C57/BL6J mice once a week for 14 consecutive weeks. Repeated club cell injury caused by naphthalene triggered regional hyperproliferation of epithelial progenitor cells, while other regions remained denuded or squamated, resulting in fibroblast proliferation and peribronchial collagen deposition associated with upregulation of the fibrogenic cytokines transforming growth factor-β and connective tissue growth factor. The total collagen content of the lung assessed by measurement of the hydroxyproline content was also increased after repeated exposure to naphthalene. These results lend support to the relevance of repeated injury of airway epithelial cells as a trigger for resting fibroblast proliferation and airway fibrosis. This model of airway fibrosis is simple and easy to reproduce, and may be expected to advance our understanding of the pathogenesis and potential treatment of airway fibrotic disorders.

  16. Uncertainty in the Utility of Immunohistochemistry in Mismatch Repair Protein Expression in Epithelial Ovarian Cancer

    PubMed Central

    Copppola, Domenico; Nicosia, Santo V.; Doty, Andrea; Sellers, Thomas A; Lee, Ji-Hyun; Fulp, Jimmy; Thompson, Zachary; Galeb, Sanja; McLaughlin, John; Narod, Steven A; Schildkraut, Joellen; Pal, Tuya

    2014-01-01

    Background Utility of immunohistochemistry (IHC) for mismatch repair (MMR) protein expression has been demonstrated in colorectal cancer but remains incompletely defined in ovarian cancer. We evaluated MMR protein expression in three population-based samples of epithelial ovarian cancers. Methods IHC staining was performed on full section (FS) or tissue microarray (TMA) slides for MLH1, MSH2, and MSH6 expression. Results Of 487 cases, 147 and 340 were performed through FS and TMA, respectively. Overall, Loss of Expression (LoE) of at least one MMR protein was observed in 12.7% based on an expression score of ≤3 (on a scale of 9). Notably, LoE was significantly higher in TMAs (17.9%) compared to FS cases (0.7%) (p <0.001). Conclusions A substantial proportion of epithelial ovarian cancers have a loss of MMR protein expression. Protein expression results vary significantly by the tissue sampling methodology utilized, raising concerns about the clinical utility of this test for ovarian tumors. PMID:23155266

  17. Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line.

    PubMed

    Tong, Zhenyue; Illek, Beate; Bhagwandin, Vikash J; Verghese, George M; Caughey, George H

    2004-11-01

    Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway. PMID:15246975

  18. Do Airway Epithelium Air–Liquid Cultures Represent the In Vivo Airway Epithelium Transcriptome?

    PubMed Central

    Dvorak, Anna; Tilley, Ann E.; Shaykhiev, Renat; Wang, Rui; Crystal, Ronald G.

    2011-01-01

    Human airway epithelial cells cultured in vitro at the air–liquid interface (ALI) form a pseudostratified epithelium that forms tight junctions and cilia, and produces mucin. These cells are widely used in models of differentiation, injury, and repair. To assess how closely the transcriptome of ALI epithelium matches that of in vivo airway epithelial cells, we used microarrays to compare the transcriptome of human large airway epithelial cells cultured at the ALI with the transcriptome of large airway epithelium obtained via bronchoscopy and brushing. Gene expression profiling showed that global gene expression correlated well between ALI cells and brushed cells, but with some differences. Gene expression patterns mirrored differences in proportions of cell types (ALIs have higher percentages of basal cells, whereas brushed cells have higher percentages of ciliated cells), that is, ALI cells expressed higher levels of basal cell–related genes, and brushed cells expressed higher levels of cilia-related genes. Pathway analysis showed that ALI cells had increased expression of cell cycle and proliferation genes, whereas brushed cells had increased expression of cytoskeletal organization and humoral immune response genes. Overall, ALI cells provide a good representation of the in vivo airway epithelial transcriptome, but for some biologic questions, the differences between in vitro and in vivo environments need to be considered. PMID:20525805

  19. Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

    PubMed

    Kelly, Catriona; Canning, Paul; Buchanan, Paul J; Williams, Mark T; Brown, Vanessa; Gruenert, Dieter C; Elborn, J Stuart; Ennis, Madeleine; Schock, Bettina C

    2013-03-01

    The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF. PMID:23316065

  20. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

    PubMed Central

    van ‘t Wout, Emily F. A.; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E.; Clarke, Hanna J.; Tommassen, Jan; Marciniak, Stefan J.; Hiemstra, Pieter S.

    2015-01-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  1. The non-antibiotic macrolide EM900 inhibits rhinovirus infection and cytokine production in human airway epithelial cells

    PubMed Central

    Lusamba Kalonji, Nadine; Nomura, Kazuhiro; Kawase, Tetsuaki; Ota, Chiharu; Kubo, Hiroshi; Sato, Takeya; Yanagisawa, Teruyuki; Sunazuka, Toshiaki; Ōmura, Satoshi; Yamaya, Mutsuo

    2015-01-01

    The anti-inflammatory effects of macrolides may be associated with a reduced frequency of exacerbation of chronic obstructive pulmonary disease (COPD). However, because the long-term use of antibiotics may promote the growth of drug-resistant bacteria, the development of a treatment to prevent COPD exacerbation with macrolides that do not exert anti-bacterial effects is necessary. Additionally, the inhibitory effects of nonantibiotic macrolides on the replication of rhinovirus (RV), which is the major cause of COPD exacerbation, have not been demonstrated. Primary cultures of human tracheal epithelial cells and nasal epithelial cells were pretreated with the nonantibiotic macrolide EM900 for 72 h prior to infection with a major group RV type 14 rhinovirus (RV14) and were further treated with EM900 after infection. Treatment with EM900 before and after infection reduced RV14 titers in the supernatants and viral RNA within the cells. Moreover, cytokine levels, including interleukin (IL)-1β and IL-6, were reduced in the supernatants following RV14 infection. Treatment with EM900 before and after infection also reduced the mRNA and protein expression of intercellular adhesion molecule-1 (ICAM-1), which is the receptor for RV14, after infection and reduced the activation of the nuclear factor kappa-B protein p50 in nuclear extracts after infection. Pretreatment with EM900 reduced the number and fluorescence intensity of the acidic endosomes through which RV RNA enters the cytoplasm. Thus, pretreatment with EM900 may inhibit RV infection by reducing the ICAM-1 levels and acidic endosomes and thus modulate the airway inflammation associated with RV infections. PMID:26462747

  2. Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle.

    PubMed

    Taha-Abdelaziz, Khaled; Wyer, Leanna; Berghuis, Lesley; Bassel, Laura L; Clark, Mary Ellen; Caswell, Jeff L

    2016-01-01

    β-defensins are an important element of the mucosal innate immune response against bacterial pathogens. Tracheal antimicrobial peptide (TAP) has microbicidal activity against the bacteria that cause bovine respiratory disease, and its expression in tracheal epithelial cells is upregulated by bacterial products including lipopolysaccharide (LPS, a TLR4 agonist), Pam3CSK4 (an agonist of Toll-like receptor 2/1), and interleukin (IL)-17A. The objectives of this study were to identify the signalling pathway by which LPS, Pam3CSK4 and IL-17A induce TAP gene expression, and to determine the effect of glucocorticoid as a model of stress on this epithelial innate immune response. In primary cultures of bovine tracheal epithelial cells (bTEC), LPS, Pam3CSK4 and IL-17A each stimulated TAP gene expression. This effect was abrogated by caffeic acid phenylester (CAPE), an inhibitor of NF-κB. Similarly, western analysis showed that LPS, Pam3CSK4 and IL-17A each induced translocation of NF-κB p65 from the cytoplasm to the nucleus, but pre-treatment with CAPE inhibited this response. Finally, pre-treatment of bTEC with the glucocorticoid dexamethasone abolished the stimulatory effect of LPS, Pam3CSK4 and IL-17A on upregulation of TAP gene expression. These findings indicate that NF-κB activation is necessary for induction of TAP gene expression by LPS (a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist), or IL-17A. Furthermore, this stimulatory response is inhibited by glucocorticoid, suggesting this as one mechanism by which stress increases the risk of bacterial pneumonia. These findings have implications for understanding the pathogenesis of stress-associated bacterial pneumonia, and for developing methods to stimulate innate immune responses in the respiratory tract of cattle. PMID:26987959

  3. A dynamic system for single and repeated exposure of airway epithelial cells to gaseous pollutants.

    PubMed

    Kastner, Pierre Edouard; Le Calvé, Stéphane; Zheng, Wuyin; Casset, Anne; Pons, Françoise

    2013-03-01

    In vitro models are promising approaches to investigate the adverse effects and the mode of action of air pollutants on the respiratory tract. We designed a dynamic system that allows the single or repeated exposure of cultured cells to two major indoor air gaseous pollutants, formaldehyde (HCHO) and nitrogen dioxide (NO2), alone or as a mixture. In this system, the Calu-3 human bronchial epithelial cell line was exposed at the air-liquid interface (ALI) or submerged by culture medium to synthetic air or to target concentrations of HCHO and/or NO2 once or on 4 consecutive days before assessment of cell viability and necrosis, IL-6 and IL-8 release and trans-epithelial electrical resistance. Our data showed that whereas the ALI method can be used for single short-term exposures only, the submerged method provides the possibility to expose Calu-3 cells in a repeated manner. As well, we found that repeated exposures of the cells to HCHO and NO2 at concentrations that can be found indoors triggered a significant decrease in cell metabolism and an increase in IL-8 release that were not evoked by a single exposure. Thus, our work highlights the fact that the development of systems and methods that allow repeated exposures of cultured cells to gaseous compounds in mixtures is of major interest to evaluate the impact of air pollution on the respiratory tract.

  4. Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.

    PubMed

    Proskocil, Becky J; Sekhon, Harmanjatinder S; Jia, Yibing; Savchenko, Valentina; Blakely, Randy D; Lindstrom, Jon; Spindel, Eliot R

    2004-05-01

    The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development. PMID:14764638

  5. The response of guinea pig airway epithelial cells and alveolar macrophages to environmental stress

    SciTech Connect

    Cohen, D.S.; Palmer, E.; Welch, W.J.; Sheppard, D. )

    1991-08-01

    Cells lining the respiratory tract form an interface between the organism and the external environment and are repeatedly exposed to physical, chemical, and metabolic stresses. The authors examined the response of cultured guinea pig tracheal epithelial cells and alveolar macrophages to various forms of stress, including clinically and environmentally relevant metabolic stresses such as ozone and acid exposure. Classic stress treatments such as heat shock and sodium arsenite treatment induced the synthesis of 28, 32, 72, 73, 90, and 110 kD stress proteins similar to those observed in other cell types. In contrast, no significant changes in the pattern of protein synthesis were detected after exposure to ambient concentrations of ozone, although ozone exposure caused significant cytotoxicity to both cell types. Another potent oxidant, hydrogen peroxide, similarly did not induce appreciable stress protein synthesis. However, surface acidification of tracheal epithelial cells and alveolar macrophages caused the induction of 72 and 78 kD stress proteins. While stress proteins may play a role in the response of respiratory cells to certain injuries such as hyperthermia and surface acidification, they may not be important in the defense against ozone or other forms of oxidative injury.

  6. TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

    SciTech Connect

    Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.; Casola, Antonella

    2011-09-30

    Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases. Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.

  7. Effects of bronchopulmonary inflammation induced by pseudomonas aeruginosa on adenovirus-mediated gene transfer to airway epithelial cells in mice.

    PubMed

    van Heeckeren, A; Ferkol, T; Tosi, M

    1998-03-01

    Cystic fibrosis (CF) patients have endobronchial inflammation caused by infection with mucoid Pseudomonas aeruginosa. Since adenovirus vectors are being studied for gene therapy for CF, we sought to determine whether bronchopulmonary inflammation would influence adenovirus-mediated gene transfer. We hypothesized that bronchopulmonary inflammation in mice inoculated with mucoid P. aeruginosa would be associated with a decrease in the efficacy of adenovirus-mediated gene transfer. Agarose beads embedded with mucoid P. aeruginosa (6 x 10(4) c.f.u. per mouse) were inoculated transtracheally into C57BL/6 mice. Control mice received sterile agarose beads. Ten days after inoculation with agarose beads, recombinant adenovirus containing the beta-galactosidase reporter gene (Ad2/beta Gal-2) was administered intranasally (1.1 x 10(9) IU per mouse), and mice were killed 3 days later. The extent of inflammation, determined by neutrophil numbers in bronchoalveolar lavage fluid and by areal lung inflammation, was significantly greater in mice inoculated with P. aeruginosa-laden agarose beads and Ad2/beta Gal-2 compared with controls. Mice that had received Pseudomonas-laden agarose beads and Ad2/beta Gal-2 had significantly fewer (P < 0.015) airway epithelial cells transduced (4.1 +/- 0.9%) compared with mice that received sterile agarose beads and Ad2/beta Gal-2 (9.4 +/- 1.4%). These results indicate that the efficacy of adenovirus-mediated gene transfer is reduced in Pseudomonas-induced bronchopulmonary inflammation.

  8. A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

    PubMed Central

    Walker, Brian; Hamilton, Robert A.; Martin, S. Lorraine

    2016-01-01

    Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aeruginosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process. PMID:27459298

  9. Extracellular ATP directly gates a cation-selective channel in rabbit airway ciliated epithelial cells

    PubMed Central

    Korngreen, Alon; Ma, Weiyuan; Priel, Zvi; Silberberg, Shai D

    1998-01-01

    A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole-cell and outside-out patch configurations of the patch-clamp technique. In solutions designed to maximize currents through voltage-gated calcium channels, there were no indications of voltage-gated Ba2+ currents. Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa:PCs:PTEA of 4:1:0.1. Permeability to Cl− was negligible. Including GDP-β-S in the intracellular solution did not inhibit the effects of ATP, nor did GTP-γ-S irreversibly activate the conductance. In outside-out membrane patches, with GDP-β-S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at −120 mV. Suramin (100 μM) inhibited the whole-cell currents activated by ATP (200 μM) by 93 ± 3 %. Similar effects of suramin were observed on ATP-activated channels in outside-out membrane patches. Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At −40 mV, the time to half-maximal current activation (t½) was 46 ± 9 s during the first exposure to 200 μM ATP and decreased to 5 ± 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP-β-S in the intracellular solution. The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 μM ATP, t½ at +40 mV was 4-fold longer than t½ at −40 mV. Half-maximal activation of the conductance shifted from 210 ± 30 to 14 ± 4 μM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0.01 mM. The Hill coefficient for ATP

  10. BRCA1/FANCD2/BRG1-Driven DNA Repair Stabilizes the Differentiation State of Human Mammary Epithelial Cells.

    PubMed

    Wang, Hua; Bierie, Brian; Li, Andrew G; Pathania, Shailja; Toomire, Kimberly; Dimitrov, Stoil D; Liu, Ben; Gelman, Rebecca; Giobbie-Hurder, Anita; Feunteun, Jean; Polyak, Kornelia; Livingston, David M

    2016-07-21

    An abnormal differentiation state is common in BRCA1-deficient mammary epithelial cells, but the underlying mechanism is unclear. Here, we report a convergence between DNA repair and normal, cultured human mammary epithelial (HME) cell differentiation. Surprisingly, depleting BRCA1 or FANCD2 (Fanconi anemia [FA] proteins) or BRG1, a mSWI/SNF subunit, caused HME cells to undergo spontaneous epithelial-to-mesenchymal transition (EMT) and aberrant differentiation. This also occurred when wild-type HMEs were exposed to chemicals that generate DNA interstrand crosslinks (repaired by FA proteins), but not in response to double-strand breaks. Suppressed expression of ΔNP63 also occurred in each of these settings, an effect that links DNA damage to the aberrant differentiation outcome. Taken together with somatic breast cancer genome data, these results point to a breakdown in a BRCA/FA-mSWI/SNF-ΔNP63-mediated DNA repair and differentiation maintenance process in mammary epithelial cells that may contribute to sporadic breast cancer development. PMID:27373334

  11. Cigarette smoking induces small airway epithelial epigenetic changes with corresponding modulation of gene expression.

    PubMed

    Buro-Auriemma, Lauren J; Salit, Jacqueline; Hackett, Neil R; Walters, Matthew S; Strulovici-Barel, Yael; Staudt, Michelle R; Fuller, Jennifer; Mahmoud, Mai; Stevenson, Christopher S; Hilton, Holly; Ho, Melisa W Y; Crystal, Ronald G

    2013-12-01

    The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared with nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared with nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 35 exhibited a correlation with gene expression, 54% with an inverse correlation of DNA methylation with gene expression and 46% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression.

  12. Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

    PubMed

    Mullins, Benjamin J; Kicic, Anthony; Ling, Kak-Ming; Mead-Hunter, Ryan; Larcombe, Alexander N

    2016-01-01

    Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size.

  13. Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine.

    PubMed

    Goncz, K K; Feeney, L; Gruenert, D C

    1999-09-01

    1. Exposure to epinephrine has been shown to have a range of effects on cells and tissues. A recent study suggested that the proliferative ability of CF epithelial cells, exposed to high concentrations of epinephrine (200 - 300 microM), was reduced when compared to that of normal cells. This approach could potentially provide a means to effectively separate cells with functional cyclic AMP-dependent Cl-ion transport from those defective in this pathway. 2. The sensitivity to killing by epinephrine is reported here for four different CF cell lines, three normal cell lines, and two CF epithelial cell lines complemented with wild-type (wt) CF transmembrane conductance regulator (CFTR) cDNA. 3. While each cell line exhibited varying sensitivity to 200 microM epinephrine, no predictable pattern was observed between the expression of wt-CFTR and cell survival following epinephrine exposure. Overall, normal cell lines did exhibit a greater resistance to epinephrine-induced cell death although, the most resistant cell line was derived from CF tracheal epithelium (SigmaCFTE29o-). 4. The expression of exogenous wt-CFTR increased the survival of one cell line (CFDEo-) when compared to the parent line, but in another complemented line, survival was reduced. 5. These findings suggest that while epinephrine induces cell killing, it is not consistently effective for preferential selection of normal over CF cells. Although CFTR may play a role in the mechanism(s) of epinephrine killing, other factors such as cell density, proliferative ability, cell type origin and phenotype are involved.

  14. Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response

    PubMed Central

    Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Groshong, Steve D.; Ito, Yoko; Travanty, Emily A.; Leete, Jennifer; Holmes, Kathryn V.; Mason, Robert J.

    2009-01-01

    The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6–8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host. PMID:19741068

  15. CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells

    PubMed Central

    Chirkova, Tatiana; Lin, Songbai; Oomens, Antonius G. P.; Gaston, Kelsey A.; Boyoglu-Barnum, Seyhan; Meng, Jia; Stobart, Christopher C.; Cotton, Calvin U.; Hartert, Tina V.; Moore, Martin L.; Ziady, Assem G.

    2015-01-01

    Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection. PMID:26297201

  16. Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells

    PubMed Central

    Moreau-Marquis, Sophie; Coutermarsh, Bonita; Stanton, Bruce A.

    2015-01-01

    Objectives Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells. Methods P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting. Results ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms. Conclusions Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI® (tobramycin) or Cayston® (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa. PMID:25213272

  17. A human esophageal epithelial cell model for study of radiation induced cancer and DNA repair

    NASA Astrophysics Data System (ADS)

    Huff, Janice; Patel, Zarana; Hada, Megumi; Cucinotta, Francis A.

    For cancer risk assessment in astronauts and for countermeasure development, it is essential to understand the molecular mechanisms of radiation carcinogenesis and how these mechanisms are influenced by exposure to the types of radiation found in space. We are developing an in vitro model system for the study of radiation-induced initiation and progression of esophageal carcinoma. Development of squamous cell carcinoma of the esophagus is associated with radiation exposure, as revealed by the significant enhanced in incidence rates for this type of cancer in the survivors of the atomic bomb detonations in Japan. It is also associated with poor nutritional status and micronutrient deficiencies, which are also important issues for long duration spaceflight. The possible synergies between nutritional issues and radiation exposure are unknown. Here we present the results of preliminary characterization of both normal and hTERT-immortalized esophageal epithelial cells grown in 2-dimensional culture. We analyzed DNA repair capacity by measuring the kinetics of formation and loss of gamma-H2AX foci following radiation exposure. Additionally, we analyzed induction of chromosomal aberrations using 3-color fluorescence in situ hybridization (FISH). Data were generated using both low LET (gamma rays) and high LET ions (1000 MeV/nucleon iron.

  18. Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells.

