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Sample records for airway epithelial cultures

  1. 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.

  2. 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...

  3. 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

  4. Culture of Airway Epithelial Cells from Neonates Sampled within 48-Hours of Birth

    PubMed Central

    Miller, David; Turner, Steve W.; Spiteri-Cornish, Daniella; McInnes, Neil; Scaife, Alison; Danielian, Peter J.; Devereux, Graham; Walsh, Garry M.

    2013-01-01

    Introduction Little is known about how neonatal airway epithelial cell phenotype impacts on respiratory disease in later life. This study aimed to establish a methodology to culture and characterise neonatal nasal epithelial cells sampled from healthy, non-sedated infants within 48 hours of delivery. Methods Nasal epithelial cells were sampled by brushing both nostrils with an interdental brush, grown to confluence and sub-cultured. Cultured cells were characterised morphologically by light and electron microscopy and by immunocytochemistry. As an exemplar pro-inflammatory chemokine, IL-8 concentrations were measured in supernatants from unstimulated monolayers and after exposure to IL-1β/TNF-α or house dust mite extract. Results Primary cultures were successfully established in 135 (91%) of 149 neonatal samples seeded, with 79% (n  =  117) successfully cultured to passage 3. The epithelial lineage of the cells was confirmed by morphological analysis and immunostaining. Constitutive IL-8 secretion was observed and was upregulated by IL-1β/TNF-α or house dust mite extract in a dose dependent manner. Conclusion We describe a safe, minimally invasive method of culturing nasal epithelial cells from neonates suitable for functional cell analysis offering an opportunity to study “naïve” cells that may prove useful in elucidating the role of the epithelium in the early origins of asthma and/or allergic rhinitis. PMID:24223790

  5. Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.

    PubMed

    S Banach, Bridget; Orenstein, Jan M; Fox, Linda M; Randell, Scott H; Rowley, Anne H; Baker, Susan C

    2009-03-01

    Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.

  6. 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...

  7. 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

  8. Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come.

    PubMed

    McLellan, Kirsty; Shields, Mike; Power, Ultan; Turner, Steve

    2015-12-01

    Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies.

  9. A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection.

    PubMed

    Schwab, Ute; Caldwell, Shannon; Matychak, Mary-Beth; Felippe, Julia

    2013-07-15

    We developed a 3-D equine bronchial epithelial cell (BEC) culture that fully differentiates into ciliary beating and mucus producing cells. Using this system, we evaluated how mucus affects the phagocytic activity of macrophages. Adult horse monocyte-derived macrophages were incubated with Rhodococcus equi for 4h either in the mucus layer of in vitro generated airway epithelium or on collagen coated membranes. Using light and electron microscopy, we noted that the number of macrophages with intracellular bacteria, and the number of intracellular bacteria per macrophage were lower in the presence of mucus. TNFα measurements revealed that the presence of BECs promoted TNFα production by R. equi-infected macrophages; a decrease in TLR-2 (involved in R. equi recognition) and an increase in EGF-R (involved in mucin production) mRNA expression were also noted. Interestingly, when foal macrophages were added to foal BECs, we made the opposite observation, i.e. many macrophages were loaded with R. equi. Our in vitro bronchial system shows great potential for the identification of mechanisms how BECs and mucus play a role in phagocyte activation and bacterial clearance. Further studies using this system will show whether the airway environment in the foal responds differently to R. equi infection.

  10. Pro-inflammatory Cytokines Impair Vitamin D-induced Host Defense in Cultured Airway Epithelial Cells.

    PubMed

    Schrumpf, Jasmijn A; Amatngalim, Gimano D; Veldkamp, Joris B; Verhoosel, Renate M; Ninaber, Dennis K; Ordonez, Soledad R; van der Does, Anne M; Haagsman, Henk P; Hiemstra, Pieter S

    2017-02-23

    Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells (PBEC) to pro-inflammatory cytokines alters their vitamin D metabolism, antibacterial activity and expression of hCAP18/LL-37. To investigate this, PBEC were differentiated at the air-liquid interphase for 14 days in presence of the pro-inflammatory cytokines TNF-α and IL-1β (TNF-α/IL-1β), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor (VDR) and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using qPCR, Western blot and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using non-typeable Haemophilus influenzae (NTHi). We found that TNF-α/IL-1β treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of NTHi. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1β, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and VDR expression remained unaffected. Furthermore, we demonstrated that the TNF-α/IL-1β-mediated induction of CYP24A1 was at least in part mediated by the transcription factor specific protein 1 (Sp1) and the EGFR-MAPK-pathway. These findings indicate that TNF-α/IL-1β decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1, and suggests that chronic inflammation impairs protective responses induced by vitamin D.