    PubMed

    Wages, Phillip A; Silbajoris, Robert; Speen, Adam; Brighton, Luisa; Henriquez, Andres; Tong, Haiyan; Bromberg, Philip A; Simmons, Steven O; Samet, James M

    2014-01-01

    Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn(2+)) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn(2+) toxicity is not fully understood. H2O2 and Zn(2+) have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn(2+) to cause cellular H2O2 production. To determine the role of Zn(2+)-induced H2O2 production in the human airway epithelial cell response to Zn(2+) exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50µM Zn(2+) for 5min in the presence of 1µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn(2+) exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn(2+)-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn(2+)-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn(2+) leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms. PMID:25462065

  19. Effects of 10 Cigarette Smoke Condensates on Primary Human Airway Epithelial Cells by Comparative Gene and Cytokine Expression Studies

    PubMed Central

    Pickett, Gavin; Seagrave, JeanClare; Boggs, Susan; Polzin, Gregory; Richter, Patricia; Tesfaigzi, Yohannes

    2010-01-01

    Cigarettes vary in tobacco blend, filter ventilation, additives, and other physical and chemical properties, but little is known regarding potential differences in toxicity to a smoker’s airway epithelia. We compared changes in gene expression and cytokine production in primary normal human bronchial epithelial cells following treatment for 18 h with cigarette smoke condensates (CSCs) prepared from five commercial and four research cigarettes, at doses of ∼4 μg/ml nicotine. Nine of the CSCs were produced under a standard International Organization for Standardization smoking machine regimen and one was produced by a more intense smoking machine regimen. Isolated messenger RNA (mRNA) was analyzed by microarray hybridization, and media was analyzed for secreted cytokines and chemokines. Twenty-one genes were differentially expressed by at least 9 of the 10 CSCs by more than twofold, including genes encoding detoxifying and antioxidant proteins. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and NAD(P)H dehydrogenase, quinone 1 (NQO-1) were selected for validation with quantitative real-time PCR (qRT-PCR) and Western blot analyses. NQO-1 expression determined with microarrays, qRT-PCR, and Western blotting differed among the CSC types, with good correlation among the different assays. CYP1A1 mRNA levels varied substantially, but there was little correlation with the protein levels. For each CSC, the three most induced and three most repressed genes were identified. These genes may be useful as markers of exposure to that particular cigarette type. Furthermore, differences in interleukin-8 secretion were observed. These studies lay the foundation for future investigations to analyze differences in the responses of in vivo systems to tobacco products marketed with claims of reduced exposure or reduced harm. PMID:20015843

  20. Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Hanrahan, John W

    2012-01-01

    Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl− and HCO3− secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3− net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl− and HCO3− net fluxes. Ieq and HCO3− fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3− is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3−. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3− secretion, suggesting that HCO3− transport under these conditions requires catalysed synthesis of carbonic acid. Cl− was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50–70% of Cl− and fluid transport was bumetanide-insensitive, suggesting basolateral Cl− loading by a sodium–potassium–chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO3− gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO3− secretion was increased by bilateral Cl− removal and therefore did not require apical Cl−/HCO3− exchange. The results suggest a model in which most HCO3− is recycled basolaterally by exchange with Cl−, and the resulting HCO3−-dependent Cl− transport provides an osmotic driving force for

  1. Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.

    PubMed

    Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Hanrahan, John W

    2012-11-01

    Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl(-) and HCO(3)(-) secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (I(sc)) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (I(eq)) calculated under open-circuit conditions. I(sc) was equivalent to the HCO(3)(-) net flux measured using the pH-stat technique, whereas I(eq) was the sum of the Cl(-) and HCO(3)(-) net fluxes. I(eq) and HCO(3)(-) fluxes were increased by bafilomycin and ZnCl(2), suggesting that some secreted HCO(3)(-) is neutralized by parallel electrogenic H(+) secretion. I(eq) and fluid secretion were dependent on the presence of both Na(+) and HCO(3)(-). The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I(eq) and HCO(3)(-) secretion, suggesting that HCO(3)(-) transport under these conditions requires catalysed synthesis of carbonic acid. Cl(-) was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl(-) and fluid transport was bumetanide-insensitive, suggesting basolateral Cl(-) loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO(3)(-) gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO(3)(-) secretion was increased by bilateral Cl(-) removal and therefore did not require apical Cl(-)/HCO(3)(-) exchange. The results suggest a model in which most HCO(3)(-) is recycled basolaterally by exchange with Cl(-), and the resulting HCO(3)(-)-dependent Cl(-) transport

  2. Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

    PubMed Central

    Johnson, Sara M.; McNally, Beth A.; Ioannidis, Ioannis; Flano, Emilio; Teng, Michael N.; Oomens, Antonius G.; Walsh, Edward E.; Peeples, Mark E.

    2015-01-01

    Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization. PMID:26658574

  3. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents.

    PubMed

    Zaccone, Eric J; Goldsmith, W Travis; Shimko, Michael J; Wells, J R; Schwegler-Berry, Diane; Willard, Patsy A; Case, Shannon L; Thompson, Janet A; Fedan, Jeffrey S

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance,we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity.We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport,without affecting Cl- transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100-360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro.

  4. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    PubMed Central

    Zaccone, Eric J.; Goldsmith, W. Travis; Shimko, Michael J.; Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A.; Fedan, Jeffrey S.

    2016-01-01

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport, without affecting Cl− transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. PMID:26454031

  5. Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

    PubMed

    Hobbs, Carey A; Blanchard, Maxime G; Alijevic, Omar; Tan, Chong Da; Kellenberger, Stephan; Bencharit, Sompop; Cao, Rui; Kesimer, Mehmet; Walton, William G; Henderson, Ashley G; Redinbo, Matthew R; Stutts, M Jackson; Tarran, Robert

    2013-12-01

    The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease. PMID:24124190

  6. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents.

    PubMed

    Zaccone, Eric J; Goldsmith, W Travis; Shimko, Michael J; Wells, J R; Schwegler-Berry, Diane; Willard, Patsy A; Case, Shannon L; Thompson, Janet A; Fedan, Jeffrey S

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance,we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity.We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport,without affecting Cl- transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100-360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. PMID:26454031

  7. Fructose transport-deficient Staphylococcus aureus reveals important role of epithelial glucose transporters in limiting sugar-driven bacterial growth in airway surface liquid.

    PubMed

    Garnett, James P; Braun, Daniela; McCarthy, Alex J; Farrant, Matthew R; Baker, Emma H; Lindsay, Jodi A; Baines, Deborah L

    2014-12-01

    Hyperglycaemia as a result of diabetes mellitus or acute illness is associated with increased susceptibility to respiratory infection with Staphylococcus aureus. Hyperglycaemia increases the concentration of glucose in airway surface liquid (ASL) and promotes the growth of S. aureus in vitro and in vivo. Whether elevation of other sugars in the blood, such as fructose, also results in increased concentrations in ASL is unknown and whether sugars in ASL are directly utilised by S. aureus for growth has not been investigated. We obtained mutant S. aureus JE2 strains with transposon disrupted sugar transport genes. NE768(fruA) exhibited restricted growth in 10 mM fructose. In H441 airway epithelial-bacterial co-culture, elevation of basolateral sugar concentration (5-20 mM) increased the apical growth of JE2. However, sugar-induced growth of NE768(fruA) was significantly less when basolateral fructose rather than glucose was elevated. This is the first experimental evidence to show that S. aureus directly utilises sugars present in the ASL for growth. Interestingly, JE2 growth was promoted less by glucose than fructose. Net transepithelial flux of D-glucose was lower than D-fructose. However, uptake of D-glucose was higher than D-fructose across both apical and basolateral membranes consistent with the presence of GLUT1/10 in the airway epithelium. Therefore, we propose that the preferential uptake of glucose (compared to fructose) limits its accumulation in ASL. Pre-treatment with metformin increased transepithelial resistance and reduced the sugar-dependent growth of S. aureus. Thus, epithelial paracellular permeability and glucose transport mechanisms are vital to maintain low glucose concentration in ASL and limit bacterial nutrient sources as a defence against infection.

  8. Influenza A virus (H1N1) increases airway epithelial cell secretion by up-regulation of potassium channel KCNN4.

    PubMed

    Waugh, Taryn; Ching, John C H; Zhou, Yan; Loewen, Matthew E

    2013-09-01

    Influenza infects the epithelial cells lining the airways. Normally epithelial cells move solutes through ion channels to create the osmotic drive to hydrate the airways. Viral alteration of this process could explain, in part, the fluid imbalance in the lungs and the resulting pulmonary edema that occurs during severe influenza infections. Using western blot and RT-qPCR, we measured ion channel and cytokine expression in the Calu3 airway cell line after infection with influenza virus (H1N1) for 48 h. We simultaneously measured chloride and potassium channel function by means of a short-circuit current (I(sc)) produced in an Ussing chamber. At a multiplicity of infection (MOI) of 10, viral M1 protein and pro-inflammatory cytokine expression was observed 24h post-infection, despite a lack of measurable change in Isc. However, we observed a decreased secretory response in cAMP- and calcium-induced Isc 48 h post-infection. This correlated with a decrease in CFTR and KCNN4 protein levels. Interestingly, a viral dose of an MOI 0.6 revealed an increased secretory response that correlated with pro-inflammatory cytokine expression. This increased secretory response seemed to be primarily driven through KCNN4. We detected an increase in KCNN4 mRNA and protein, while CFTR function and expression remained unchanged. Furthermore, inhibition of the KCNN4-stimulated I(sc) with TRAM-34, a specific inhibitor, ameliorated the response, implicating KCNN4 as the main driving force behind the secretory phenotype.

  9. Differential susceptibility of inbred mouse strains to chlorine-induced airway fibrosis.

    PubMed

    Mo, Yiqun; Chen, Jing; Schlueter, Connie F; Hoyle, Gary W

    2013-01-15

    Chlorine is a reactive gas that is considered a chemical threat agent. Humans who develop acute lung injury from chlorine inhalation typically recover normal lung function; however, a subset can experience chronic airway disease. To examine pathological changes following chlorine-induced lung injury, mice were exposed to a single high dose of chlorine, and repair of the lung was analyzed at multiple times after exposure. In FVB/NJ mice, chlorine inhalation caused pronounced fibrosis of larger airways that developed by day 7 after exposure and was associated with airway hyperreactivity. In contrast, A/J mice had little or no airway fibrosis and had normal lung function at day 7. Unexposed FVB/NJ mice had less keratin 5 staining (basal cell marker) than A/J mice in large intrapulmonary airways where epithelial repair was poor and fibrosis developed after chlorine exposure. FVB/NJ mice had large areas devoid of epithelium on day 1 after exposure leading to fibroproliferative lesions on days 4 and 7. A/J mice had airways covered by squamous keratin 5-stained cells on day 1 that transitioned to a highly proliferative reparative epithelium by day 4 followed by the reappearance of ciliated and Clara cells by day 7. The data suggest that lack of basal cells in the large intrapulmonary airways and failure to effect epithelial repair at these sites are factors contributing to the development of airway fibrosis in FVB/NJ mice. The observed differences in susceptibility to chlorine-induced airway disease provide a model in which mechanisms and treatment of airway fibrosis can be investigated.

  10. Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair

    PubMed Central

    Quantius, Jennifer; Schmoldt, Carole; Vazquez-Armendariz, Ana I.; Becker, Christin; El Agha, Elie; Wilhelm, Jochen; Morty, Rory E.; Vadász, István; Mayer, Konstantin; Gattenloehner, Stefan; Fink, Ludger; Matrosovich, Mikhail; Li, Xiaokun; Seeger, Werner; Lohmeyer, Juergen; Bellusci, Saverio; Herold, Susanne

    2016-01-01

    Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(β4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of β-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10

  11. Branching Morphogenesis of Immortalized Human Bronchial Epithelial Cells in Three-Dimensional Culture

    PubMed Central

    Kaisani, Aadil; Delgado, Oliver; Fasciani, Gail; Kim, Sang Bum; Wright, Woodring E.; Minna, John D.; Shay, Jerry W.

    2014-01-01

    While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases. PMID:24830354

  12. Low-level laser irradiation promotes the proliferation and maturation of keratinocytes during epithelial wound repair

    PubMed Central

    Sperandio, Felipe F.; Simões, Alyne; Corrêa, Luciana; Aranha, Ana Cecília C.; Giudice, Fernanda S.; Hamblin, Michael R.; Sousa, Suzana C.O.M.

    2015-01-01

    Low-level laser therapy (LLLT) has been extensively employed to improve epithelial wound healing, though the exact response of epithelium maturation and stratification after LLLT is unknown. Thus, this study aimed to assess the in vitro growth and differentiation of keratinocytes (KCs) and in vivo wound healing response when treated with LLLT. Human KCs (HaCaT cells) showed an enhanced proliferation with all the employed laser energy densities (3, 6 and 12 J/cm2, 660nm, 100mW), together with an increased expression of Cyclin D1. Moreover, the immunoexpression of proteins related to epithelial proliferation and maturation (p63, CK10, CK14) all indicated a faster maturation of the migrating KCs in the LLLT-treated wounds. In that way, an improved epithelial healing was promoted by LLLT with the employed parameters; this improvement was confirmed by changes in the expression of several proteins related to epithelial proliferation and maturation. PMID:25411997

  13. 7,12-dimethylbenz(a)anthracene-induced DNA binding and repair synthesis in susceptible and nonsusceptible mammary epithelial cells in culture. [Rats

    SciTech Connect

    Tay, L.K.; Russo, J.

    1981-07-01

    The effect of age and parity on the binding of 7,12-dimethylbenz(a)anthracene (DMBA) to DNA and the repair of DMBA-damaged DNA have been demonstrated in logarithmic phase and confluent mammary epithelial cell cultures from young virgin (YV), old virgin (OV), and parous (P) noninbred and inbred Sprague-Dawley rats. Excision repair was determined by measuring, in the presence of hydroxyurea and 5-bromodeoxyuridine, tritiated thymidine incorporation into DNA during the repair process. These results suggest that age and parity not only lower the binding of DMBA to mammary epithelial cell DNA but also increase the efficiency of DNA repair processes, which may explain the lower susceptibility of OV and P rats to DMBA-induced mammary carcinogenesis.

  14. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  15. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) increases the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells by phosphorylating Shank2E protein.

    PubMed

    Koeppen, Katja; Coutermarsh, Bonita A; Madden, Dean R; Stanton, Bruce A

    2014-06-13

    The glucocorticoid dexamethasone increases cystic fibrosis transmembrane conductance regulator (CFTR) abundance in human airway epithelial cells by a mechanism that requires serum- and glucocorticoid-induced protein kinase 1 (SGK1) activity. The goal of this study was to determine whether SGK1 increases CFTR abundance by phosphorylating Shank2E, a PDZ domain protein that contains two SGK1 phosphorylation consensus sites. We found that SGK1 phosphorylates Shank2E as well as a peptide containing the first SGK1 consensus motif of Shank2E. The dexamethasone-induced increase in CFTR abundance was diminished by overexpression of a dominant-negative Shank2E in which the SGK1 phosphorylation sites had been mutated. siRNA-mediated reduction of Shank2E also reduced the dexamethasone-induced increase in CFTR abundance. Taken together, these data demonstrate that the glucocorticoid-induced increase in CFTR abundance requires phosphorylation of Shank2E at an SGK1 consensus site.

  16. The impact of oil spill to lung health--Insights from an RNA-seq study of human airway epithelial cells.

    PubMed

    Liu, Yao-Zhong; Roy-Engel, Astrid M; Baddoo, Melody C; Flemington, Erik K; Wang, Guangdi; Wang, He

    2016-03-01

    The Deepwater Horizon oil spill (BP oil spill) in the Gulf of Mexico was a unique disaster event, where a huge amount of oil spilled from the sea bed and a large volume of dispersants were applied to clean the spill. The operation lasted for almost 3 months and involved >50,000 workers. The potential health hazards to these workers may be significant as previous research suggested an association of persistent respiratory symptoms with exposure to oil and oil dispersants. To reveal the potential effects of oil and oil dispersants on the respiratory system at the molecular level, we evaluated the transcriptomic profile of human airway epithelial cells grown under treatment of crude oil, the dispersants Corexit 9500 and Corexit 9527, and oil-dispersant mixtures. We identified a very strong effect of Corexit 9500 treatment, with 84 genes (response genes) differentially expressed in treatment vs. control samples. We discovered an interactive effect of oil-dispersant mixtures; while no response gene was found for Corexit 9527 treatment alone, cells treated with Corexit 9527+oil mixture showed an increased number of response genes (46 response genes), suggesting a synergic effect of 9527 with oil on airway epithelial cells. Through GO (gene ontology) functional term and pathway-based analysis, we identified upregulation of gene sets involved in angiogenesis and immune responses and downregulation of gene sets involved in cell junctions and steroid synthesis as the prevailing transcriptomic signatures in the cells treated with Corexit 9500, oil, or Corexit 9500+oil mixture. Interestingly, these key molecular signatures coincide with important pathological features observed in common lung diseases, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Our study provides mechanistic insights into the detrimental effects of oil and oil dispersants to the respiratory system and suggests significant health impacts of the recent BP oil spill to those people

  17. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model.

    PubMed

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4(+)CD25(+) regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4(+) or CD8(+) cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3(DTR) mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  18. The impact of oil spill to lung health--Insights from an RNA-seq study of human airway epithelial cells.

    PubMed

    Liu, Yao-Zhong; Roy-Engel, Astrid M; Baddoo, Melody C; Flemington, Erik K; Wang, Guangdi; Wang, He

    2016-03-01

    The Deepwater Horizon oil spill (BP oil spill) in the Gulf of Mexico was a unique disaster event, where a huge amount of oil spilled from the sea bed and a large volume of dispersants were applied to clean the spill. The operation lasted for almost 3 months and involved >50,000 workers. The potential health hazards to these workers may be significant as previous research suggested an association of persistent respiratory symptoms with exposure to oil and oil dispersants. To reveal the potential effects of oil and oil dispersants on the respiratory system at the molecular level, we evaluated the transcriptomic profile of human airway epithelial cells grown under treatment of crude oil, the dispersants Corexit 9500 and Corexit 9527, and oil-dispersant mixtures. We identified a very strong effect of Corexit 9500 treatment, with 84 genes (response genes) differentially expressed in treatment vs. control samples. We discovered an interactive effect of oil-dispersant mixtures; while no response gene was found for Corexit 9527 treatment alone, cells treated with Corexit 9527+oil mixture showed an increased number of response genes (46 response genes), suggesting a synergic effect of 9527 with oil on airway epithelial cells. Through GO (gene ontology) functional term and pathway-based analysis, we identified upregulation of gene sets involved in angiogenesis and immune responses and downregulation of gene sets involved in cell junctions and steroid synthesis as the prevailing transcriptomic signatures in the cells treated with Corexit 9500, oil, or Corexit 9500+oil mixture. Interestingly, these key molecular signatures coincide with important pathological features observed in common lung diseases, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Our study provides mechanistic insights into the detrimental effects of oil and oil dispersants to the respiratory system and suggests significant health impacts of the recent BP oil spill to those people

  19. Induction of regulator of G-protein signaling 2 expression by long-acting β2-adrenoceptor agonists and glucocorticoids in human airway epithelial cells.

    PubMed

    Holden, Neil S; George, Tresa; Rider, Christopher F; Chandrasekhar, Ambika; Shah, Suharsh; Kaur, Manminder; Johnson, Malcolm; Siderovski, David P; Leigh, Richard; Giembycz, Mark A; Newton, Robert

    2014-01-01

    In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting β2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and

  20. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

    PubMed Central

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4+CD25+ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4+ or CD8+ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  1. Primary in vitro culture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium and Aspergillus fumigatus interactions.

    PubMed

    Khoufache, Khaled; Cabaret, Odile; Farrugia, Cécile; Rivollet, Danièle; Alliot, Annie; Allaire, Eric; Cordonnier, Catherine; Bretagne, Stéphane; Botterel, Françoise

    2010-12-01

    Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus. PMID:20608777

  2. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

    SciTech Connect

    Kan-o, Keiko; Matsumoto, Koichiro; Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi; Inoue, Hiromasa

    2013-05-31

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

  3. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells

    PubMed Central

    Volckens, John; Dailey, Lisa; Walters, Glenn; Devlin, Robert B.

    2010-01-01

    Exposure of cultured cells to particulate matter air pollution is usually accomplished by collecting particles on a solid matrix, extracting the particles from the matrix, suspending them in liquid, and applying the suspension to cells grown on plastic and submerged in medium. The objective of this work was to develop a more physiologically and environmentally relevant model of air pollutant deposition on cultures of human primary airway epithelial cells. We hypothesize that the toxicology of inhaled particulate matter depends strongly on both the particulate dispersion state and the mode of delivery to cells. Our exposure system employs a combination of unipolar charging and electrostatic force to deposit particles directly from the air onto cells grown at an air-liquid interface in a heated, humidified exposure chamber. Normal human bronchial epithelial cells exposed to concentrated, coarse ambient particulate matter in this system expressed increased levels of inflammatory biomarkers at 1 hour following exposure and relative to controls exposed to particle-free air. More importantly, these effects are seen at particulate loadings that are 1-2 orders of magnitude lower than levels applied using traditional in vitro systems. PMID:19603682

  4. The deubiquitinating enzyme USP10 regulates the post-endocytic sorting of cystic fibrosis transmembrane conductance regulator in airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Barnaby, Roxanna L; Stanton, Bruce A

    2009-07-10

    The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.

  5. An airway epithelial iNOS-DUOX2-thyroid peroxidase metabolome drives Th1/Th2 nitrative stress in human severe asthma.

    PubMed

    Voraphani, N; Gladwin, M T; Contreras, A U; Kaminski, N; Tedrow, J R; Milosevic, J; Bleecker, E R; Meyers, D A; Ray, A; Ray, P; Erzurum, S C; Busse, W W; Zhao, J; Trudeau, J B; Wenzel, S E

    2014-09-01

    Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H(2)O(2). Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H(2)O(2), but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H(2)O(2) formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H(2)O(2) to nitrogen dioxide radical (NO(2)(•)) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS-DUOX2-TPO-NO(2)(•) metabolome drives nitrative stress in HAEC and likely in SA.

  6. The 2009 pandemic A/Wenshan/01/2009 H1N1 induces apoptotic cell death in human airway epithelial cells.

    PubMed

    Yang, Ning; Hong, Xiaoxu; Yang, Penghui; Ju, Xiangwu; Wang, Yuguo; Tang, Jun; Li, Chenggang; Fan, Quanshui; Zhang, Fuqiang; Chen, Zhongwei; Xing, Li; Zhao, Zhongpeng; Gao, Xiao; Liao, Guoyang; Li, Qihan; Wang, Xiliang; Li, Dangsheng; Jiang, Chengyu

    2011-08-01

    In 2009, a novel swine-origin H1N1 influenza virus emerged in Mexico and quickly spread to other countries, including China. This 2009 pandemic H1N1 can cause human respiratory disease, but its pathogenesis remains poorly understood. Here, we studied the infection and pathogenesis of a new 2009 pandemic strain, A/Wenshan/01/2009 H1N1, in China in human airway epithelial cell lines compared with contemporary seasonal H1N1 influenza virus. Our results showed that viral infection by the A/Wenshan H1N1 induced significant apoptotic cell death in both the human nasopharyngeal carcinoma cell line CNE-2Z and the human lung adenocarcinoma cell line A549. The A/Wenshan H1N1 virus enters both of these cell types more efficiently than the seasonal influenza virus. Viral entry in both cell lines was shown to be mediated by clathrin- and dynamin-dependent endocytosis. Therefore, we discovered that the 2009 pandemic H1N1 strain, A/Wenshan/01/2009, can induce apoptotic cell death in epithelial cells of the human respiratory tract, suggesting a molecular pathogenesis for the 2009 pandemic H1N1. PMID:21816972

  7. An Airway Epithelial iNOS-DUOX2-Thyroid Peroxidase Metabolome Drives Th1/Th2 Nitrative Stress in Human Severe Asthma

    PubMed Central

    Voraphani, N; Gladwin, MT; Contreras, AU; Kaminski, N; Tedrow, JR; Milosevic, J; Bleecker, ER; Meyers, DA; Ray, A; Ray, P; Erzurum, SC; Busse, WW; Zhao, J; Trudeau, JB; Wenzel, SE

    2014-01-01

    Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma, 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In severe asthma, high 3NT levels were associated with high IFN-γ and low IL-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAEC). We found IL-13 and IFN-γ synergistically enhanced iNOS, nitrite and 3NT, corresponding with increased H2O2. Catalase inhibited while superoxide dismutase enhanced 3NT formation, supporting a critical role for H2O2 but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H2O2 formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H2O2 to nitrogen dioxide radical (NO2•) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of severe asthma from controls. IFN-γ induced TPO in HAEC and siRNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO and iNOS were higher in severe asthma and correlated with 3NT. Thus a novel iNOS-DUOX2-TPO-NO2• metabolome drives nitrative stress in HAEC and likely in severe asthma. PMID:24518246

  8. Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells

    PubMed Central

    Nassour, Joe; Martien, Sébastien; Martin, Nathalie; Deruy, Emeric; Tomellini, Elisa; Malaquin, Nicolas; Bouali, Fatima; Sabatier, Laure; Wernert, Nicolas; Pinte, Sébastien; Gilson, Eric; Pourtier, Albin; Pluquet, Olivier; Abbadie, Corinne

    2016-01-01

    The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis. PMID:26822533

  9. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

    PubMed

    Mallet, Justin D; Dorr, Marie M; Drigeard Desgarnier, Marie-Catherine; Bastien, Nathalie; Gendron, Sébastien P; Rochette, Patrick J

    2016-01-01

    Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced. PMID:27611318

  10. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes

    PubMed Central

    Mallet, Justin D.; Bastien, Nathalie; Gendron, Sébastien P.; Rochette, Patrick J.

    2016-01-01

    Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced. PMID:27611318

  11. Drosophila Imaginal Discs as a Model of Epithelial Wound Repair and Regeneration

    PubMed Central

    Smith-Bolton, Rachel

    2016-01-01

    Significance: The Drosophila larval imaginal discs, which form the adult fly during metamorphosis, are an established model system for the study of epithelial tissue damage. The disc proper is a simple columnar epithelium, but it contains complex patterning and cell-fate specification, and is genetically tractable. These features enable unbiased genetic screens to identify genes involved in all aspects of the wound response, from sensing damage to wound closure, initiation of regeneration, and re-establishment of proper cell fates. Identification of the genes that facilitate epithelial wound closure and regeneration will enable development of more sophisticated wound treatments for clinical use. Recent Advances: Imaginal disc epithelia can be damaged in many different ways, including fragmentation, induction of cell death, and irradiation. Recent work has demonstrated that the tissue's response to damage varies depending on how the wound was induced. Here, we summarize the different responses activated in these epithelial tissues after the different types of damage. Critical Issues: These studies highlight that not all wounds elicit the same response from the surrounding tissue. A complete understanding of the various wound-healing mechanisms in Drosophila will be a first step in understanding how to manage damaged human tissues and optimize healing in different clinical contexts. Future Directions: Further work is necessary to understand the similarities and differences among an epithelial tissue's responses to different insults. Ongoing studies will identify the genes and pathways employed by injured imaginal discs. Thus, work in this genetically tractable system complements work in more conventional wound-healing models. PMID:27274435

  12. Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3.

    PubMed

    Kim, Dusik; Kim, Juyeon; Burghardt, Beáta; Best, Len; Steward, Martin C

    2014-07-15

    Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.

  13. Pregnane X receptor agonists enhance intestinal epithelial wound healing and repair of the intestinal barrier following the induction of experimental colitis.

    PubMed

    Terc, Joshua; Hansen, Ashleigh; Alston, Laurie; Hirota, Simon A

    2014-05-13

    The intestinal epithelial barrier plays a key role in the maintenance of homeostasis within the gastrointestinal tract. Barrier dysfunction leading to increased epithelial permeability is associated with a number of gastrointestinal disorders including the inflammatory bowel diseases (IBD) - Crohn's disease and ulcerative colitis. It is thought that the increased permeability in patients with IBD may be driven by alterations in the epithelial wound healing response. To this end considerable study has been undertaken to identify signaling pathways that may accelerate intestinal epithelial wound healing and normalize the barrier dysfunction observed in IBD. In the current study we examined the role of the pregnane X receptor (PXR) in modulating the intestinal epithelial wound healing response. Mutations and reduced mucosal expression of the PXR are associated with IBD, and others have reported that PXR agonists can dampen intestinal inflammation. Furthermore, stimulation of the PXR has been associated with increased cell migration and proliferation, two of the key processes involved in wound healing. We hypothesized that PXR agonists would enhance intestinal epithelial repair. Stimulation of Caco-2 intestinal epithelial cells with rifaximin, rifampicin and SR12813, all potent agonists of the PXR, significantly increased wound closure. This effect was driven by p38 MAP kinase-dependent cell migration, and occurred in the absence of cell proliferation. Treating mice with a rodent specific PXR agonist, pregnenolone 16α-carbonitrile (PCN), attenuated the intestinal barrier dysfunction observed in the dextran sulphate sodium (DSS) model of experimental colitis, an effect that occurred independent of the known anti-inflammatory effects of PCN. Taken together our data indicate that the activation of the PXR can enhance intestinal epithelial repair and suggest that targeting the PXR may help to normalize intestinal barrier dysfunction observed in patients with IBD

  14. Nucleotide excision repair is reduced in oral epithelial tissues compared with skin.

    PubMed

    Mitchell, David; Paniker, Lakshmi; Godar, Dianne

    2012-01-01

    Ultraviolet radiation (UVR) exposure to internal tissues for diagnostic, therapeutic and cosmetic procedures has increased dramatically over the past decade. The greatest increase in UVR exposure of internal tissues occurs in the cosmetic industry where it is combined with oxidizing agents for teeth whitening, often in conjunction with indoor tanning. To address potential carcinogenic risks of these procedures, we analyzed the formation and repair of the DNA photoproducts associated with the signature mutations of UVR. Radioimmunoassay was used to quantify the induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in DNA purified from three reconstructed tissues, EpiDerm(TM) , EpiGingival(TM) and EpiOral(TM) . We observed comparable levels of DNA damage in all tissues immediately after UVR exposure. In contrast, repair was significantly reduced in both oral tissues compared with EpiDerm(TM) . Our data suggest that UVR exposure of oral tissues can result in accumulation of DNA damage and increase the risk for carcinoma and melanoma of the mouth. Because NER is a broad-spectrum defense against DNA damage caused by a variety of agents in addition to UVR, our data suggest that the relatively low NER efficiency observed in oral tissues may have wide-ranging consequences in this highly exposed environment.

  15. Nucleotide excision repair is reduced in oral epithelial tissues compared to skin‡

    PubMed Central

    Mitchell, David; Paniker, Lakshmi; Godar, Dianne

    2012-01-01

    Ultraviolet radiation (UVR) exposure to internal tissues for diagnostic, therapeutic and cosmetic procedures has increased dramatically over the past decade. The greatest increase in UVR exposure of internal tissues occurs in the cosmetic industry where it is combined with oxidizing agents for teeth whitening, often in conjunction with indoor tanning. To address potential carcinogenic risks of these procedures, we analyzed the formation and repair of the DNA photoproducts associated with the signature mutations of UVR. Radioimmunoassay was used to quantify the induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in DNA purified from three reconstructed tissues, EpiDerm™, EpiGingival™, and EpiOral™ (MatTek Corp.). We observed comparable levels of DNA damage in all tissues immediately after UVR exposure. In contrast, repair was significantly reduced in both oral tissues compared to EpiDerm™. Our data suggest that UVR exposure of oral tissues can result in accumulation of DNA damage and increase the risk for carcinoma and melanoma of the mouth. Because NER is a broad-spectrum defense against DNA damage caused by a variety of agents in addition to UVR, our data suggest that the relatively low NER efficiency observed in oral tissues may have wide-ranging consequences in this highly exposed environment. PMID:22519509

  16. Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

    PubMed Central

    Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

    2014-01-01

    As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

  17. Effects of human Parvovirus B19 and Bocavirus VP1 unique region on tight junction of human airway epithelial A549 cells.

    PubMed

    Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

    2014-01-01

    As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity.

  18. Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier

    PubMed Central

    Bashir, Mohamed Elfatih H.; Ward, Jason M.; Cummings, Matthew; Karrar, Eltayeb E.; Root, Michael; Mohamed, Abu Bekr A.; Naclerio, Robert M.; Preuss, Daphne

    2013-01-01

    Background The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored. Methodology/Principal Findings Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. Conclusions/Significance Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is

  19. Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Huang, Junwei; Shan, Jiajie; Kim, Dusik; Liao, Jie; Evagelidis, Alexandra; Alper, Seth L; Hanrahan, John W

    2012-01-01

    Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl−/HCO3− exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (Isc). We have studied the role of AE2 in Cl− and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced ≥90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na+–K+–2Cl− cotransporter) or NBCe1 (Na+–nHCO3− cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO3−] as compared with the control lines. Unstimulated equivalent short-circuit current (Ieq) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both Ieq and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl−/HCO3− exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl− removal was reduced by ∼80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl− loading during cAMP-stimulated secretion of Cl− and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway

  20. Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3.

    PubMed

    Huang, Junwei; Shan, Jiajie; Kim, Dusik; Liao, Jie; Evagelidis, Alexandra; Alper, Seth L; Hanrahan, John W

    2012-11-01

    Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl(-)/HCO(3)(-) exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (I(sc)). We have studied the role of AE2 in Cl(-) and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced 90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na(+)-K(+)-2Cl(-) cotransporter) or NBCe1 (Na(+)-nHCO(3)(-) cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO(3)(-)] as compared with the control lines. Unstimulated equivalent short-circuit current (I(eq)) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both I(eq) and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl(-)/HCO(3)(-) exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl(-) removal was reduced by ∼80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl(-) loading during cAMP-stimulated secretion of Cl(-) and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in

  1. Function and repair of dental enamel - Potential role of epithelial transport processes of ameloblasts.

    PubMed

    Varga, Gábor; Kerémi, Beáta; Bori, Erzsébet; Földes, Anna

    2015-07-01

    The hardest mammalian tissue, dental enamel is produced by ameloblasts, which are electrolyte-transporting epithelial cells. Although the end product is very different, they show many similarities to transporting epithelia of the pancreas, salivary glands and kidney. Enamel is produced in a multi-step epithelial secretory process that features biomineralization which is an interplay of secreted ameloblast specific proteins and the time-specific transport of minerals, protons and bicarbonate. First, "secretory" ameloblasts form the entire thickness of the enamel layer, but with low mineral content. Then they differentiate into "maturation" ameloblasts, which remove organic matrix from the enamel and in turn further build up hydroxyapatite crystals. The protons generated by hydroxyapatite formation need to be buffered, otherwise enamel will not attain full mineralization. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. The whole process has been the focus of many immunohistochemical and gene knock-out studies, but, perhaps surprisingly, no functional data existed for mineral ion transport by ameloblasts. However, recent studies including ours provided a better insight for molecular mechanism of mineral formation. The secretory regulation is not completely known as yet, but its significance is crucial. Impairing regulation retards or prevents completion of enamel mineralization and results in the development of hypomineralized enamel that easily erodes after dental eruption. Factors that impair this function are fluoride and disruption of pH regulators. Revealing these factors may eventually lead to the treatment of enamel hypomineralization related to genetic or environmentally induced malformation.

  2. Let-7a modulates particulate matter (≤ 2.5 μm)-induced oxidative stress and injury in human airway epithelial cells by targeting arginase 2.

    PubMed

    Song, Lei; Li, Dan; Gu, Yue; Li, Xiaoping; Peng, Liping

    2016-10-01

    Epidemiological studies show that particulate matter (PM) with an aerodynamic diameter ≤ 2.5 μm (PM2.5) is associated with cardiorespiratory diseases via the induction of excessive oxidative stress. However, the precise mechanism underlying PM2.5-mediated oxidative stress injury has not been fully elucidated. Accumulating evidence has indicated the microRNA let-7 family might play a role in PM-mediated pathological processes. In this study, we investigated the role of let-7a in oxidative stress and cell injury in human bronchial epithelial BEAS2B (B2B) cells after PM2.5 exposure. The let-7a level was the most significantly decreased in B2B cells after PM2.5 exposure. The overexpression of let-7a suppressed intracellular reactive oxygen species levels and the percentage of apoptotic cells after PM2.5 exposure, while the let-7a level decreased arginase 2 (ARG2) mRNA and protein levels in B2B cells by directly targeting the ARG2 3'-untranslated region. ARG2 expression was upregulated in B2B cells during PM2.5 treatment, and ARG2 knockdown could remarkably reduce oxidative stress and cellular injury. Moreover, its restoration could abrogate the protective effects of let-7a against PM2.5-induced injury. In conclusion, let-7a decreases and ARG2 increases resulting from PM2.5 exposure may exacerbate oxidative stress, cell injury and apoptosis of B2B cells. The let-7a/ARG2 axis is a likely therapeutic target for PM2.5-induced airway epithelial injury. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Human tryptase epsilon (PRSS22), a new member of the chromosome 16p13.3 family of human serine proteases expressed in airway epithelial cells.

    PubMed

    Wong, G W; Yasuda, S; Madhusudhan, M S; Li, L; Yang, Y; Krilis, S A; Sali, A; Stevens, R L

    2001-12-28

    Probing of the GenBank expressed sequence tag (EST) data base with varied human tryptase cDNAs identified two truncated ESTs that subsequently were found to encode overlapping portions of a novel human serine protease (designated tryptase epsilon or protease, serine S1 family member 22 (PRSS22)). The tryptase epsilon gene resides on chromosome 16p13.3 within a 2.5-Mb complex of serine protease genes. Although at least 7 of the 14 genes in this complex encode enzymatically active proteases, only one tryptase epsilon-like gene was identified. The trachea and esophagus were found to contain the highest steady-state levels of the tryptase epsilon transcript in adult humans. Although the tryptase epsilon transcript was scarce in adult human lung, it was present in abundance in fetal lung. Thus, the tryptase epsilon gene is expressed in the airways in a developmentally regulated manner that is different from that of other human tryptase genes. At the cellular level, tryptase epsilon is a major product of normal pulmonary epithelial cells, as well as varied transformed epithelial cell lines. Enzymatically active tryptase epsilon is also constitutively secreted from these cells. The amino acid sequence of human tryptase epsilon is 38-44% identical to those of human tryptase alpha, tryptase beta I, tryptase beta II, tryptase beta III, transmembrane tryptase/tryptase gamma, marapsin, and Esp-1/testisin. Nevertheless, comparative protein structure modeling and functional studies using recombinant material revealed that tryptase epsilon has a substrate preference distinct from that of its other family members. These data indicate that the products of the chromosome 16p13.3 complex of tryptase genes evolved to carry out varied functions in humans.

  4. Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells.

    PubMed

    Lam, W Y; Tang, Julian W; Yeung, Apple C M; Chiu, Lawrence C M; Sung, Joseph J Y; Chan, Paul K S

    2008-03-01

    Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells, which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay, flow cytometric measurement of propidium iodide, annexin V staining, and Western blot analyses with antibodies specific for proapoptotic and antiapoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase and activation of caspase-3, caspase-7, and caspase-8 during the NS1-induced apoptosis. The results of caspase inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1 NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (the time posttransfection required to reach >30% apoptosis was reduced from 24 to 6 h). Furthermore, 24 h posttransfection, an increase in Fas ligand mRNA expression of about 5.6-fold was detected in cells transfected with H5N1 NS1. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A virus H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.

  5. The effect of nedocromil sodium on human airway epithelial cell-induced eosinophil chemotaxis and adherence to human endothelial cell in vitro.

    PubMed

    Abdelaziz, M M; Devalia, J L; Khair, O A; Rusznak, C; Calderon, M; Sapsford, R J; Bayram, H; Davies, R J

    1997-04-01

    Although some studies have shown that long-term treatment of asthmatics with nedocromil sodium can reduce airway hyperresponsiveness and improve symptoms and lung function, the mechanisms underlying its effects are not well understood. We have investigated the effect of nedocromil sodium on eosinophil chemotaxis, eosinophil adherence to human endothelial cells and release of soluble intercellular adhesion molecule-1 (sICAM-1) from endothelial cells, induced by conditioned medium collected from cultured human bronchial epithelial cells. Conditioned medium significantly increased eosinophil chemotaxis from a baseline median value of 2.1 (range 1.9-4.5) cells-high power field(-1) (HPF) to 10.5 (range 7.8-12.3) cells-HPF(-1) (p<0.05). Similarly, conditioned medium significantly increased eosinophil adherence to endothelial cells from a baseline value of 9 (range 8-12)% to 23 (range 21-30)% (p<0.05). Nedocromil sodium, at 10(-5) M concentration, significantly attenuated the eosinophil chemotaxis and adherence induced by conditioned medium. Conditioned medium also significantly increased the release of sICAM-1 from endothelial cells, from a baseline value of 11.5 (range 8.1-15.4) pg x microg(-1) protein to 67.6 (range 55.6-73.5) pg x microg(-1) protein (p<0.05). This was significantly attenuated by anti-tumour necrosis factor-alpha (TNF-alpha), anti-interleukin-1beta (IL-1beta) and 10(-5) M nedocromil sodium. These findings suggest that human bronchial epithelial cell-derived mediators may potentiate eosinophil activity, and that this can be modulated by nedocromil sodium, suggesting a possible mechanism underlying its anti-inflammatory effect.