  11. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

  12. Tissue inhibitor of metalloproteinase-1 moderates airway re-epithelialization by regulating matrilysin activity.

    PubMed

    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

    2008-05-01

    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.

  13. 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

  14. Cultured Human Airway Epithelial Cells (Calu-3): A Model of Human Respiratory Function, Structure, and Inflammatory Responses

    PubMed Central

    Zhu, Yan; Chidekel, Aaron; Shaffer, Thomas H.

    2010-01-01

    This article reviews the application of the human airway Calu-3 cell line as a respiratory model for studying the effects of gas concentrations, exposure time, biophysical stress, and biological agents on human airway epithelial cells. Calu-3 cells are grown to confluence at an air-liquid interface on permeable supports. To model human respiratory conditions and treatment modalities, monolayers are placed in an environmental chamber, and exposed to specific levels of oxygen or other therapeutic modalities such as positive pressure and medications to assess the effect of interventions on inflammatory mediators, immunologic proteins, and antibacterial outcomes. Monolayer integrity and permeability and cell histology and viability also measure cellular response to therapeutic interventions. Calu-3 cells exposed to graded oxygen concentrations demonstrate cell dysfunction and inflammation in a dose-dependent manner. Modeling positive airway pressure reveals that pressure may exert a greater injurious effect and cytokine response than oxygen. In experiments with pharmacological agents, Lucinactant is protective of Calu-3 cells compared with Beractant and control, and perfluorocarbons also protect against hyperoxia-induced airway epithelial cell injury. The Calu-3 cell preparation is a sensitive and efficient preclinical model to study human respiratory processes and diseases related to oxygen- and ventilator-induced lung injury. PMID:20948883

  15. 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...

  16. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  17. 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.

  18. 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

  19. 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...

  20. 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.

  1. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    PubMed

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  2. The Diacetyl-exposed Human Airway Epithelial Secretome: New Insights Into Flavoring Induced Airways Disease.

    PubMed

    Brass, David M; Gwinn, William M; Valente, Ashlee M; Kelly, Francine L; Brinkley, Christie D; Nagler, Andrew E; Moseley, M Arthur; Morgan, Daniel L; Palmer, Scott M; Foster, Matthew W

    2017-03-01

    Bronchiolitis obliterans (BO) is an increasingly important lung disease characterized by fibroproliferative airway lesions and decrements in lung function. Occupational exposure to the artificial food flavoring ingredient diacetyl, commonly used to impart a buttery flavor to microwave popcorn, has been associated with BO development. In the occupational setting, diacetyl vapor is first encountered by the airway epithelium. To better understand the effects of diacetyl vapor on the airway epithelium we used an unbiased proteomic approach to characterize both the apical and basolateral secretomes of air liquid interface cultures of primary human airway epithelial cells from four unique donors after exposure to an occupationally relevant ~1100 ppm of diacetyl vapor or PBS as a control on alternating days. Basolateral and apical supernatants collected 48 hours after the third exposure were analyzed using one-dimensional liquid chromatography tandem mass spectrometry. Paired t-tests adjusted for multiple comparisons were used to assess differential expression between diacetyl and PBS exposure. Of the significantly differentially expressed proteins identified, 61 were unique to the apical secretome, 81 were unique to the basolateral secretome and there were an additional 11 present in both. Pathway enrichment analysis using publicly available databases reveals that proteins associated with matrix remodeling including degradation, assembly and new matrix organization were over-represented in the data sets. Similarly, protein modifiers of epidermal growth factor receptor signaling were significantly altered. The ordered changes in protein expression suggest that the airway epithelial response to diacetyl may contribute to BO pathogenesis.