  6. Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

    PubMed Central

    Johnson, L G; Boyles, S E; Wilson, J; Boucher, R C

    1995-01-01

    Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis. Images PMID:7533790

  7. Analysis of aberrant methylation in DNA repair genes during malignant transformation of human bronchial epithelial cells induced by cadmium.

    PubMed

    Zhou, Zhi-heng; Lei, Yi-xiong; Wang, Cai-xia

    2012-02-01

    Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood yet. Aberrant methylation was investigated in order to obtain insight into the DNA repair-related epigenetic mechanisms underlying CdCl(2)-induced malignant transformation of human bronchial epithelial cells (16HBE). Gene expression and DNA methylation were assessed in untreated control cells; 5th, 15th, and 35th passage of CdCl2-treated cells and tumorigenic cells (TCs) from nude mice by using high-performance liquid chromatography, real-time PCR, Western blot analysis, and methylation-specific PCR assay. During Cd-induced malignant transformation, global DNA methylation progressively increased and was associated with the overexpression of the DNA methyltransferase genes DNMT1 and DNMT3a but not DNMT3b. Expression of both the messenger RNA and proteins of the DNA repair genes (hMSH2, ERCC1, XRCC1, and hOGG1) progressively reduced and DNA damage increased with Cd-induced transformation. The promoter regions of hMSH2, ERCC1, XRCC1, and hOGG1 were heavily methylated in the 35th passage transformed cells and the TCs. The DNA demethylating agent 5-aza-2'-deoxycytidine could reverse the Cd-induced global DNA hypermethylation, DNMT hyperactivity, and the silencing of hMSH2, ERCC1, XRCC1, and hOGG1 in a time-dependent manner. The results indicate that DNMT1 and DNMT3a overexpression can result in global DNA hypermethylation and silencing of the hMSH2, ERCC1, XRCC1, and hOGG1 genes. They may partly explain the epigenetic mechanisms underlying the carcinogenesis due to Cd.

  8. Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

    PubMed

    Chen, Mei; Rajapakse, Dinusha; Fraczek, Monika; Luo, Chang; Forrester, John V; Xu, Heping

    2016-06-01

    Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

  9. Store-Operated Ca2+ Release-Activated Ca2+ Channels Regulate PAR2-Activated Ca2+ Signaling and Cytokine Production in Airway Epithelial Cells.

    PubMed

    Jairaman, Amit; Yamashita, Megumi; Schleimer, Robert P; Prakriya, Murali

    2015-09-01

    The G-protein-coupled protease-activated receptor 2 (PAR2) plays an important role in the pathogenesis of various inflammatory and auto-immune disorders. In airway epithelial cells (AECs), stimulation of PAR2 by allergens and proteases triggers the release of a host of inflammatory mediators to regulate bronchomotor tone and immune cell recruitment. Activation of PAR2 turns on several cell signaling pathways of which the mobilization of cytosolic Ca(2+) is likely a critical but poorly understood event. In this study, we show that Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by stromal interaction molecule 1 and Orai1 are a major route of Ca(2+) entry in primary human AECs and drive the Ca(2+) elevations seen in response to PAR2 activation. Activation of CRAC channels induces the production of several key inflammatory mediators from AECs including thymic stromal lymphopoietin, IL-6, and PGE2, in part through stimulation of gene expression via nuclear factor of activated T cells (NFAT). Furthermore, PAR2 stimulation induces the production of many key inflammatory mediators including PGE2, IL-6, IL-8, and GM-CSF in a CRAC channel-dependent manner. These findings indicate that CRAC channels are the primary mechanism for Ca(2+) influx in AECs and a vital checkpoint for the induction of PAR2-induced proinflammatory cytokines.

  10. Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.

    PubMed

    Song, Yuhu; Lou, Howard H; Boyer, Julie L; Limberis, Maria P; Vandenberghe, Luk H; Hackett, Neil R; Leopold, Philip L; Wilson, James M; Crystal, Ronald G

    2009-03-01

    Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.

  11. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    PubMed

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  12. THE EFFECTS OF COMBINATORIAL EXPOSURE OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES ON AIRWAY EPITHELIAL CELL RELEASE OF CHEMOTACTIC MEDIATORS

    EPA Science Inventory

    Asthma is a chronic inflammatory disorder of the airways affecting nearly 15 million individuals nationally. Within the inflamed asthmatic airway there exist complex interactions between many cells and the cytokines they release, in particular mast cells, eosinophils, T-lymphocy...

  13. Effect of anticholinesterase agents on airway epithelial function. Annual report, 15 July 1986-14 July 1987

    SciTech Connect

    Marin, M.G.

    1987-08-01

    Irreversible anticholinesterase compounds have potential serious deleterious health effects when employed as chemical warfare agents. Intoxication with these agents causes an accumulation of acetylcholine at nerve muscle and nerve gland junctions. Because tracheal submucosal glands have rich cholinergic innervation, we hypothesized that exposure to anticholinesterase agents, such as soman, would stimulate glandular secretion. This would cause pathological changes in the important lung defense mechanism of mucociliary clearance. Despite the potential importance of anticholinesterase agents on lung function, little information was available concerning the effects of these agents on mucociliary transport. During the past year, studies were completed designed to determine the effect of soman and its antidotes on mucociliary transport. Mucociliary transport was measured on radiopertechnetate-tagged microaggregated albumin in anesthetized ferrets, utilizing two sodium-iodized crystals, collimated to detect the rate of tracheal movement over a 19-mm length of airway. In experimental animals, the author injected intravenously either soman or soman plus an antidote or an antidote alone, between the two periods. Soman resulted in a significant dose-related increase in mucociliary transport relative to control. Neither atropine nor pralidoxime alone had a significant effect on transport rate. While atropine injected simultaneously with soman blocked the stimulatory effects of soman on transport, pralidoxime failed to alter this increase in transport. Studies performed were also to determine the effect of soman and its antidotes on submucosal glandular secretion in vivo.

  14. Mucolytic treatment with N-acetylcysteine L-lysinate metered dose inhaler in dogs: airway epithelial function changes.

    PubMed

    Tomkiewicz, R P; App, E M; Coffiner, M; Fossion, J; Maes, P; King, M

    1994-01-01

    N-acetylcysteine L-lysinate Nacystelyn (L-NAC) is a newly synthesized mucolytic agent, of which the action in vivo has not been well defined. In six healthy mongrel dogs, the rheological properties of mucus, its mucociliary and cough clearability, and the transepithelial potential difference (PD) of the tracheobronchial epithelium were evaluated after placebo and L-NAC metered dose inhaler (MDI) aerosols. The principal index of mucus rigidity, log G*, decreased at all airway sites with L-NAC administration, i.e. the mucus became less rigid and more deformable (the overall change in G* was 0.29 log units, i.e. ca. twofold decrease). The viscoelasticity-derived mucus transportability parameters, mucociliary (MCI) and cough (CCI) clearability indices, increased with L-NAC MDI, particularly CCI, which predicts the effect of mucus rheology on cough clearability. PD increased significantly with L-NAC administration at all measurement sites, which appears to be a novel effect for a direct acting mucolytic agent. Tracheal mucus linear velocity (TMV) increased after L-NAC compared with placebo, as did the normalized frog palate transport rate (NFPTR). The increase in NFPTR was greater than that predicted from the mucus rheological properties alone, suggesting that L-NAC still resident in the collected mucus stimulated the frog palate cilia. The index of mucus flux, the collection rate in mg.min-1, was higher with L-NAC compared with placebo. From our results, we conclude that L-NAC shows potential benefit in terms of improving mucus rheological properties and clearability. It may act, in part, by stimulating the fresh secretion of mucus of lower viscoelasticity. The stimulation of mucociliary clearance could be related to ion flux changes, as indicated by the increase in PD. PMID:8143836

  15. Mucolytic treatment with N-acetylcysteine L-lysinate metered dose inhaler in dogs: airway epithelial function changes.

    PubMed

    Tomkiewicz, R P; App, E M; Coffiner, M; Fossion, J; Maes, P; King, M

    1994-01-01

    N-acetylcysteine L-lysinate Nacystelyn (L-NAC) is a newly synthesized mucolytic agent, of which the action in vivo has not been well defined. In six healthy mongrel dogs, the rheological properties of mucus, its mucociliary and cough clearability, and the transepithelial potential difference (PD) of the tracheobronchial epithelium were evaluated after placebo and L-NAC metered dose inhaler (MDI) aerosols. The principal index of mucus rigidity, log G*, decreased at all airway sites with L-NAC administration, i.e. the mucus became less rigid and more deformable (the overall change in G* was 0.29 log units, i.e. ca. twofold decrease). The viscoelasticity-derived mucus transportability parameters, mucociliary (MCI) and cough (CCI) clearability indices, increased with L-NAC MDI, particularly CCI, which predicts the effect of mucus rheology on cough clearability. PD increased significantly with L-NAC administration at all measurement sites, which appears to be a novel effect for a direct acting mucolytic agent. Tracheal mucus linear velocity (TMV) increased after L-NAC compared with placebo, as did the normalized frog palate transport rate (NFPTR). The increase in NFPTR was greater than that predicted from the mucus rheological properties alone, suggesting that L-NAC still resident in the collected mucus stimulated the frog palate cilia. The index of mucus flux, the collection rate in mg.min-1, was higher with L-NAC compared with placebo. From our results, we conclude that L-NAC shows potential benefit in terms of improving mucus rheological properties and clearability. It may act, in part, by stimulating the fresh secretion of mucus of lower viscoelasticity. The stimulation of mucociliary clearance could be related to ion flux changes, as indicated by the increase in PD.

  16. Effects of aluminium chloride and aluminium chlorohydrate on DNA repair in MCF10A immortalised non-transformed human breast epithelial cells.

    PubMed

    Farasani, A; Darbre, P D

    2015-11-01

    Use of underarm aluminium (Al)-based antiperspirant salts may be a contributory factor in breast cancer development. At the 10th Keele meeting, Al was reported to cause anchorage-independent growth and double strand DNA breaks in MCF10A immortalised non-transformed human breast epithelial cells. We now report that exposure of MCF10A cells to Al chloride or Al chlorohydrate also compromised DNA repair systems. Long-term (19-21 weeks) exposure to Al chloride or Al chlorohydrate at a 10(-4) M concentration resulted in reduced levels of BRCA1 mRNA as determined by real-time RT-PCR and BRCA1 protein as determined by Western immunoblotting. Reduced levels of mRNA for other DNA repair genes (BRCA2, CHK1, CHK2, Rad51, ATR) were also observed using real-time RT-PCR. Loss of BRCA1 or BRCA2 gene function has long been associated with inherited susceptibility to breast cancer but these results suggest that exposure to aluminium-based antiperspirant salts may also reduce levels of these key components of DNA repair in breast epithelial cells. If Al can not only damage DNA but also compromise DNA repair systems, then there is the potential for Al to impact on breast carcinogenesis.

  17. Effects of aluminium chloride and aluminium chlorohydrate on DNA repair in MCF10A immortalised non-transformed human breast epithelial cells.

    PubMed

    Farasani, A; Darbre, P D

    2015-11-01

    Use of underarm aluminium (Al)-based antiperspirant salts may be a contributory factor in breast cancer development. At the 10th Keele meeting, Al was reported to cause anchorage-independent growth and double strand DNA breaks in MCF10A immortalised non-transformed human breast epithelial cells. We now report that exposure of MCF10A cells to Al chloride or Al chlorohydrate also compromised DNA repair systems. Long-term (19-21 weeks) exposure to Al chloride or Al chlorohydrate at a 10(-4) M concentration resulted in reduced levels of BRCA1 mRNA as determined by real-time RT-PCR and BRCA1 protein as determined by Western immunoblotting. Reduced levels of mRNA for other DNA repair genes (BRCA2, CHK1, CHK2, Rad51, ATR) were also observed using real-time RT-PCR. Loss of BRCA1 or BRCA2 gene function has long been associated with inherited susceptibility to breast cancer but these results suggest that exposure to aluminium-based antiperspirant salts may also reduce levels of these key components of DNA repair in breast epithelial cells. If Al can not only damage DNA but also compromise DNA repair systems, then there is the potential for Al to impact on breast carcinogenesis. PMID:26319584

  18. Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge.

    PubMed

    Hartwig, Stacey M; Ketterer, Margaret; Apicella, Michael A; Varga, Steven M

    2016-11-01

    Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Co-culture of 16HBE14o- cells with NTHi resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSV-specific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection.

  19. Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge.

    PubMed

    Hartwig, Stacey M; Ketterer, Margaret; Apicella, Michael A; Varga, Steven M

    2016-11-01

    Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Co-culture of 16HBE14o- cells with NTHi resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSV-specific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection. PMID:27573069

  20. Physiological impact of abnormal lipoxin A₄ production on cystic fibrosis airway epithelium and therapeutic potential.

    PubMed

    Higgins, Gerard; Ringholz, Fiona; Buchanan, Paul; McNally, Paul; Urbach, Valérie

    2015-01-01

    Lipoxin A4 has been described as a major signal for the resolution of inflammation and is abnormally produced in the lungs of patients with cystic fibrosis (CF). In CF, the loss of chloride transport caused by the mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel gene results in dehydration, mucus plugging, and reduction of the airway surface liquid layer (ASL) height which favour chronic lung infection and neutrophil based inflammation leading to progressive lung destruction and early death of people with CF. This review highlights the unique ability of LXA4 to restore airway surface hydration, to stimulate airway epithelial repair, and to antagonise the proinflammatory program of the CF airway, circumventing some of the most difficult aspects of CF pathophysiology. The report points out novel aspects of the cellular mechanism involved in the physiological response to LXA4, including release of ATP from airway epithelial cell via pannexin channel and subsequent activation of and P2Y11 purinoreceptor. Therefore, inadequate endogenous LXA4 biosynthesis reported in CF exacerbates the ion transport abnormality and defective mucociliary clearance, in addition to impairing the resolution of inflammation, thus amplifying the vicious circle of airway dehydration, chronic infection, and inflammation.

  1. Physiological Impact of Abnormal Lipoxin A4 Production on Cystic Fibrosis Airway Epithelium and Therapeutic Potential

    PubMed Central

    Higgins, Gerard; McNally, Paul; Urbach, Valérie

    2015-01-01

    Lipoxin A4 has been described as a major signal for the resolution of inflammation and is abnormally produced in the lungs of patients with cystic fibrosis (CF). In CF, the loss of chloride transport caused by the mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel gene results in dehydration, mucus plugging, and reduction of the airway surface liquid layer (ASL) height which favour chronic lung infection and neutrophil based inflammation leading to progressive lung destruction and early death of people with CF. This review highlights the unique ability of LXA4 to restore airway surface hydration, to stimulate airway epithelial repair, and to antagonise the proinflammatory program of the CF airway, circumventing some of the most difficult aspects of CF pathophysiology. The report points out novel aspects of the cellular mechanism involved in the physiological response to LXA4, including release of ATP from airway epithelial cell via pannexin channel and subsequent activation of and P2Y11 purinoreceptor. Therefore, inadequate endogenous LXA4 biosynthesis reported in CF exacerbates the ion transport abnormality and defective mucociliary clearance, in addition to impairing the resolution of inflammation, thus amplifying the vicious circle of airway dehydration, chronic infection, and inflammation. PMID:25866809

  2. Macrophages promote benzopyrene-induced tumor transformation of human bronchial epithelial cells by activation of NF-κB and STAT3 signaling in a bionic airway chip culture and in animal models.

    PubMed

    Li, Encheng; Xu, Zhiyun; Zhao, Hui; Sun, Zhao; Wang, Lei; Guo, Zhe; Zhao, Yang; Gao, Zhancheng; Wang, Qi

    2015-04-20

    We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis.

  3. Macrophages promote benzopyrene-induced tumor transformation of human bronchial epithelial cells by activation of NF-κB and STAT3 signaling in a bionic airway chip culture and in animal models

    PubMed Central

    Sun, Zhao; Wang, Lei; Guo, Zhe; Zhao, Yang; Gao, Zhancheng; Wang, Qi

    2015-01-01

    We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis. PMID:25823926

  4. Resolvin D1 Attenuates Poly(I:C)-Induced Inflammatory Signaling in Human Airway Epithelial Cells via TAK1

    PubMed Central

    Hsiao, Hsi-Min; Thatcher, Thomas H.; Levy, Elizabeth P.; Fulton, Robert A.; Owens, Kristina M.; Phipps, Richard P.; Sime, Patricia J.

    2014-01-01

    The respiratory epithelium are lung sentinel cells and are the first to contact inhaled inflammatory insults including air pollutants, smoke and microorganisms. To avoid damaging exuberant or chronic inflammation, the inflammatory process must be tightly controlled and terminated once the insult is mitigated. Inflammation-resolution is now known to be an active process involving a new genus of lipid mediators called “specialized pro-resolving lipid mediators” (SPMs) that includes resolvin D1 (RvD1). We and others have reported that RvD1 counteracts pro-inflammatory signaling and promotes resolution. A knowledge gap is that the specific cellular targets and mechanisms of action for RvD1 remain largely unknown. Here, we identified the mechanism whereby RvD1 disrupts inflammatory mediator production induced by the viral mimic poly(I:C) in primary human lung epithelial cells. RvD1 strongly suppressed the viral mimic poly(I:C)-induced IL-6 and IL-8 production and pro-inflammatory signaling involving MAP kinases and NF-κB. Most importantly, we found that RvD1 inhibited the phosphorylation of TAK1, a key upstream regulatory kinase common to both the MAP kinase and NF-κB pathways, by inhibiting the formation of a poly(I:C)-induced signaling complex composed of TAK1, TAB1 and TRAF6. We confirmed that ALX/FPR2 and GPR32, two RvD1 receptors, were expressed on hSAEC. Furthermore, blocking these receptors abrogated the inhibitory action of RvD1. Herein, we present the idea that RvD1 has the potential to be used as an anti-inflammatory and pro-resolving agent, possibly in the context of exuberant host responses to damaging respirable agents such as viruses. PMID:25320283

  5. Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells.

    PubMed

    Boisvieux-Ulrich, E; Le Pechon-Vallée, C; Million, K; Baeza-Squiban, A; Houcine, O; Guennou, C; Reichert, U; Marano, F

    2000-04-01

    The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.

  6. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    PubMed

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense. PMID:27496896

  7. Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

    PubMed

    Krishna, Venkatramana D; Roach, Erin; Zaidman, Nathan A; Panoskaltsis-Mortari, Angela; Rotschafer, Jessica H; O'Grady, Scott M; Cheeran, Maxim C-J

    2015-01-01

    Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection. PMID:26384331

  8. Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc

    SciTech Connect

    Wu Weidong Silbajoris, Robert A.; Cao Dongsun; Bromberg, Philip A.; Zhang Qiao; Peden, David B.; Samet, James M.

    2008-09-01

    Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional and posttranscriptional events that regulate COX-2 expression in a human bronchial epithelial cell line BEAS-2B exposed to Zn{sup 2+}. Zn{sup 2+} exposure resulted in pronounced increases in COX-2 mRNA and protein expression, which were prevented by pretreatment with the transcription inhibitor actinomycin D, implying the involvement of transcriptional regulation. This was supported by the observation of increased COX-2 promoter activity in Zn{sup 2+}-treated BEAS-2B cells. Mutation of the cAMP response element (CRE), but not the {kappa}B-binding sites in the COX-2 promoter markedly reduced COX-2 promoter activity induced by Zn{sup 2+}. Inhibition of NF{kappa}B activation did not block Zn{sup 2+}-induced COX-2 expression. Measurement of mRNA stability demonstrated that Zn{sup 2+} exposure impaired the degradation of COX-2 mRNA in BEAS-2B cells. This message stabilization effect of Zn{sup 2+} exposure was shown to be dependent on the integrity of the 3'-untranslated region found in the COX-2 transcript. Taken together, these data demonstrate that the CRE and mRNA stability regulates COX-2 expression induced in BEAS-2B cells exposed to extracellular Zn{sup 2+}.

  9. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    PubMed

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.

  10. Higher AgNOR Expression in Metaplastic and Dysplastic Airway Epithelial Cells Predicts the Risk of Developing Lung Cancer in Women Chronically Exposed to Biomass Smoke.