  3. Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

    PubMed Central

    Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C.; Hershenson, Marc B.

    2008-01-01

    Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function. PMID:18787220

  4. Airway Epithelial miRNA Expression Is Altered in Asthma

    PubMed Central

    Solberg, Owen D.; Ostrin, Edwin J.; Love, Michael I.; Peng, Jeffrey C.; Bhakta, Nirav R.; Nguyen, Christine; Solon, Margaret; Nguyen, Cindy; Barczak, Andrea J.; Zlock, Lorna T.; Blagev, Denitza P.; Finkbeiner, Walter E.; Ansel, K. Mark; Arron, Joseph R.; Erle, David J.

    2012-01-01

    Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13–regulated miRNAs. Methods: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway

  5. Induction of CFTR gene expression by 1,25(OH)2 vitamin D3, 25OH vitamin D3, and vitamin D3 in cultured human airway epithelial cells and in mouse airways.

    PubMed

    DiFranco, Kristina M; Mulligan, Jennifer K; Sumal, Aman S; Diamond, Gill

    2017-01-24

    Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which often leads to protein misfolding and no CFTR surface localization. This then leads to chronic airway infections, inflammation, and tissue damage. Although vitamin D has been explored as a therapy to treat CF due to its antimicrobial-inducing and anti-inflammatory properties, the effect of 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on CFTR directly has not been studied. We treated cultured healthy and diseased bronchial epithelial cells (BEC) with 10nM 1α,25(OH)2D3 for 6 and 24h and found that 1α,25(OH)2D3 increases both mRNA and protein CFTR levels using RT-qPCR, flow cytometry and fluorescence immunohistochemistry. Treatment of CF cells with 10nM 1α,25(OH)2D3 led to an increase in both total and surface CFTR expression, suggesting 1α,25(OH)2D3 could be used to increase properly localized CFTR in airway cells. To determine if BEC could convert the more clinically relevant cholecalciferol to 25OHD3, cultured non-CF and CF BECs were treated with a range of cholecalciferol concentrations, and 25OHD3 levels were quantified by ELISA. We found that 25OHD3 levels increased in a concentration-dependent manner. Treatment of BEC with 10μM cholecalciferol led to increases in both CYP24A1 and CFTR mRNA levels, even when added to the apical surface of cells grown in an air-liquid interface, suggesting that topical administration of vitamin D could be used therapeutically. To demonstrate this in vivo, we intranasally delivered 1μM 1α,25(OH)2D3 into mice. After 6h, we observed induction of both Cyp24A1 and CFTR expression in the tracheas of treated mice. The major findings of this study are that vitamin D can be converted to the active form when topically administered to the airway, and this could be used to increase CFTR levels in patients with CF. This could potentially be useful as an adjunctive therapy, together with

  6. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans.

  7. 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...

  8. 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.

  9. 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

  10. Airway epithelial IL-15 transforms monocytes into dendritic cells.

    PubMed

    Regamey, Nicolas; Obregon, Carolina; Ferrari-Lacraz, Sylvie; van Leer, Coretta; Chanson, Marc; Nicod, Laurent P; Geiser, Thomas

    2007-07-01

    IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.

  11. Cigarette smoke extract inhibits expression of peroxiredoxin V and increases airway epithelial permeability.

    PubMed

    Serikov, Vladimir B; Leutenegger, Christian; Krutilina, Raisa; Kropotov, Andrei; Pleskach, Nadezhda; Suh, Jung H; Tomilin, Nikolay V