    PubMed

    Mondal, Nandan Kumar; Roychoudhury, Sanghita; Ray, Manas Ranjan

    2015-01-01

    We evaluated AgNOR expression in airway epithelial cells (AECs) as a risk factor of lung carcinogenesis in 228 nonsmoking women exposed to biomass fuel (BMF). A total of 185 age-matched women who cooked with cleaner fuel (liquefied petroleum gas [LPG]) were enrolled as study controls. Compared with controls, Papanicolaou-stained sputum samples showed 4 and 8 times higher prevalence of metaplasia and dysplasia, respectively, in AECs of BMF users. AgNOR staining showed significantly larger numbers of dots and larger size and percentage of AgNOR-occupied nuclear area in normal AECs of BMF users than in controls. Interestingly, AgNOR parameters increased dramatically when the cells were transformed from normalcy to metaplasia and dysplasia. Compared with LPG users, BMF users showed a marked rise in reactive oxygen species (ROS) generation and a depletion of superoxide dismutase (SOD), indicating oxidative stress. Indoor air of BMF-using households had 2-5 times more particulate pollutants (PM10 and PM2.5), 73% more nitrogen dioxide (NO2), and 4 times more particulate-laden benzo(a)pyrene [B(a)P], but no difference in sulfur dioxide was observed. A high-performance liquid chromatography (HPLC) study estimated a 6-fold rise in benzene metabolite trans, trans-muconic acid (t,t-MA) in urine of BMF users. After controlling confounding factors using multivariate logistic regression, positive associations were observed between cellular changes, AgNOR parameters, and PM10, PM2.5, NO2, B(a)P, and t,t-MA levels, especially the concentration of B(a)P. In conclusion, cumulative exposure to biomass smoke causes oxidative stress and enhances AgNOR expression in precancerous metaplastic and dysplastic AECs and appears to be a risk factor for developing lung cancer. PMID:25746830

  11. Nicotine reduces the levels of surfactant proteins A and D via Wnt/β-catenin and PKC signaling in human airway epithelial cells.

    PubMed

    Zou, Weifeng; Liu, Sha; Hu, Jinxing; Sheng, Qing; He, Fang; Li, Bing; Ran, Pixin

    2016-01-15

    A deficiency of surfactant proteins A and D has been proposed as a mechanism in airway remodeling, which is one characteristic of chronic obstructive pulmonary disease (COPD). We recently showed that in vitro nicotine exposure induces Wnt3a/β-catenin activation, which is a pathway that has also been implicated in altering levels of SP-A and SP-D. Nicotine induced activation of protein kinase C(PKC), and the involvement of PKC in mediating Wnt signaling has been demonstrated previously. The main aim of this study was to investigate whether human bronchial epithelial cells reduce levels of SP-A and SP-D in vitro following nicotine stimulation via the Wnt3a/β-catenin and PKC signaling pathway. We showed that nicotine activated the Wnt3a/β-catenin and PKC signaling pathway, and this activation was accompanied by a decrease in SP-A and SP-D expression. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented translocation of β-catenin into the nucleus and reduction levels of SP-A and SP-D. Furthermore, a PKC inhibitor partially prevented these effects,which suggests in HBECs, Wnt3a/β-catenin and PKC pathways interact during nicotine-reduced levels of SP-A and SP-D. These results suggest that HBECs reduce the levels of surfactant proteins A and D in vitro via the Wnt3a/β-catenin and PKC signaling pathway upon nicotine stimulation.

  12. Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells

    PubMed Central

    Krishna, Venkatramana D.; Roach, Erin; Zaidman, Nathan A.; Panoskaltsis-Mortari, Angela; Rotschafer, Jessica H.; O’Grady, Scott M.; Cheeran, Maxim C-J.

    2015-01-01

    Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection. PMID:26384331

  13. Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

    PubMed

    Krishna, Venkatramana D; Roach, Erin; Zaidman, Nathan A; Panoskaltsis-Mortari, Angela; Rotschafer, Jessica H; O'Grady, Scott M; Cheeran, Maxim C-J

    2015-01-01

    Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection.

  14. The long-acting β2-adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M A

    2015-01-01

    Background and Purpose Inhaled glucocorticoid (ICS)/long-acting β2-adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. Experimental Approach BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. Key Results Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. Conclusions and Implications These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues. PMID:25598440

  15. Effects of nitrogen-doped multi-walled carbon nanotubes compared to pristine multi-walled carbon nanotubes on human small airway epithelial cells.

    PubMed

    Mihalchik, Amy L; Ding, Weiqiang; Porter, Dale W; McLoughlin, Colleen; Schwegler-Berry, Diane; Sisler, Jennifer D; Stefaniak, Aleksandr B; Snyder-Talkington, Brandi N; Cruz-Silva, Rodolfo; Terrones, Mauricio; Tsuruoka, Shuji; Endo, Morinobu; Castranova, Vincent; Qian, Yong

    2015-07-01

    Nitrogen-doped multi-walled carbon nanotubes (ND-MWCNTs) are modified multi-walled carbon nanotubes (MWCNTs) with enhanced electrical properties that are used in a variety of applications, including fuel cells and sensors; however, the mode of toxic action of ND-MWCNT has yet to be fully elucidated. In the present study, we compared the interaction of ND-MWCNT or pristine MWCNT-7 with human small airway epithelial cells (SAEC) and evaluated their subsequent bioactive effects. Transmission electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and X-ray diffraction suggested the presence of N-containing defects in the lattice of the nanotube. The ND-MWCNTs were determined to be 93.3% carbon, 3.8% oxygen, and 2.9% nitrogen. A dose-response cell proliferation assay showed that low doses of ND-MWCNT (1.2μg/ml) or MWCNT-7 (0.12μg/ml) increased cellular proliferation, while the highest dose of 120μg/ml of either material decreased proliferation. ND-MWCNT and MWCNT-7 appeared to interact with SAEC at 6h and were internalized by 24h. ROS were elevated at 6 and 24h in ND-MWCNT exposed cells, but only at 6h in MWCNT-7 exposed cells. Significant alterations to the cell cycle were observed in SAEC exposed to either 1.2μg/ml of ND-MWCNT or MWCNT-7 in a time and material-dependent manner, possibly suggesting potential damage or alterations to cell cycle machinery. Our results indicate that ND-MWCNT induce effects in SAEC over a time and dose-related manner which differ from MWCNT-7. Therefore, the physicochemical characteristics of the materials appear to alter their biological effects.

  16. Effects of nitrogen-doped multi-walled carbon nanotubes compared to pristine multi-walled carbon nanotubes on human small airway epithelial cells

    PubMed Central

    Mihalchik, Amy L.; Ding, Weiqiang; Porter, Dale W.; McLoughlin, Colleen; Schwegler-Berry, Diane; Sisler, Jennifer D.; Stefaniak, Aleksandr B.; Snyder-Talkington, Brandi N.; Cruz-Silva, Rodolfo; Terrones, Mauricio; Tsuruoka, Shuji; Endo, Morinobu; Castranova, Vincent; Qian, Yong

    2015-01-01

    Nitrogen-doped multi-walled carbon nanotubes (ND-MWCNTs) are modified multi-walled carbon nanotubes (MWCNTs) with enhanced electrical properties that are used in a variety of applications, including fuel cells and sensors; however, the mode of toxic action of ND-MWCNT has yet to be fully elucidated. In the present study, we compared the interaction of ND-MWCNT or pristine MWCNT-7 with human small airway epithelial cells (SAEC) and evaluated their subsequent bioactive effects. Transmission electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and X-ray diffraction suggested the presence of N-containing defects in the lattice of the nanotube. The ND-MWCNTs were determined to be 93.3% carbon, 3.8% oxygen, and 2.9% nitrogen. A dose–response cell proliferation assay showed that low doses of ND-MWCNT (1.2 mg/ml) or MWCNT-7 (0.1 mg/ml) increased cellular proliferation, while the highest dose of 120 mg/ml of either material decreased proliferation. ND-MWCNT and MWCNT-7 appeared to interact with SAEC at 6 h and were internalized by 24 h. ROS were elevated at 6 and 24 h in ND-MWCNT exposed cells, but only at 6 h in MWCNT-7 exposed cells. Significant alterations to the cell cycle were observed in SAEC exposed to either 1.2 mg/ml of ND-MWCNT or MWCNT-7 in a time and material-dependent manner, possibly suggesting potential damage or alterations to cell cycle machinery. Our results indicate that ND-MWCNT induce effects in SAEC over a time and dose-related manner which differ from MWCNT-7. Therefore, the physicochemical characteristics of the materials appear to alter their biological effects. PMID:25797581

  17. Potentiation of NF-κB-dependent transcription and inflammatory mediator release by histamine in human airway epithelial cells

    PubMed Central

    Holden, N S; Gong, W; King, E M; Kaur, M; Giembycz, M A; Newton, R

    2007-01-01

    Background and purpose: In asthma, histamine contributes to bronchoconstriction, vasodilatation and oedema, and is associated with the late phase response. The current study investigates possible inflammatory effects of histamine acting on nuclear factor κB (NF-κB)-dependent transcription and cytokine release. Experimental approach: Using BEAS-2B bronchial epithelial cells, NF-κB-dependent transcription and both release and mRNA expression of IL-6 and IL-8 were examined by reporter assay, ELISA and quantitative RT-PCR. Histamine receptors were detected using qualitative RT-PCR and function examined using selective agonists and antagonists. Key results: Addition of histamine to TNFα-stimulated BEAS-2B cells maximally potentiated NF-κB-dependent transcription 1.8 fold, whereas IL-6 and IL-8 protein release were enhanced 7.3- and 2.7-fold respectively. These responses were, in part, NF-κB-dependent and were associated with 2.6- and 1.7-fold enhancements of IL-6 and IL-8 mRNA expression. The H1 receptor antagonist, mepyramine, caused a rightward shift in the concentration-response curves of TNFα-induced NF-κB-dependent transcription (pA2=9.91) and release of IL-6 (pA2=8.78) and IL-8 (pA2=8.99). Antagonists of histamine H2, H3 and H4 receptors were without effect. Similarly, H3 and H4 receptor agonists did not affect TNFα-induced NF-κB-dependent transcription, or IL-6 and IL-8 release at concentrations below 10 μM. The anti-inflammatory glucocorticoid, dexamethasone, inhibited the histamine enhanced NF-κB-dependent transcription and IL-6 and IL-8 release. Conclusions and implications: Potentiation of NF-κB-dependent transcription and inflammatory cytokine release by histamine predominantly involves receptors of the H1 receptor subtype. These data support an anti-inflammatory role for H1 receptor antagonists by preventing the transcription and release of pro-inflammatory cytokines. PMID:17891168

  18. Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses

    PubMed Central

    Wang, Qiong; Nagarkar, Deepti R.; Bowman, Emily R.; Schneider, Dina; Gosangi, Babina; Lei, Jing; Zhao, Ying; McHenry, Christina L.; Burgens, Richai V.; Miller, David J.; Sajjan, Umadevi; Hershenson, Marc B.

    2010-01-01

    Rhinovirus (RV), a single-stranded RNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral double-stranded RNA produced during replication may be recognized by the host pattern recognition receptors Toll-like receptor (TLR)-3, retinoic acid inducible gene (RIG)-I and melanoma-differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV double-stranded (ds)-RNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and interferon (IFN) and IFN-stimulated gene expression determined by quantitative PCR. The separate requirements of RIG-I, MDA5 and IFN response factor (IRF)-3 were determined using their respective siRNAs. The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule TRIF. Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β̤, IFN-λ̣1, IFN-λ2/3, IRF7, RIG-I, MDA5, IP-10/CXCL10, IL-8/CXCL8 and GM-CSF. siRNA against IRF3, MDA5 and TRIF, but not RIG-I, decreased RV1B-induced expression of IFN-β̤ IFN-λ̣1, IFN-λ2/3, IRF7, RIG-I, MDA5 and IP-10/CXCL10, but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA, and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3. PMID:19890046

  19. Bioelectric and Morphological Response of Liquid-Covered Human Airway Epithelial Calu-3 Cell Monolayer to Periodic Deposition of Colloidal 3-Mercaptopropionic-Acid Coated CdSe-CdS/ZnS Core-Multishell Quantum Dots.

    PubMed

    Turdalieva, Aizat; Solandt, Johan; Shambetova, Nestan; Xu, Hao; Blom, Hans; Brismar, Hjalmar; Zelenina, Marina; Fu, Ying

    2016-01-01

    Lung epithelial cells are extensively exposed to nanoparticles present in the modern urban environment. Nanoparticles, including colloidal quantum dots (QDs), are also considered to be potentially useful carriers for the delivery of drugs into the body. It is therefore important to understand the ways of distribution and the effects of the various types of nanoparticles in the lung epithelium. We use a model system of liquid-covered human airway epithelial Calu-3 cell cultures to study the immediate and long-term effects of repeated deposition of colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs on the lung epithelial cell surface. By live confocal microscope imaging and by QD fluorescence measurements we show that the QD permeation through the mature epithelial monolayers is very limited. At the time of QD deposition, the transepithelial electrical resistance (TEER) of the epithelial monolayers transiently decreased, with the decrement being proportional to the QD dose. Repeated QD deposition, once every six days for two months, lead to accumulation of only small amounts of the QDs in the cell monolayer. However, it did not induce any noticeable changes in the long-term TEER and the molecular morphology of the cells. The colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs could therefore be potentially used for the delivery of drugs intended for the surface of the lung epithelia during limited treatment periods. PMID:26913754

  20. Bioelectric and Morphological Response of Liquid-Covered Human Airway Epithelial Calu-3 Cell Monolayer to Periodic Deposition of Colloidal 3-Mercaptopropionic-Acid Coated CdSe-CdS/ZnS Core-Multishell Quantum Dots.

    PubMed

    Turdalieva, Aizat; Solandt, Johan; Shambetova, Nestan; Xu, Hao; Blom, Hans; Brismar, Hjalmar; Zelenina, Marina; Fu, Ying

    2016-01-01

    Lung epithelial cells are extensively exposed to nanoparticles present in the modern urban environment. Nanoparticles, including colloidal quantum dots (QDs), are also considered to be potentially useful carriers for the delivery of drugs into the body. It is therefore important to understand the ways of distribution and the effects of the various types of nanoparticles in the lung epithelium. We use a model system of liquid-covered human airway epithelial Calu-3 cell cultures to study the immediate and long-term effects of repeated deposition of colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs on the lung epithelial cell surface. By live confocal microscope imaging and by QD fluorescence measurements we show that the QD permeation through the mature epithelial monolayers is very limited. At the time of QD deposition, the transepithelial electrical resistance (TEER) of the epithelial monolayers transiently decreased, with the decrement being proportional to the QD dose. Repeated QD deposition, once every six days for two months, lead to accumulation of only small amounts of the QDs in the cell monolayer. However, it did not induce any noticeable changes in the long-term TEER and the molecular morphology of the cells. The colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs could therefore be potentially used for the delivery of drugs intended for the surface of the lung epithelia during limited treatment periods.

  1. Bioelectric and Morphological Response of Liquid-Covered Human Airway Epithelial Calu-3 Cell Monolayer to Periodic Deposition of Colloidal 3-Mercaptopropionic-Acid Coated CdSe-CdS/ZnS Core-Multishell Quantum Dots

    PubMed Central

    Turdalieva, Aizat; Solandt, Johan; Shambetova, Nestan; Xu, Hao; Blom, Hans; Brismar, Hjalmar; Zelenina, Marina; Fu, Ying

    2016-01-01

    Lung epithelial cells are extensively exposed to nanoparticles present in the modern urban environment. Nanoparticles, including colloidal quantum dots (QDs), are also considered to be potentially useful carriers for the delivery of drugs into the body. It is therefore important to understand the ways of distribution and the effects of the various types of nanoparticles in the lung epithelium. We use a model system of liquid-covered human airway epithelial Calu-3 cell cultures to study the immediate and long-term effects of repeated deposition of colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs on the lung epithelial cell surface. By live confocal microscope imaging and by QD fluorescence measurements we show that the QD permeation through the mature epithelial monolayers is very limited. At the time of QD deposition, the transepithelial electrical resistance (TEER) of the epithelial monolayers transiently decreased, with the decrement being proportional to the QD dose. Repeated QD deposition, once every six days for two months, lead to accumulation of only small amounts of the QDs in the cell monolayer. However, it did not induce any noticeable changes in the long-term TEER and the molecular morphology of the cells. The colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs could therefore be potentially used for the delivery of drugs intended for the surface of the lung epithelia during limited treatment periods. PMID:26913754

  2. Impaired recovery from naphthalene-induced bronchiolar epithelial injury in mice exposed to aged and diluted sidestream cigarette smoke.

    PubMed

    Van Winkle, Laura S; Brown, Collette D; Shimizu, Judith A; Gunderson, Andrew D; Evans, Michael J; Plopper, Charles G

    2004-12-01

    The effect of sidestream tobacco smoke combined with other pollutants is largely unknown. Previously, we found that distal airway epithelial repair was inhibited in mice exposed to sidestream tobacco smoke (TS) for 5 days followed by single exposure to naphthalene (NA), a common polycyclic aromatic hydrocarbon found in cigarette smoke, diesel exhaust, and pesticide formulations. The main injury target of NA is the nonciliated (Clara) bronchiolar cell. NA injury normally resolves in two weeks. Repair in mice exposed to TS and NA was unresolved in the distal bronchioles 14 days post-NA injury. We hypothesized that repair inhibition persisted as a first step towards long-term airway remodeling and expanded the previous study by evaluating repair 21 days after acute NA injury. Repair was evaluated using high resolution histopathology, TEM, and quantitative morphometry. In animals exposed to TS and NA, repair was still impaired; re-differentiation of Clara cells at the bronchoalveolar duct junction was incomplete, indicating repair was continuing. Compared to 14 days post-NA-injury, repair at 21 days post-NA treatment was more extensive. Animals exposed only to TS had epithelium similar to controls. While TS exposure impairs bronchiolar epithelial repair after NA exposure, this effect appears to be slowly resolving over time. PMID:15475173

  3. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  4. Airway and lung pathology due to mucosal surface dehydration in β-Epithelial Na+ Channel-overexpressing mice: role of TNFα and IL-4Rα signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment

    PubMed Central

    Livraghi, Alessandra; Grubb, Barbara R.; Hudson, Elizabeth J.; Wilkinson, Kristen J.; Sheehan, John K.; Mall, Marcus A.; O'Neal, Wanda K.; Boucher, Richard C.; Randell, Scott H.

    2009-01-01

    Overexpression of the epithelial Na+ channel β subunit (Scnn1b gene, βENaC protein) in transgenic (Tg) mouse airways dehydrates mucosal surfaces, producing mucus obstruction, inflammation, and neonatal mortality. Airway inflammation includes macrophage activation, neutrophil and eosinophil recruitment, and elevated KC, TNFα and chitinase levels. These changes recapitulate aspects of complex human obstructive airway diseases, but their molecular mechanisms are poorly understood. We used genetic and pharmacologic approaches to identify pathways relevant to the development of Scnn1b-Tg mouse lung pathology. Genetic deletion of tumor necrosis factor alpha (TNFα) or its receptor, TNFR1, had no measurable effect on the phenotype. Deletion of the interleukin-4 receptor alpha subunit (IL-4Rα) abolished transient mucous secretory cell (MuSC) abundance and eosinophilia normally observed in neonatal wild-type (WT) mice. Similarly, IL-4Rα deficiency decreased MuSC and eosinophils in neonatal Scnn1b-Tg mice, which correlated with improved neonatal survival. However, chronic lung pathology in adult Scnn1b-Tg mice was not affected by IL-4Rα status. Prednisolone treatment ablated eosinophilia and MuSC in adult Scnn1b-Tg mice, but did not decrease mucus plugging or neutrophilia. These studies demonstrate that: 1) normal neonatal mouse airway development entails an IL-4Rα-dependent, transient abundance of MuSC and eosinophils; 2) absence of IL-4Rα improved neonatal survival of Scnn1b-Tg mice, likely reflecting decreased formation of asphyxiating mucus plugs; and 3) in Scnn1b-Tg mice, neutrophilia, mucus obstruction, and airspace enlargement are IL-4Rα- and TNFα-independent, and only MuSC and eosinophilia are sensitive to glucocorticoids. Thus, manipulation of multiple pathways will likely be required to treat the complex pathogenesis caused by airway surface dehydration. PMID:19299736

  5. Anti-Inflammatory Activity of a Novel Family of Aryl Ureas Compounds in an Endotoxin-Induced Airway Epithelial Cell Injury Model

    PubMed Central

    Cabrera-Benitez, Nuria E.; Pérez-Roth, Eduardo; Casula, Milena; Ramos-Nuez, Ángela; Ríos-Luci, Carla; Rodríguez-Gallego, Carlos; Sologuren, Ithaisa; Jakubkiene, Virginija; Slutsky, Arthur S.; Padrón, José M.; Villar, Jesús

    2012-01-01

    Background Despite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS. Methodology/Principal Findings After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings. Conclusions/Significance Using a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and

  6. Size-partitioning of an urban aerosol to identify particle determinants involved in the proinflammatory response induced in airway epithelial cells

    PubMed Central

    Ramgolam, Kiran; Favez, Olivier; Cachier, Hélène; Gaudichet, Annie; Marano, Francelyne; Martinon, Laurent; Baeza-Squiban, Armelle

    2009-01-01

    Background The contribution of air particles in human cardio-respiratory diseases has been enlightened by several epidemiological studies. However the respective involvement of coarse, fine and ultrafine particles in health effects is still unclear. The aim of the present study is to determine which size fraction from a chemically characterized background aerosol has the most important short term biological effect and to decipher the determinants of such a behaviour. Results Ambient aerosols were collected at an urban background site in Paris using four 13-stage low pressure cascade impactors running in parallel (winter and summer 2005) in order to separate four size-classes (PM0.03–0.17 (defined here as ultrafine particles), PM0.17–1 (fine), PM1–2.5(intermediate) and PM2.5–10 (coarse)). Accordingly, their chemical composition and their pro-inflammatory potential on human airway epithelial cells were investigated. Considering isomass exposures (same particle concentrations for each size fractions) the pro-inflammatory response characterized by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) release was found to decrease with aerosol size with no seasonal dependency. When cells were exposed to isovolume of particle suspensions in order to respect the particle proportions observed in ambient air, the GM-CSF release was maximal with the fine fraction. In presence of a recombinant endotoxin neutralizing protein, the GM-CSF release induced by particles is reduced for all size-fractions, with exception of the ultra-fine fraction which response is not modified. The different aerosol size-fractions were found to display important chemical differences related to the various contributing primary and secondary sources and aerosol age. The GM-CSF release was correlated to the organic component of the aerosols and especially its water soluble fraction. Finally, Cytochrome P450 1A1 activity that reflects PAH bioavailability varied as a function of the season

  7. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    SciTech Connect

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. )

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  8. Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc

    EPA Science Inventory

    Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional...

  9. Ozone enhances diesel exhaust particles (DEP)-induced interleukin-8 (IL-8) gene expression in human airway epithelial cells through activation of nuclear factors- kappaB (NF-kappaB) and IL-6 (NF-IL6).

    PubMed

    Kafoury, Ramzi M; Kelley, James

    2005-12-01

    Ozone, a highly reactive oxidant gas is a major component of photochemical smog. As an inhaled toxicant, ozone induces its adverse effects mainly on the lung. Inhalation of particulate matter has been reported to cause airway inflammation in humans and animals. Furthermore, epidemiological evidence has indicated that exposure to particulate matter (PM[2.5-10]), including diesel exhaust particles (DEP) has been correlated with increased acute and chronic respiratory morbidity and exacerbation of asthma. Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems. Ozone and particulate matter may be chemically coupled in the ambient air. In the present study we determined whether ozone exposure enhances DEP effect on interleukin-8 (IL-8) gene expression in human airway epithelial cells. We report that ozone exposure (0.5 ppm x 1 hr) significantly increased DEP-induced IL-8 gene expression in A549 cells (117 +/- 19 pg/ml, n = 6, p < 0.05) as compared to cultures treated with DEP (100 microg/ml x 4 hr) alone (31 +/- 3 pg/ml, n = 6), or cultures exposed to purified air (24 +/- 6 pg/ml, n = 6). The increased DEP-induced IL-8 gene expression following ozone exposure was attributed to ozone-induced increase in the activity of the transcription factors NF-kappaB and NF-IL6. The results of the present study indicate that ozone exposure enhances the toxicity of DEP in human airway epithelial cells by augmenting IL-8 gene expression, a potent chemoattractant of neutrophils in the lung. PMID:16819095

  10. Activation of transcription factor IL-6 (NF-IL-6) and nuclear factor-kappaB (NF-kappaB) by lipid ozonation products is crucial to interleukin-8 gene expression in human airway epithelial cells.

    PubMed

    Kafoury, Ramzi M; Hernandez, Jazmir M; Lasky, Joseph A; Toscano, William A; Friedman, Mitchell

    2007-04-01

    Ozone (O(3)) is a major component of smog and an inhaled toxicant to the lung. O(3) rapidly reacts with the airway epithelial cell membrane phospholipids to generate lipid ozonation products (LOP). 1-Hydroxy-1-hydroperoxynonane (HHP-C9) is an important LOP, produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine. This LOP, at a biologically relevant concentration (100 microM), increases the activity of phospholipase C, nuclear factors-kappaB (NF-kappaB), and interleukin-6 (NF-IL-6) and the expression of the inflammatory gene, interleukin-8 (IL-8) in a cultured human bronchial epithelial cell line (BEAS-2B). The signaling pathways of ozone and its biologically-active products are as yet undefined. In the present study, we report that the HHP LOP, HHP-C9 (100 microM x 4 h), activated the expression of IL-8 (218 +/- 26% increase over control, n = 4, P < 0.01) through an apparent interaction between the two transcription factors, NF-kappaB and NF-IL-6. Transfection studies using luciferase reporter assays demonstrated that HHP-C9 induced a significant increase in NF-kappaB-DNA binding activity (37 +/- 7% increase over control, n = 6, P < 0.05). Inhibition of NF-kappaB showed a statistically significant but modest decrease in IL-8 release, which suggested a role for another transcription factor, NF-IL-6. Exposure of BEAS-2B cells to HHP-C9 induced a significant increase in the DNA binding activity of NF-IL-6 (45 +/- 11% increase over control, n = 6, P < 0.05). The results of the present study indicate that NF-IL-6 interacts with NF-kappaB in regulating the expression of IL-8 in cultured human airway epithelial cells exposed to LOP, the biological products of ozone in the lung. PMID:17366569

  11. Spontaneous production of transforming growth factor-beta 2 by primary cultures of bronchial epithelial cells. Effects on cell behavior in vitro.

    PubMed Central

    Sacco, O; Romberger, D; Rizzino, A; Beckmann, J D; Rennard, S I; Spurzem, J R

    1992-01-01

    The ability of airway epithelial cells to produce transforming growth factor-beta (TGF-beta) may be an important mechanism for the control of growth, differentiation, and repair of the airway epithelium. To determine whether airway epithelial cells are capable of producing TGF-beta, we examined primary cultures of bovine bronchial epithelial cells. Using a bioassay, TGF-beta activity was detected readily in media conditioned by bovine bronchial epithelial cells. Neutralizing antisera to TGF-beta 1 and TGF-beta 2 were used to demonstrate that the majority of the activity was of the TGF-beta 2 isoform. Interestingly, some of the TGF-beta activity was present in the conditioned media as "active" TGF-beta, not requiring acid activation. The production of TGF-beta was variable, depending on cell density and the presence of retinoic acid. The presence of endogenously produced active TGF-beta in the culture media was shown to modulate the behavior of the cell cultures as evidenced by the effects of TGF-beta-neutralizing antisera on cell size and fibronectin production. Our results suggest that active TGF-beta produced by airway epithelial cells may function in an autocrine or paracrine manner to modulate epithelial cell behavior. PMID:1401072

  12. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    PubMed

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  13. Differential Roles of Hydrogen Peroxide in Adaptive and Inflammatory Gene Expression Induced by Exposure of Human Airway Epithelial Cells to Zn2+

    EPA Science Inventory

    Oxidant stress is believed to play an important role in particulate matter (PM)–mediated toxicity in the respiratory tract. Zinc (Zn2+) is a ubiquitous component of PM that has been shown to induce adverse responses such as inflammatory and adaptive gene expression in airway epit...

  14. Phenotypic modification of human airway epithelial cells in air-liquid interface culture induced by exposure to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

    PubMed

    Carson, Johnny L; Brighton, Luisa E; Jaspers, Ilona

    2015-04-01

    The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.

  15. Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

    PubMed Central

    Pirela, Sandra V.; Miousse, Isabelle R.; Lu, Xiaoyan; Castranova, Vincent; Thomas, Treye; Qian, Yong; Bello, Dhimiter; Kobzik, Lester; Koturbash, Igor; Demokritou, Philip

    2015-01-01

    Background Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives We assessed the biological responses of a panel of human cell lines to PEPs. Methods Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in

  16. Surgical Airway

    PubMed Central

    Patel, Sapna A; Meyer, Tanya K

    2014-01-01

    Close to 3% of all intubation attempts are considered difficult airways, for which a plan for a surgical airway should be considered. Our article provides an overview of the different types of surgical airways. This article provides a comprehensive review of the main types of surgical airways, relevant anatomy, necessary equipment, indications and contraindications, preparation and positioning, technique, complications, and tips for management. It is important to remember that the placement of a surgical airway is a lifesaving procedure and should be considered in any setting when one “cannot intubate, cannot ventilate”. PMID:24741501

  17. Activation of ERK accelerates repair of renal tubular epithelial cells, whereas it inhibits progression of fibrosis following ischemia/reperfusion injury.

    PubMed

    Jang, Hee-Seong; Han, Sang Jun; Kim, Jee In; Lee, Sanggyu; Lipschutz, Joshua H; Park, Kwon Moo

    2013-12-01

    Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.

  18. Lower nasopharyngeal epithelial cell repair and diminished innate inflammation responses contribute to the onset of acute otitis media in otitis-prone children.

    PubMed

    Verhoeven, David; Nesselbush, Monica; Pichichero, Michael E

    2013-08-01

    About 30 % of young children experience excessive, frequent episodes of middle ear infection and are classified as acute otitis media prone (OP). Streptococcus pneumoniae (Spn) is a predominant otopathogen in OP and non-OP (NOP) children. The pathogenesis of middle ear infection involves otopathogen nasopharyngeal (NP) colonization followed by an upper respiratory viral infection that modifies the NP environment to allow a sufficient inoculum of bacteria to reflux via the Eustachian tube into the middle ear space. Here, we analyzed the NP mucosal repair response between age-matched stringently defined OP (sOP) and NOP children who progressed to middle ear infection caused by Spn. We found lower epidermal growth factor, epidermal growth factor receptor, and angiogenin cytokine concentrations in nasal washes of sOP compared with NOP children. Despite higher expression of TLR2/4 transcript expression in nasal epithelium and in polymorphonuclear cells present in nasal secretions in sOP children, sOP children had lower expression of proinflammatory cytokines such as IL-6 and IL-8 in the NP. Chemotaxis-associated cytokine expression at onset of AOM in sOP children was also lower compared with NOP children, possibly indicating a lower capacity to signal the innate immune system. We conclude that lower epithelial cell repair responses during viral infection in the NP combined with diminished innate inflammatory responses potentiate Spn pathogenesis in the sOP child.

  19. MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment.

    PubMed

    Poole, Jill A; Wyatt, Todd A; Romberger, Debra J; Staab, Elizabeth; Simet, Samantha; Reynolds, Stephen J; Sisson, Joseph H; Kielian, Tammy

    2015-01-01

    Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement. PMID:26376975

  20. A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine: airway epithelial function and mucus changes in dog.

    PubMed

    Tomkiewicz, R P; App, E M; De Sanctis, G T; Coffiner, M; Maes, P; Rubin, B K; King, M

    1995-12-01

    A newly synthesized mucolytic agent, N-acetylcysteine L-lysinate (Nacystelyn) was studied. Tracheal mucus velocity (TMV), transepithelial potential difference (PD), rheological properties, and ion content of collected airway secretions were evaluated in six healthy mongrel dogs after placebo, Nacystelyn (NAL) and acetylcysteine (NAC) metered dose inhaler (MDI) aerosols. Although TMV was increased and viscoelasticity decreased after both treatments, the treatment effect with NAL was significantly greater. Furthermore, NAL increased the negative PD and CI- content of secretions in the trachea, an effect not observed after NAC. Both compounds increased ciliary beat frequency (CBF) on the frog palate at a concentration range similar to that approximated in dog airways. The increased mucociliary clearance could be partially explained by favourable rheological changes combined with stimulation of CBF. Since both compounds break disulfide bonds in mucus polymers, the greater change in mucus rheology and clearance rate after NAL, without change in water content, could be explained by the increase in CI- content. Nacystelyn appears to combine different modes of action which synergistically cause an increase in the clearance rate of airway secretions. PMID:8819180

  1. A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine: airway epithelial function and mucus changes in dog.

    PubMed

    Tomkiewicz, R P; App, E M; De Sanctis, G T; Coffiner, M; Maes, P; Rubin, B K; King, M

    1995-12-01

    A newly synthesized mucolytic agent, N-acetylcysteine L-lysinate (Nacystelyn) was studied. Tracheal mucus velocity (TMV), transepithelial potential difference (PD), rheological properties, and ion content of collected airway secretions were evaluated in six healthy mongrel dogs after placebo, Nacystelyn (NAL) and acetylcysteine (NAC) metered dose inhaler (MDI) aerosols. Although TMV was increased and viscoelasticity decreased after both treatments, the treatment effect with NAL was significantly greater. Furthermore, NAL increased the negative PD and CI- content of secretions in the trachea, an effect not observed after NAC. Both compounds increased ciliary beat frequency (CBF) on the frog palate at a concentration range similar to that approximated in dog airways. The increased mucociliary clearance could be partially explained by favourable rheological changes combined with stimulation of CBF. Since both compounds break disulfide bonds in mucus polymers, the greater change in mucus rheology and clearance rate after NAL, without change in water content, could be explained by the increase in CI- content. Nacystelyn appears to combine different modes of action which synergistically cause an increase in the clearance rate of airway secretions.

  2. Respiratory health of elite athletes – preventing airway injury: a critical review

    PubMed Central

    Kippelen, Pascale; Fitch, Kenneth D; Anderson, Sandra Doreen; Bougault, Valerie; Boulet, Louis-Philippe; Rundell, Kenneth William; Sue-Chu, Malcolm; McKenzie, Donald C

    2012-01-01

    Elite athletes, particularly those engaged in endurance sports and those exposed chronically to airborne pollutants/irritants or allergens, are at increased risk for upper and lower airway dysfunction. Airway epithelial injury may be caused by dehydration and physical stress applied to the airways during severe exercise hyperpnoea and/or by inhalation of noxious agents. This is thought to initiate an inflammatory cascade/repair process that, ultimately, could lead to airway hyperresponsiveness (AHR) and asthma in susceptible athletes. The authors review the evidence relating to prevention or reduction of the risk of AHR/asthma development. Appropriate measures should be implemented when athletes exercise strenuously in an attempt to attenuate the dehydration stress and reduce the exposure to noxious airborne agents. Environmental interventions are the most important. Non-pharmacological strategies can assist, but currently, pharmacological measures have not been demonstrated to be effective. Whether early prevention of airway injury in elite athletes can prevent or reduce progression to AHR/asthma remains to be established. PMID:22522585

  3. Bronchial epithelial cells: The key effector cells in the pathogenesis of chronic obstructive pulmonary disease?

    PubMed

    Gao, Wei; Li, Lingling; Wang, Yujie; Zhang, Sini; Adcock, Ian M; Barnes, Peter J; Huang, Mao; Yao, Xin

    2015-07-01

    The primary function of the bronchial epithelium is to act as a defensive barrier aiding the maintenance of normal airway function. Bronchial epithelial cells (BEC) form the interface between the external environment and the internal milieu, making it a major target of inhaled insults. However, BEC can also serve as effectors to initiate and orchestrate immune and inflammatory responses by releasing chemokines and cytokines, which recruit and activate inflammatory cells. They also produce excess reactive oxygen species as a result of an oxidant/antioxidant imbalance that contributes to chronic pulmonary inflammation and lung tissue damage. Accumulated mucus from hyperplastic BEC obstructs the lumen of small airways, whereas impaired cell repair, squamous metaplasia and increased extracellular matrix deposition underlying the epithelium is associated with airway remodelling particularly fibrosis and thickening of the airway wall. These alterations in small airway structure lead to airflow limitation, which is critical in the clinical diagnosis of chronic obstructive pulmonary disease (COPD). In this review, we discuss the abnormal function of BEC within a disturbed immune homeostatic environment consisting of ongoing inflammation, oxidative stress and small airway obstruction. We provide an overview of recent insights into the function of the bronchial epithelium in the pathogenesis of COPD and how this may provide novel therapeutic approaches for a number of chronic lung diseases.

  4. Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes

    PubMed Central

    Sun, Xiaoru; Zheng, Minghuan; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Dragos; Chen, Chengshui; Chen, Luonan; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC-specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL) and CD8+ T cells from lungs (T-LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up-regulated and 70% down-regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over-expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types. PMID:24826900

  5. An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M

    2015-01-01

    Background and Purpose International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. Experimental Approach A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Key Results Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression ‘fingerprint’ where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even ‘super agonist’. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. Conclusions and Implications The generation of gene expression ‘fingerprints’ in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. PMID:25393397

  6. Verproside inhibits TNF-α-induced MUC5AC expression through suppression of the TNF-α/NF-κB pathway in human airway epithelial cells.

    PubMed

    Lee, Su Ui; Sung, Min Hee; Ryu, Hyung Won; Lee, Jinhyuk; Kim, Hui-Seong; In, Hyun Ju; Ahn, Kyung-Seop; Lee, Hyun-Jun; Lee, Hyeong-Kyu; Shin, Dae-Hee; Lee, Yongnam; Hong, Sung-Tae; Oh, Sei-Ryang

    2016-01-01

    Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of MUC5AC, are significant risk factors in asthma and chronic obstructive pulmonary disease (COPD) patients. Previously, we reported that verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a potent anti-asthmatic candidate drug in vivo. However, the molecular mechanisms underlying the pharmacological actions of verproside remain unknown. Here, we found that verproside significantly reduces the expression levels of tumor necrosis factor alpha (TNF-α)-induced MUC5AC mRNA and protein by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors such as IκB kinase (IKK)β, IκBα, and TGF-β-activated kinase 1 (TAK1) in NCI-H292 cells. Moreover, verproside attenuated TNF-α-induced MUC5AC transcription more effectively when combined with an IKK (BAY11-7082) or a TAK1 (5z-7-oxozeaenol) inhibitor than when administered alone. Importantly, we demonstrated that verproside negatively modulates the formation of the TNF-α-receptor (TNFR) 1 signaling complex [TNF-RSC; TNFR1-recruited TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein kinase 1 (RIP1), and TAK1], the most upstream signaling factor of NF-κB signaling. In silico molecular docking studies show that verproside binds between TRADD and TRAF2 subunits. Altogether, these results suggest that verproside could be a good therapeutic candidate for treatment of inflammatory airway diseases such as asthma and COPD by blocking the TNF-α/NF-κB signaling pathway. PMID:26318254

  7. Verproside inhibits TNF-α-induced MUC5AC expression through suppression of the TNF-α/NF-κB pathway in human airway epithelial cells.

    PubMed

    Lee, Su Ui; Sung, Min Hee; Ryu, Hyung Won; Lee, Jinhyuk; Kim, Hui-Seong; In, Hyun Ju; Ahn, Kyung-Seop; Lee, Hyun-Jun; Lee, Hyeong-Kyu; Shin, Dae-Hee; Lee, Yongnam; Hong, Sung-Tae; Oh, Sei-Ryang

    2016-01-01

    Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of MUC5AC, are significant risk factors in asthma and chronic obstructive pulmonary disease (COPD) patients. Previously, we reported that verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a potent anti-asthmatic candidate drug in vivo. However, the molecular mechanisms underlying the pharmacological actions of verproside remain unknown. Here, we found that verproside significantly reduces the expression levels of tumor necrosis factor alpha (TNF-α)-induced MUC5AC mRNA and protein by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors such as IκB kinase (IKK)β, IκBα, and TGF-β-activated kinase 1 (TAK1) in NCI-H292 cells. Moreover, verproside attenuated TNF-α-induced MUC5AC transcription more effectively when combined with an IKK (BAY11-7082) or a TAK1 (5z-7-oxozeaenol) inhibitor than when administered alone. Importantly, we demonstrated that verproside negatively modulates the formation of the TNF-α-receptor (TNFR) 1 signaling complex [TNF-RSC; TNFR1-recruited TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein kinase 1 (RIP1), and TAK1], the most upstream signaling factor of NF-κB signaling. In silico molecular docking studies show that verproside binds between TRADD and TRAF2 subunits. Altogether, these results suggest that verproside could be a good therapeutic candidate for treatment of inflammatory airway diseases such as asthma and COPD by blocking the TNF-α/NF-κB signaling pathway.

  8. An uptake of cationized ferritin by alveolar type I cells in airway-instilled goat lung: distribution of anionic sites on the epithelial surface.