    2006-01-01

    Inhaled cigarette smoke induces oxidative stress in the epithelium of airways. Peroxiredoxin V (PRXV) is a potent antioxidant protein, highly expressed in cells of the airway epithelium. The goal of our study was to determine whether cigarette smoke extract (CSE) influenced expression of this protein in airway epithelia in vivo and in vitro. In Sprague-Dawley rats, we determined effects of CSE on airway epithelial permeability, mRNA levels and expression of PRXV protein. Exposure of isolated tracheal segment in vitro to 20% CSE for 4 h resulted in development of increased permeability to albumin, significantly reduced mRNA levels for PRXV, and reduced amounts of PRXV protein in the epithelium. In cultures of the airway epithelial cell lines (Calu-3, JME), primary airway cell culture (cow), and alveolar epithelial cells A549, CSE also significantly decreased transepithelial electrical resistance and expression of PRXV protein, and induced glutathione and protein oxidation. To demonstrate functional importance of PRXV, we exposed clones of HeLa cells with siRNA-downregulated PRXV to hydrogen peroxide, which resulted in increased rate of cell death and protein oxidation. CSE directly downregulates expression of functionally important antioxidant enzyme PRXV in the epithelial cells of airways, which represents one pathophysiological mechanism of cigarette smoke toxicity.

  12. Culture and differentiation of mouse tracheal epithelial cells.

    PubMed

    You, Yingjian; Brody, Steven L

    2013-01-01

    Airway epithelial cell biology has been greatly advanced by studies of genetically defined and modified mice; however it is often difficult to isolate, manipulate, and assay epithelial cell-specific responses in vivo. In vitro proliferation and differentiation of mouse airway epithelial cells are made possible by a high-fidelity system for primary culture of mouse tracheal epithelial cells described in this chapter. Using this method, epithelial cells purified from mouse tracheas proliferate in growth factor-enriched medium. Subsequent culture in defined medium and the use of the air-liquid interface condition result in the development of well-differentiated epithelia composed of ciliated and non-ciliated cells with characteristics of native airways. Methods are also provided for manipulation of differentiation and analysis of differentiation and gene expression. These approaches allow the assessment of global responses and those of specific cell subpopulations within the airway epithelium.

  13. Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

    PubMed Central

    Okada, Seiko F.; Ribeiro, Carla M. P.; Sesma, Juliana I.; Seminario-Vidal, Lucia; Abdullah, Lubna H.; van Heusden, Catharina; Lazarowski, Eduardo R.

    2013-01-01

    ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion. PMID:23763446

  14. Inflammation promotes airway epithelial ATP release via calcium-dependent vesicular pathways.

    PubMed

    Okada, Seiko F; Ribeiro, Carla M P; Sesma, Juliana I; Seminario-Vidal, Lucia; Abdullah, Lubna H; van Heusden, Catharina; Lazarowski, Eduardo R; Boucher, Richard C

    2013-11-01

    ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.

  15. Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

    PubMed

    Ruffin, Manon; Bilodeau, Claudia; Maillé, Émilie; LaFayette, Shantelle L; McKay, Geoffrey A; Trinh, Nguyen Thu Ngan; Beaudoin, Trevor; Desrosiers, Martin-Yvon; Rousseau, Simon; Nguyen, Dao; Brochiero, Emmanuelle

    2016-09-01

    Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa-secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non-cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.-Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact

  16. 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.

  17. 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...

  18. Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma

    PubMed Central

    Singhania, Akul; Rupani, Hitasha; Jayasekera, Nivenka; Lumb, Simon; Hales, Paul; Gozzard, Neil; Davies, Donna E.

    2017-01-01

    Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy. PMID:28045928

  19. Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma.

    PubMed

    Singhania, Akul; Rupani, Hitasha; Jayasekera, Nivenka; Lumb, Simon; Hales, Paul; Gozzard, Neil; Davies, Donna E; Woelk, Christopher H; Howarth, Peter H

    2017-01-01

    Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

  20. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    PubMed

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  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. Breaking barriers. New insights into airway epithelial barrier function in health and disease.

    PubMed

    Rezaee, Fariba; Georas, Steve N

    2014-05-01

    Epithelial permeability is a hallmark of mucosal inflammation, but the molecular mechanisms involved remain poorly understood. A key component of the epithelial barrier is the apical junctional complex that forms between neighboring cells. Apical junctional complexes are made of tight junctions and adherens junctions and link to the cellular cytoskeleton via numerous adaptor proteins. Although the existence of tight and adherens junctions between epithelial cells has long been recognized, in recent years there have been significant advances in our understanding of the molecular regulation of junctional complex assembly and disassembly. Here we review the current thinking about the structure and function of the apical junctional complex in airway epithelial cells, emphasizing the translational aspects of relevance to cystic fibrosis and asthma. Most work to date has been conducted using cell culture models, but technical advancements in imaging techniques suggest that we are on the verge of important new breakthroughs in this area in physiological models of airway diseases.