    PubMed

    Atwal, O S; Viel, L; Minhas, K J

    1990-07-01

    The present study has investigated ultrastructural localization of anionic sites on the luminal surface of the alveolar epithelium of goat lung by direct airway instillation of cationized ferritin (CF) in the cranial lobe of the right lung through a bronchoscope. The cationic probe decorated preferentially the luminal plasmalemmal vesicles and plasmalemma proper of alveolar type I cell. This indicated the presence of highly charged anionic microdomains at these binding sites. The ligand was internalized in the free plasmalemmal vesicles of alveolar type I cell within 2 min. Heavy decoration of vesicles at 5 min of perfusion indicated that the amount of CF internalization increased with its concentration in the alveoli. It is suggested that exposure of alveolar surface to several gases of ruminal-origin induces changes in the surface charge of luminal plasmalemma of alveolar type I cells. The significance of these anionic plasmalemmal sites is discussed in relation to the adjustment of osmotic pressure gradient across the alveolar-capillary membrane of the ruminant lung.

  9. An uptake of cationized ferritin by alveolar type I cells in airway-instilled goat lung: distribution of anionic sites on the epithelial surface.

    PubMed

    Atwal, O S; Viel, L; Minhas, K J

    1990-07-01

    The present study has investigated ultrastructural localization of anionic sites on the luminal surface of the alveolar epithelium of goat lung by direct airway instillation of cationized ferritin (CF) in the cranial lobe of the right lung through a bronchoscope. The cationic probe decorated preferentially the luminal plasmalemmal vesicles and plasmalemma proper of alveolar type I cell. This indicated the presence of highly charged anionic microdomains at these binding sites. The ligand was internalized in the free plasmalemmal vesicles of alveolar type I cell within 2 min. Heavy decoration of vesicles at 5 min of perfusion indicated that the amount of CF internalization increased with its concentration in the alveoli. It is suggested that exposure of alveolar surface to several gases of ruminal-origin induces changes in the surface charge of luminal plasmalemma of alveolar type I cells. The significance of these anionic plasmalemmal sites is discussed in relation to the adjustment of osmotic pressure gradient across the alveolar-capillary membrane of the ruminant lung. PMID:2390765

  10. Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung.

    PubMed

    Persinger, Rebecca L; Poynter, Matthew E; Ckless, Karna; Janssen-Heininger, Yvonne M W

    2002-01-01

    The lung can be exposed to a variety of reactive nitrogen intermediates through the inhalation of environmental oxidants and those produced during inflammation. Reactive nitrogen species (RNS) include, nitrogen dioxide (.NO2) and peroxynitrite (ONOO-). Classically known as a major component of both indoor and outdoor air pollution, .NO2 is a toxic free radical gas. .NO2 can also be formed during inflammation by the decomposition of ONOO- or through peroxidase-catalyzed reactions. Due to their reactive nature, RNS may play an important role in disease pathology. Depending on the dose and the duration of administration, .NO, has been documented to cause pulmonary injury in both animal and human studies. Injury to the lung epithelial cells following exposure to .NO2 is characterized by airway denudation followed by compensatory proliferation. The persistent injury and repair process may contribute to airway remodeling, including the development of fibrosis. To better understand the signaling pathways involved in epithelial cell death by .NO2 or otherRNS, we routinely expose cells in culture to continuous gas-phase .NO2. Studies using the .NO2 exposure system revealed that lung epithelial cell death occurs in a density dependent manner. In wound healing experiments, .NO2 induced cell death is limited to cells localized in the leading edge of the wound. Importantly, .NO2-induced death does not appear to be dependent on oxidative stress per se. Potential cell signaling mechanisms will be discussed, which include the mitogen activated protein kinase, c-Jun N-terminal Kinase and the Fas/Fas ligand pathways. During periods of epithelial loss and regeneration that occur in diseases such as asthma or during lung development, epithelial cells in the lung may be uniquely susceptible to death. Understanding the molecular mechanisms of epithelial cell death associated with the exposure to .NO2 will be important in designing therapeutics aimed at protecting the lung from

  11. Induction of CXC chemokines in A549 airway epithelial cells by trypsin and staphylococcal proteases − a possible route for neutrophilic inflammation in chronic rhinosinusitis

    PubMed Central

    Sachse, F; von Eiff, C; Stoll, W; Becker, K; Rudack, C

    2006-01-01

    While various microorganisms have been recovered from patients with chronic rhinosinusitis, the inflammatory impact of virulence factors, in particular proteases from Staphylococcus aureus and coagulase negative staphylococci on the nasal epithelium, has not yet been investigated. Expression of CXC chemokines was determined in the epithelium of patients with chronic rhinosinusitis by immunohistochemistry. In a cell culture system of A549 respiratory epithelial cells, chemokine levels were quantified by enzyme-linked immunosorbent assay (ELISA) after stimulation with supernatants originating from three different staphylococcal strains or with trypsin, representing a serine protease. Inhibition experiments were performed with prednisolone, with the serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonylfluoride (AEBSF) and with the nuclear transcription factor (NF)-κΒ inhibitor (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulphonyl]-2-propenenitrite (BAY) 11–7085. Electromobility shift assays (EMSA) were used to demonstrate NF-κB-dependent protein synthesis. CXC chemokines interleukin (IL)-8, growth-related oncogene alpha (GRO-α) and granulocyte chemotactic protein-2 (GCP-2) were expressed in the patients’ epithelium whereas epithelial cell-derived neutrophil attractant 78 (ENA-78) was rarely detected. In A549 cells, chemokines IL-8, ENA-78 and GRO-α but not GCP-2 were induced by trypsin and almost equal levels were induced by staphylococcal supernatants. IL-8, GRO-α and ENA-78 synthesis was suppressed almost completely by AEBSF and BAY 11–7085, whereas prednisolone reduced chemokine levels differentially dependent on the supernatant added. CXC chemokines were detectable in the epithelium of patients with chronic rhinosinusitis. Staphylococcal serine proteases induced CXC chemokines in A549 cells, probably by the activation of proteases activated receptors, and thus might potentially be involved in neutrophilic inflammation in chronic sinusitis. PMID:16734624

  12. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    PubMed

    Ferru-Clément, Romain; Fresquet, Fleur; Norez, Caroline; Métayé, Thierry; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  13. NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling*

    PubMed Central

    Lillehoj, Erik P.; Hyun, Sang Won; Feng, Chiguang; Zhang, Lei; Liu, Anguo; Guang, Wei; Nguyen, Chinh; Luzina, Irina G.; Atamas, Sergei P.; Passaniti, Antonino; Twaddell, William S.; Puché, Adam C.; Wang, Lai-Xi; Cross, Alan S.; Goldblum, Simeon E.

    2012-01-01

    Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6–1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7–1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38–56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli. PMID:22247545

  14. Repair and regeneration of tracheal surface epithelium and submucosal glands in a mouse model of hypoxic-ischemic injury

    PubMed Central

    HEGAB, AHMED E.; NICKERSON, DEREK W.; HA, VI LUAN; DARMAWAN, DAPHNE O.; GOMPERTS, BRIGITTE N.

    2012-01-01

    Background and objective The heterotopic syngeneic tracheal transplant mouse model is an acute hypoxic-ischemic injury model that undergoes complete repair and regeneration. We hypothesized that the repair and regeneration process of the surface epithelium and submucosal glands would occur in a reproducible pattern that could be followed by the expression of specific markers of epithelial cell types. Methods We used the syngeneic heterotopic tracheal transplant model to develop a temporal and spatial map of cellular repair and regeneration by examining the tracheal grafts at post-transplant days 1, 3, 5, 7, 10 and 14. We used pulsed BrdU and immunofluorescent staining to identify and follow proliferating and repairing cell populations. Results We confirmed the reproducibility of the injury and repair in the model and we found a distinct sequence of reappearance of the various stem/ progenitor and differentiated cell populations of the tracheal surface epithelium and submucosal glands. In the initial phase, the basal and duct cells that survived the injury proliferated to re-epithelialize the basement membrane with K5 and K14 expressing cells. Then these cells proliferated further and differentiated to restore the function of the epithelium. During this repair process, TROP-2 marked all repairing submucosal gland tubules and ducts. Non-CCSP-expressing serous cells were found to differentiate 4–5 days before Clara, mucus and ciliated cells. Conclusions Improving our understanding of the reparative process of the airway epithelium will allow us to identify cell-specific mechanisms of repair that could be used as novel therapeutic approaches for abnormal repair leading to airway diseases. PMID:22617027

  15. Stem cell therapy to protect and repair the developing brain: a review of mechanisms of action of cord blood and amnion epithelial derived cells

    PubMed Central

    Castillo-Melendez, Margie; Yawno, Tamara; Jenkin, Graham; Miller, Suzanne L.

    2013-01-01

    In the research, clinical, and wider community there is great interest in the use of stem cells to reduce the progression, or indeed repair brain injury. Perinatal brain injury may result from acute or chronic insults sustained during fetal development, during the process of birth, or in the newborn period. The most readily identifiable outcome of perinatal brain injury is cerebral palsy, however, this is just one consequence in a spectrum of mild to severe neurological deficits. As we review, there are now clinical trials taking place worldwide targeting cerebral palsy with stem cell therapies. It will likely be many years before strong evidence-based results emerge from these trials. With such trials underway, it is both appropriate and timely to address the physiological basis for the efficacy of stem-like cells in preventing damage to, or regenerating, the newborn brain. Appropriate experimental animal models are best placed to deliver this information. Cell availability, the potential for immunological rejection, ethical, and logistical considerations, together with the propensity for native cells to form teratomas, make it unlikely that embryonic or fetal stem cells will be practical. Fortunately, these issues do not pertain to the use of human amnion epithelial cells (hAECs), or umbilical cord blood (UCB) stem cells that are readily and economically obtained from the placenta and umbilical cord discarded at birth. These cells have the potential for transplantation to the newborn where brain injury is diagnosed or even suspected. We will explore the novel characteristics of hAECs and undifferentiated UCB cells, as well as UCB-derived endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs), and how immunomodulation and anti-inflammatory properties are principal mechanisms of action that are common to these cells, and which in turn may ameliorate the cerebral hypoxia and inflammation that are final pathways in the pathogenesis of perinatal brain

  16. Stem cell therapy to protect and repair the developing brain: a review of mechanisms of action of cord blood and amnion epithelial derived cells.

    PubMed

    Castillo-Melendez, Margie; Yawno, Tamara; Jenkin, Graham; Miller, Suzanne L

    2013-10-24

    In the research, clinical, and wider community there is great interest in the use of stem cells to reduce the progression, or indeed repair brain injury. Perinatal brain injury may result from acute or chronic insults sustained during fetal development, during the process of birth, or in the newborn period. The most readily identifiable outcome of perinatal brain injury is cerebral palsy, however, this is just one consequence in a spectrum of mild to severe neurological deficits. As we review, there are now clinical trials taking place worldwide targeting cerebral palsy with stem cell therapies. It will likely be many years before strong evidence-based results emerge from these trials. With such trials underway, it is both appropriate and timely to address the physiological basis for the efficacy of stem-like cells in preventing damage to, or regenerating, the newborn brain. Appropriate experimental animal models are best placed to deliver this information. Cell availability, the potential for immunological rejection, ethical, and logistical considerations, together with the propensity for native cells to form teratomas, make it unlikely that embryonic or fetal stem cells will be practical. Fortunately, these issues do not pertain to the use of human amnion epithelial cells (hAECs), or umbilical cord blood (UCB) stem cells that are readily and economically obtained from the placenta and umbilical cord discarded at birth. These cells have the potential for transplantation to the newborn where brain injury is diagnosed or even suspected. We will explore the novel characteristics of hAECs and undifferentiated UCB cells, as well as UCB-derived endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs), and how immunomodulation and anti-inflammatory properties are principal mechanisms of action that are common to these cells, and which in turn may ameliorate the cerebral hypoxia and inflammation that are final pathways in the pathogenesis of perinatal brain

  17. Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces.

    PubMed

    Haisma, Elisabeth M; Göblyös, Anikó; Ravensbergen, Bep; Adriaans, Alwin E; Cordfunke, Robert A; Schrumpf, Jasmijn; Limpens, Ronald W A L; Schimmel, Kirsten J M; den Hartigh, Jan; Hiemstra, Pieter S; Drijfhout, Jan Wouter; El Ghalbzouri, Abdoelwaheb; Nibbering, Peter H

    2016-07-01

    We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and

  18. Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces.

    PubMed

    Haisma, Elisabeth M; Göblyös, Anikó; Ravensbergen, Bep; Adriaans, Alwin E; Cordfunke, Robert A; Schrumpf, Jasmijn; Limpens, Ronald W A L; Schimmel, Kirsten J M; den Hartigh, Jan; Hiemstra, Pieter S; Drijfhout, Jan Wouter; El Ghalbzouri, Abdoelwaheb; Nibbering, Peter H

    2016-07-01

    We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and

  19. APO-9'-Fucoxanthinone Extracted from Undariopsis peteseniana Protects Oxidative Stress-Mediated Apoptosis in Cigarette Smoke-Exposed Human Airway Epithelial Cells.

    PubMed

    Jang, Jun-Ho; Lee, Ji-Hyeok; Chand, Hitendra S; Lee, Jong-Soo; Lin, Yong; Weathington, Nathaniel; Mallampalli, Rama; Jeon, You-Jin; Nyunoya, Toru

    2016-01-01

    Long-term cigarette smoking increases the risk for chronic obstructive pulmonary disease (COPD), characterized by irreversible expiratory airflow limitation. The pathogenesis of COPD involves oxidative stress and chronic inflammation. Various natural marine compounds possess both anti-oxidant and anti-inflammatory properties, but few have been tested for their efficacy in COPD models. In this study, we conducted an in vitro screening test to identify natural compounds isolated from various brown algae species that might provide protection against cigarette smoke extract (CSE)-induced cytotoxicity. Among nine selected natural compounds, apo-9'-fucoxanthinone (Apo9F) exhibited the highest protection against CSE-induced cytotoxicity in immortalized human bronchial epithelial cells (HBEC2). Furthermore, the protective effects of Apo9F were observed to be associated with a significant reduction in apoptotic cell death, DNA damage, and the levels of mitochondrial reactive oxygen species (ROS) released from CSE-exposed HBEC2 cells. These results suggest that Apo9F protects against CSE-induced DNA damage and apoptosis by regulating mitochondrial ROS production. PMID:27455285

  20. APO-9'-Fucoxanthinone Extracted from Undariopsis peteseniana Protects Oxidative Stress-Mediated Apoptosis in Cigarette Smoke-Exposed Human Airway Epithelial Cells.

    PubMed

    Jang, Jun-Ho; Lee, Ji-Hyeok; Chand, Hitendra S; Lee, Jong-Soo; Lin, Yong; Weathington, Nathaniel; Mallampalli, Rama; Jeon, You-Jin; Nyunoya, Toru

    2016-07-21

    Long-term cigarette smoking increases the risk for chronic obstructive pulmonary disease (COPD), characterized by irreversible expiratory airflow limitation. The pathogenesis of COPD involves oxidative stress and chronic inflammation. Various natural marine compounds possess both anti-oxidant and anti-inflammatory properties, but few have been tested for their efficacy in COPD models. In this study, we conducted an in vitro screening test to identify natural compounds isolated from various brown algae species that might provide protection against cigarette smoke extract (CSE)-induced cytotoxicity. Among nine selected natural compounds, apo-9'-fucoxanthinone (Apo9F) exhibited the highest protection against CSE-induced cytotoxicity in immortalized human bronchial epithelial cells (HBEC2). Furthermore, the protective effects of Apo9F were observed to be associated with a significant reduction in apoptotic cell death, DNA damage, and the levels of mitochondrial reactive oxygen species (ROS) released from CSE-exposed HBEC2 cells. These results suggest that Apo9F protects against CSE-induced DNA damage and apoptosis by regulating mitochondrial ROS production.

  1. APO-9′-Fucoxanthinone Extracted from Undariopsis peteseniana Protects Oxidative Stress-Mediated Apoptosis in Cigarette Smoke-Exposed Human Airway Epithelial Cells

    PubMed Central

    Jang, Jun-Ho; Lee, Ji-Hyeok; Chand, Hitendra S.; Lee, Jong-Soo; Lin, Yong; Weathington, Nathaniel; Mallampalli, Rama; Jeon, You-Jin; Nyunoya, Toru

    2016-01-01

    Long-term cigarette smoking increases the risk for chronic obstructive pulmonary disease (COPD), characterized by irreversible expiratory airflow limitation. The pathogenesis of COPD involves oxidative stress and chronic inflammation. Various natural marine compounds possess both anti-oxidant and anti-inflammatory properties, but few have been tested for their efficacy in COPD models. In this study, we conducted an in vitro screening test to identify natural compounds isolated from various brown algae species that might provide protection against cigarette smoke extract (CSE)-induced cytotoxicity. Among nine selected natural compounds, apo-9′-fucoxanthinone (Apo9F) exhibited the highest protection against CSE-induced cytotoxicity in immortalized human bronchial epithelial cells (HBEC2). Furthermore, the protective effects of Apo9F were observed to be associated with a significant reduction in apoptotic cell death, DNA damage, and the levels of mitochondrial reactive oxygen species (ROS) released from CSE-exposed HBEC2 cells. These results suggest that Apo9F protects against CSE-induced DNA damage and apoptosis by regulating mitochondrial ROS production. PMID:27455285

  2. Infection of human airway epithelial cells by different subtypes of Dobrava-Belgrade virus reveals gene expression patterns corresponding to their virulence potential.

    PubMed

    Witkowski, Peter T; Bourquain, Daniel; Bankov, Katrin; Auste, Brita; Dabrowski, Piotr W; Nitsche, Andreas; Krüger, Detlev H; Schaade, Lars

    2016-06-01

    Dobrava-Belgrade virus (DOBV) is a pathogen causing hemorrhagic fever with renal syndrome in Europe. Virulence and case fatality rate are associated with virus genotype; however the reasons for these differences are not well understood. In this work we present virus-specific effects on the gene expression profiles of human lung epithelial cells (A549) infected with different genotypes of DOBV (Dobrava, Kurkino, and Sochi), as well as the low-virulent Tula virus (TULV). The data was collected by whole-genome gene expression microarrays and confirmed by quantitative real-time PCR. Despite their close genetic relationship, the expression profiles induced by infection with different hantaviruses are significantly varying. Major differences were observed in regulation of immune response genes, which were especially induced by highly virulent DOBV genotypes Dobrava and Sochi in contrast to less virulent DOBV-Kurkino and TULV. This work gives first insights into the differences of virus - host interactions of DOBV on genotype level. PMID:27058765

  3. A Multi-Omics Approach Identifies Key Hubs Associated with Cell Type-Specific Responses of Airway Epithelial Cells to Staphylococcal Alpha-Toxin

    PubMed Central

    Richter, Erik; Harms, Manuela; Ventz, Katharina; Gierok, Philipp; Chilukoti, Ravi Kumar; Hildebrandt, Jan-Peter; Mostertz, Jörg; Hochgräfe, Falko

    2015-01-01

    Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects. PMID:25816343

  4. Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

    PubMed Central

    An, S.S.; Bai, T.R.; Bates, J.H.T.; Black, J.L.; Brown, R.H.; Brusasco, V.; Chitano, P.; Deng, L.; Dowell, M.; Eidelman, D.H.; Fabry, B.; Fairbank, N.J.; Ford, L.E.; Fredberg, J.J.; Gerthoffer, W.T.; Gilbert, S.H.; Gosens, R.; Gunst, S.J.; Halayko, A.J.; Ingram, R.H.; Irvin, C.G.; James, A.L.; Janssen, L.J.; King, G.G.; Knight, D.A.; Lauzon, A.M.; Lakser, O.J.; Ludwig, M.S.; Lutchen, K.R.; Maksym, G.N.; Martin, J.G.; Mauad, T.; McParland, B.E.; Mijailovich, S.M.; Mitchell, H.W.; Mitchell, R.W.; Mitzner, W.; Murphy, T.M.; Paré, P.D.; Pellegrino, R.; Sanderson, M.J.; Schellenberg, R.R.; Seow, C.Y.; Silveira, P.S.P.; Smith, P.G.; Solway, J.; Stephens, N.L.; Sterk, P.J.; Stewart, A.G.; Tang, D.D.; Tepper, R.S.; Tran, T.; Wang, L.

    2008-01-01

    Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored. PMID:17470619

  5. Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn{sup 2+}

    SciTech Connect

    Tal, T.L.; Graves, L.M.; Silbajoris, R.; Bromberg, P.A.; Wu, W.; Samet, J.M. . E-mail: samet.james@epa.gov

    2006-07-01

    Epidemiological studies have implicated zinc (Zn{sup 2+}) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn{sup 2+}-induced EGFR activation in HAEC, we treated HAEC with 500 {mu}M ZnSO{sub 4} for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn{sup 2+} results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn{sup 2+}-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn{sup 2+} treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn{sup 2+}. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn{sup 2+} or V{sup 4+} was significantly diminished. Moreover, exposure of HAEC to Zn{sup 2+} also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn{sup 2+}-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn{sup 2+} exposure.

  6. Phospholipase D1 is threonine-phosphorylated in human-airway epithelial cells stimulated by sphingosine-1-phosphate by a mechanism involving Src tyrosine kinase and protein kinase Cdelta.

    PubMed Central

    Ghelli, Anna; Porcelli, Anna M; Facchini, Annalisa; Hrelia, Silvana; Flamigni, Flavio; Rugolo, Michela

    2002-01-01

    The regulatory role of protein kinase C (PKC) delta isoform in the stimulation of phospholipase D (PLD) by sphingosine-1-phosphate (SPP) in a human-airway epithelial cell line (CFNPE9o(-)) was revealed by using antisense oligodeoxynucleotide to PKCdelta, in combination with the specific inhibitor rottlerin. Cell treatment with antisense oligodeoxynucleotide, but not with sense oligodeoxynucleotide, completely eliminated PKCdelta expression and resulted in the strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKCalpha, beta, delta, epsilon and zeta isoforms expressed in these cells, only PKCdelta was activated on cell stimulation with SPP, as indicated by translocation into the membrane fraction. Furthermore, pertussis toxin and genistein eliminated both PKCdelta translocation and PLD activation. In particular, a significant reduction in phosphatidylbutanol formation by SPP was observed in the presence of 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1), an inhibitor of Src tyrosine kinase. Furthermore, the activity of Src kinase was slightly increased by SPP and inhibited by PP1. However, the level of PKCdelta tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCdelta. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas the PLD1 isoform alone was threonine-phosphorylated in SPP-treated cells. PLD1 threonine phosphorylation was strongly inhibited by rottlerin, by anti-PKCdelta oligodeoxynucleotide and by PP1. In conclusion, in CFNPE9o(-) cells, SPP interacts with a membrane receptor linked to a G(i) type of G-protein, leading to activation of PLD, probably the PLD1 isoform, by a signalling pathway involving Src and PKCdelta. PMID:12014986

  7. Ozone-induced acute tracheobronchial epithelial injury: relationship to granulocyte emigration in the lung

    SciTech Connect

    Hyde, D.M.; Hubbard, W.C.; Wong, V.; Wu, R.; Pinkerton, K.; Plopper, C.G. )

    1992-05-01

    To investigate the relationship between granulocyte emigration and epithelial injury in specific airway generations of the tracheobronchial tree following short-term ozone exposure, we exposed rhesus monkeys for 8 h to 0.00 (controls) or 0.96 ppm ozone with post-exposure periods of 1, 12, 24, 72, and 168 h in filtered air before necropsy. There were five control and three exposed monkeys for each of the post-exposure times for a total of 20 monkeys. Neutrophils isolated from peripheral blood and labeled with 111In-tropolonate were infused in the cephalic vein in unanesthetized monkeys (except the 1-h group) 4 to 5 h before necropsy. The trachea and microdissected bronchi (fourth and ninth generations) and respiratory bronchioles (fifteenth generation) from the right upper lobe of each monkey were examined by electron microscopy. Labeled neutrophil influx into lung tissue and bronchoalveolar lavage fluid (BALF) was maximal at 12 h and returned to baseline by 24 h after exposure. This was in contrast to total neutrophils in BALF, which were significantly elevated through 24 h after exposure but returned to baseline by 72 h. Lavage protein was significantly elevated at 24 h after exposure but was at control levels at all other times. Morphometric observations showed epithelial necrosis at 1 and 12 h in the trachea and bronchioles but continued to be observed in significant numbers at 24 h after exposure in bronchi. A significant increase in the labeling index of epithelial cells was observed at 12 h only in bronchi. Epithelial necrosis and repair was associated with the presence of granulocytes in the epithelium and interstitium of all airway levels. However, eosinophils were maximally increased in the epithelium and interstitium of bronchi at 24 h after exposure when epithelial necrosis was maximal in these airways and when lavage protein was significantly elevated.

  8. Respiratory Syncytial Virus Infection Upregulates NLRC5 and Major Histocompatibility Complex Class I Expression through RIG-I Induction in Airway Epithelial Cells

    PubMed Central

    Guo, Xuancheng; Liu, Taixiang; Shi, Hengfei; Wang, Jingjing; Ji, Ping; Wang, Hongwei; Hou, Yayi; Tan, Ren Xiang

    2015-01-01

    ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid-inducible gene I (RIG-I). We show here that RSV infection upregulates major histocompatibility complex class I (MHC-I) expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via beta interferon (IFN-β) production, since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFN-β, but not by antibody to IFN-γ. RSV infection resulted in RIG-I upregulation and induction of NLRC5 and MHC-I. Suppression of RIG-I induction significantly blocked NLRC5, as well as MHC-I, upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC-I following introduction of the RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore proposed in which the host upregulates MHC-I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC-I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease. IMPORTANCE Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Infection early in life is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I (MHC-I) on the cell surface, which facilitates cytotoxic T cell activation and antiviral immunity. Here, we show that RSV infection of lung epithelial

  9. Allergen-induced airway responses.

    PubMed

    Gauvreau, Gail M; El-Gammal, Amani I; O'Byrne, Paul M

    2015-09-01

    Environmental allergens are an important cause of asthma and can contribute to loss of asthma control and exacerbations. Allergen inhalation challenge has been a useful clinical model to examine the mechanisms of allergen-induced airway responses and inflammation. Allergen bronchoconstrictor responses are the early response, which reaches a maximum within 30 min and resolves by 1-3 h, and late responses, when bronchoconstriction recurs after 3-4 h and reaches a maximum over 6-12 h. Late responses are followed by an increase in airway hyperresponsiveness. These responses occur when IgE on mast cells is cross-linked by an allergen, causing degranulation and the release of histamine, neutral proteases and chemotactic factors, and the production of newly formed mediators, such as cysteinyl leukotrienes and prostaglandin D2. Allergen-induced airway inflammation consists of an increase in airway eosinophils, basophils and, less consistently, neutrophils. These responses are mediated by the trafficking and activation of myeloid dendritic cells into the airways, probably as a result of the release of epithelial cell-derived thymic stromal lymphopoietin, and the release of pro-inflammatory cytokines from type 2 helper T-cells. Allergen inhalation challenge has also been a widely used model to study potential new therapies for asthma and has an excellent negative predictive value for this purpose. PMID:26206871

  10. Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females.

    PubMed

    Hall, Olivia J; Limjunyawong, Nathachit; Vermillion, Meghan S; Robinson, Dionne P; Wohlgemuth, Nicholas; Pekosz, Andrew; Mitzner, Wayne; Klein, Sabra L

    2016-09-01

    Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women's health. PMID:27631986

  11. Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females

    PubMed Central

    Vermillion, Meghan S.; Robinson, Dionne P.; Pekosz, Andrew; Mitzner, Wayne

    2016-01-01

    Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women’s health. PMID:27631986

  12. Difficult Airway Management in Field Conditions: Somalia Experience.

    PubMed

    Özkan, Ahmet Selim; Nasır, Serdar Nazif

    2015-10-01

    Difficult airway is defined as having the patient's mask ventilation or difficult tracheal intubation of an experienced anaesthesiologist. A number of reasons, such as congenital or acquired anatomical anomalies, can cause difficult intubation and difficult ventilation. Keeping all equipment ready for airway management of patients will reduce mortality and complications. In this case, it is intended that the submission of difficult airway management who encountered in mandibular reconstruction for mandible bone defect repairing with reconstruction plates before at the field conditions in Somalia.

  13. Airway and Extracellular Matrix Mechanics in COPD.

    PubMed

    Bidan, Cécile M; Veldsink, Annemiek C; Meurs, Herman; Gosens, Reinoud

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases worldwide, and is characterized by airflow obstruction that is not fully reversible with treatment. Even though airflow obstruction is caused by airway smooth muscle contraction, the extent of airway narrowing depends on a range of other structural and functional determinants that impact on active and passive tissue mechanics. Cells and extracellular matrix in the airway and parenchymal compartments respond both passively and actively to the mechanical stimulation induced by smooth muscle contraction. In this review, we summarize the factors that regulate airway narrowing and provide insight into the relative contributions of different constituents of the extracellular matrix and their biomechanical impact on airway obstruction. We then review the changes in extracellular matrix composition in the airway and parenchymal compartments at different stages of COPD, and finally discuss how these changes impact airway narrowing and the development of airway hyperresponsiveness. Finally, we position these data in the context of therapeutic research focused on defective tissue repair. As a conclusion, we propose that future works should primarily target mild or early COPD, prior to the widespread structural changes in the alveolar compartment that are more characteristic of severe COPD.

  14. Airway and Extracellular Matrix Mechanics in COPD

    PubMed Central

    Bidan, Cécile M.; Veldsink, Annemiek C.; Meurs, Herman; Gosens, Reinoud

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases worldwide, and is characterized by airflow obstruction that is not fully reversible with treatment. Even though airflow obstruction is caused by airway smooth muscle contraction, the extent of airway narrowing depends on a range of other structural and functional determinants that impact on active and passive tissue mechanics. Cells and extracellular matrix in the airway and parenchymal compartments respond both passively and actively to the mechanical stimulation induced by smooth muscle contraction. In this review, we summarize the factors that regulate airway narrowing and provide insight into the relative contributions of different constituents of the extracellular matrix and their biomechanical impact on airway obstruction. We then review the changes in extracellular matrix composition in the airway and parenchymal compartments at different stages of COPD, and finally discuss how these changes impact airway narrowing and the development of airway hyperresponsiveness. Finally, we position these data in the context of therapeutic research focused on defective tissue repair. As a conclusion, we propose that future works should primarily target mild or early COPD, prior to the widespread structural changes in the alveolar compartment that are more characteristic of severe COPD. PMID:26696894

  15. The loss of Hoxa5 function promotes Notch-dependent goblet cell metaplasia in lung airways

    PubMed Central

    Boucherat, Olivier; Chakir, Jamila; Jeannotte, Lucie

    2012-01-01

    Summary Hox genes encode transcription factors controlling complex developmental processes in various organs. Little is known, however, about how HOX proteins control cell fate. Herein, we demonstrate that the goblet cell metaplasia observed in lung airways from Hoxa5−/− mice originates from the transdifferentiation of Clara cells. Reduced CC10 expression in Hoxa5−/− embryos indicates that altered cell specification occurs prior to birth. The loss of Hoxa5 function does not preclude airway repair after naphthalene exposure, but the regenerated epithelium presents goblet cell metaplasia and less CC10-positive cells, demonstrating the essential role of Hoxa5 for correct differentiation. Goblet cell metaplasia in Hoxa5−/− mice is a FOXA2-independent process. However, it is associated with increased Notch signaling activity. Consistent with these findings, expression levels of activated NOTCH1 and the effector gene HEY2 are enhanced in patients with chronic obstructive pulmonary disease. In vivo administration of a γ-secretase inhibitor attenuates goblet cell metaplasia in Hoxa5−/− mice, highlighting the contribution of Notch signaling to the phenotype and suggesting a potential therapeutic strategy to inhibit goblet cell differentiation and mucus overproduction in airway diseases. In summary, the loss of Hoxa5 function in lung mesenchyme impacts on epithelial cell fate by modulating Notch signaling. PMID:23213461

  16. Vascular airway compression management in a case of aortic arch and descending thoracic aortic aneurysm

    PubMed Central

    Kumar, Alok; Dutta, Vikas; Negi, Sunder; Puri, G. D.

    2016-01-01

    Airway compression due to distal aortic arch and descending aortic aneurysm repair has been documented. This case of tracheal and left main stem bronchus compression due to aortic aneurysm occurred in a 42-year-old man. The airway compression poses a challenge for the anesthesiologist in airway management during aortic aneurysm repair surgery. The fiber-optic bronchoscope is very helpful in decision-making both preoperatively and postoperatively in such cases. We report a case of airway compression in a 42-year-old patient who underwent elective distal aortic arch and descending aortic aneurysm repair. PMID:27397474

  17. Gamma delta T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corneal epithelial abrasion in C57BL/6 mice induces an inflammatory response, with peak accumulation of neutrophils in the corneal stroma within 12 hours. Platelets localize in the limbal vessels throughout the same time course as neutrophils and contribute to wound healing because antibody-dependen...

  18. Recent advances in understanding inflammation and remodeling in the airways in chronic obstructive pulmonary disease.

    PubMed

    Sohal, Sukhwinder Singh; Ward, Chris; Danial, Wan; Wood-Baker, Richard; Walters, Eugene Haydn

    2013-06-01

    The authors have reviewed the current literature on airway inflammation and remodeling in smoking-related chronic obstructive pulmonary disease (COPD). Detailed data on airway remodeling in COPD are especially sparse and how these changes lead to decline in lung function is not well understood. Small airway fibrosis and obliteration are likely to be the main contributors to physiological airway dysfunction and occur earlier than any subsequent development of emphysema. One potential mechanism contributing to small airway fibrosis/obliteration and change in extracellular matrix is epithelial-mesenchymal transition. When associated with angiogenesis (so-called epithelial-mesenchymal transition type 3) it may well also be the link with the development of cancer, which is closely associated with COPD, predominantly in large airways. The authors have focused on our recent publications in these areas. Further investigations teasing out these mechanisms will help improve our understanding of key airway disease processes in COPD, which may have major therapeutic implications.

  19. Increased expression of senescence markers in cystic fibrosis airways.

    PubMed

    Fischer, Bernard M; Wong, Jessica K; Degan, Simone; Kummarapurugu, Apparao B; Zheng, Shuo; Haridass, Prashamsha; Voynow, Judith A

    2013-03-15

    Cystic Fibrosis (CF) is a chronic lung disease characterized by chronic neutrophilic airway inflammation and increased levels of neutrophil elastase (NE) in the airways. We have previously reported that NE treatment triggers cell cycle arrest. Cell cycle arrest can lead to senescence, a complete loss of replicative capacity. Importantly, senescent cells can be proinflammatory and would perpetuate CF chronic inflammation. By immunohistochemistry, we evaluated whether airway sections from CF and control subjects expressed markers of senescence, including p16(INK4a) (p16), a cyclin-dependent kinase inhibitor, phospho-Histone H2A.X (γH2A.X), and phospho-checkpoint 2 kinase (phospho-Chk2), which are also DNA damage response markers. Compared with airway epithelium from control subjects, CF airway epithelium had increased levels of expression of all three senescence markers. We hypothesized that the high load of NE in the CF airway triggers epithelial senescence by upregulating expression of p16, which inhibits cyclin-dependent kinase 4 (CDK4). Normal human bronchial epithelial (NHBE) cells, cultured in air-liquid interface were treated with NE (0, 200, and 500 nM) to induce visible injury. Total cell lysates were collected and evaluated by Western analysis for p16 protein expression and CDK4 kinase activity. NE significantly increased p16 expression and decreased CDK4 kinase activity in NHBE cells. These results support the concept that NE triggers expression of senescence markers in CF airway epithelial cells. PMID:23316069

  20. Mitochondrial Transplantation Attenuates Airway Hyperresponsiveness by Inhibition of Cholinergic Hyperactivity

    PubMed Central

    Su, Yuan; Zhu, Liping; Yu, Xiangyuan; Cai, Lei; Lu, Yankai; Zhang, Jiwei; Li, Tongfei; Li, Jiansha; Xia, Jingyan; Xu, Feng; Hu, Qinghua

    2016-01-01

    Increased cholinergic activity has been highlighted in the pathogenesis of airway hyperresponsiveness, and alternations of mitochondrial structure and function appear to be involved in many lung diseases including airway hyperresponsiveness. It is crucial to clarify the cause-effect association between mitochondrial dysfunction and cholinergic hyperactivity in the pathogenesis of airway hyperresponsiveness. Male SD rats and cultured airway epithelial cells were exposed to cigarette smoke plus lipopolysaccharide administration; mitochondria isolated from airway epithelium were delivered into epithelial cells in vitro and in vivo. Both the cigarette smoke plus lipopolysaccharide-induced cholinergic hyperactivity in vitro and the airway hyperresponsiveness to acetylcholine in vivo were reversed by the transplantation of exogenous mitochondria. The rescue effects of exogenous mitochondria were imitated by the elimination of excessive reactive oxygen species or blockage of muscarinic M3 receptor, but inhibited by M receptor enhancer. Mitochondrial transplantation effectively attenuates cigarette smoke plus lipopolysaccharide-stimulated airway hyperresponsiveness through the inhibition of ROS-enhanced epithelial cholinergic hyperactivity. PMID:27279915

  1. Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

    PubMed Central

    Ren, Xiaoyun

    2014-01-01

    Neisseria meningitidis is the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen, Neisseria meningitidis is frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using an in vitro assay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis. PMID:25225250

  2. Disease-associated Neisseria meningitidis isolates inhibit wound repair in respiratory epithelial cells in a type IV pilus-independent manner.

    PubMed

    Ren, Xiaoyun; MacKichan, Joanna K

    2014-12-01

    Neisseria meningitidis is the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen, Neisseria meningitidis is frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using an in vitro assay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis. PMID:25225250

  3. Retinal pigment epithelial cell proliferation

    PubMed Central

    Temple, Sally

    2015-01-01

    The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most in vitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration. PMID:26041390

  4. RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

    PubMed

    Lee, Ye-Ji; Park, Hyun-Jung; Woo, So-Youn; Park, Eun-Mi; Kang, Jihee Lee

    2014-09-01

    Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

  5. Triggers of airway inflammation.

    PubMed

    Kerrebijn, K F

    1986-01-01

    Most asthmatics have hyperresponsive airways. This makes them more sensitive than non-asthmatics to bronchoconstricting environmental exposures which, in their turn, may enhance responsiveness. Airway inflammation is considered to be a key determinant of airway hyperresponsiveness: the fact that chronic airway inflammation in cystic fibrosis does not lead to airway hyperresponsiveness of any importance indicates, however, that the role of airway inflammation is complex and incompletely elucidated. The main inducers of airway inflammation are viral infections, antigens, occupational stimuli and pollutants. Although exercise, airway cooling and hyper- or hypotonic aerosols are potent stimuli of bronchoconstriction, it is questionable if airway inflammation is involved in their mode of action. Each of the above-mentioned stimuli is discussed, with emphasis laid on the relation of symptoms to mechanisms. PMID:3533597

  6. Respiratory epithelial cells orchestrate pulmonary innate immunity

    PubMed Central

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and ‘instruct’ the professional immune system to protect the lungs from infection and injury. PMID:25521682

  7. Relation of smoking to immunoreactive endothelin in the bronchiolar epithelial cells.

    PubMed Central

    Shokeir, M. O.; Paré, P.; Wright, J. L.

    1994-01-01

    BACKGROUND--Endothelin is a potent bronchoconstrictor which appears to be important in asthma. To ascertain whether cigarette smoking is associated with any alteration in the proportion of bronchiolar epithelial cells which express endothelin immunoreactivity, the airways in the lungs of non-smokers and smokers were analysed. Since an increase in immunoreactivity has been found in the bronchial epithelial cells of asthmatic subjects, cigarette smokers with and without evidence of airway hyperresponsiveness were also selected. METHODS--A point counting method which examined the proportion of endothelin immunoreactive epithelial cells in membranous and respiratory bronchioles was used. RESULTS--Neither smoking itself nor evidence of airway hyperresponsiveness altered the percentage of endothelin immunoreactive epithelial cells in the membraneous and respiratory bronchioles. CONCLUSIONS--Cigarette smoke does not induce endothelin production in bronchiolar epithelial cells, and the airway hyperresponsiveness seen in some patients with lung disease induced by cigarette smoking is not related to exaggerated endothelin production in epithelial cells. PMID:8091324

  8. Airway Hydration and COPD

    PubMed Central

    Ghosh, Arunava; Boucher, R.C.; Tarran, Robert

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the prevalent causes of worldwide mortality and encompasses two major clinical phenotypes, i.e., chronic bronchitis (CB) and emphysema. The most common cause of COPD is chronic tobacco inhalation. Research focused on the chronic bronchitic phenotype of COPD has identified several pathological processes that drive disease initiation and progression. For example, the lung’s mucociliary clearance (MCC) system performs the critical task of clearing inhaled pathogens and toxic materials from the lung. MCC efficiency is dependent on: (i) the ability of apical plasma membrane ion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC) to maintain airway hydration; (ii) ciliary beating; and, (iii) appropriate rates of mucin secretion. Each of these components is impaired in CB and likely contributes to the mucus stasis/accumulation seen in CB patients. This review highlights the cellular components responsible for maintaining MCC and how this process is disrupted following tobacco exposure and with CB. We shall also discuss existing therapeutic strategies for the treatment of chronic bronchitis and how components of the MCC can be used as biomarkers for the evaluation of tobacco or tobacco-like-product exposure. PMID:26068443