  3. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

    PubMed Central

    Gowers, Kate H. C.; Lee, Dani Do Hyang; Brown, James M.; Crowley, Claire; Teixeira, Vitor H.; Smith, Claire M.; Urbani, Luca; Hamilton, Nicholas J.; Thakrar, Ricky M.; Booth, Helen L.; Birchall, Martin A.; De Coppi, Paolo; Giangreco, Adam; O’Callaghan, Christopher

    2016-01-01

    Rationale: Stem cell–based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell–seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air–liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air–liquid interface cultures. Measurements and Main Results: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Conclusions: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical

  4. Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells.

    PubMed

    Olsen, Colin E; Liguori, Andrew E; Zong, Yue; Lantz, R Clark; Burgess, Jefferey L; Boitano, Scott

    2008-08-01

    As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

  5. 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...

  6. 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

  7. 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.

  8. Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells.

    PubMed

    Kolesnikova, Larissa; Heck, Sonja; Matrosovich, Tatyana; Klenk, Hans-Dieter; Becker, Stephan; Matrosovich, Mikhail

    2013-05-01

    The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy. Budding of spherical and filamentous virions was observed on the apical surfaces of cells with no association with cilia and secretory granules. Quantitative analysis of the distribution of viral buds on the cell surface indicated that the tips of the microvilli represented a prominent site of influenza virus budding in the human airway epithelium. As the microvilli of differentiated cells are involved in many fundamental cell functions, these data will prompt further studies on the biological significance of microvilli-associated budding for virus replication, transmission and pathogenicity.

  9. Airway epithelial-derived factor relaxes pulmonary vascular smooth muscle.

    PubMed

    Farah, Omar R; Li, Dongge; McIntyre, Brendan A S; Pan, Jingyi; Belik, Jaques

    2009-01-01

    The factors controlling the pulmonary vascular resistance under physiological conditions are poorly understood. We have previously reported on an apparent cross talk between the airway and adjacent pulmonary arterial bed where a factor likely derived from the bronchial epithelial cells reduced the magnitude of agonist-stimulated force in the vascular smooth muscle. The main purpose of this investigation was to evaluate whether bronchial epithelial cells release a pulmonary arterial smooth muscle relaxant factor. Conditioned media from SPOC-1 or BEAS-2B, a rat- and a human-derived bronchial epithelial cell line, respectively, were utilized. This media significantly relaxed precontracted adult but not fetal pulmonary arterial muscle in an oxygen tension-dependent manner. This response was mediated via soluble guanylate cyclase, involving AKT/PI3-kinase and neuronal nitric oxide synthase. Airway epithelial cell-conditioned media increased AKT phosphorylation in pulmonary smooth muscle cells (SMC) and reduced intracellular calcium change following ATP stimulation to a significantly greater extent than observed for bronchial SMC. The present data strongly support the evidence for bronchial epithelial cells releasing a stable and soluble factor capable of inducing pulmonary arterial SMC relaxation. We speculate that under physiological conditions, the maintenance of a low pulmonary vascular resistance, postnatally, is in part modulated by the airway epithelium.

  10. 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.

  11. Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double‐stranded RNA

    PubMed Central

    Reale, Riccardo; Held, Marie; Loxham, Matthew; Millar, Timothy M.; Collins, Jane E.; Swindle, Emily J.; Morgan, Hywel; Davies, Donna E.

    2017-01-01

    Abstract Introduction The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross‐talk are not fully understood. Methods In a close‐contact co‐culture model of human airway epithelial and endothelial cells, cellular crosstalk was analyzed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy, and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double‐stranded RNA (Poly(I:C)). Using a microfluidic culture system, the temporal release of mediators was analyzed in the co‐culture model. Results Within 4 h of challenge, double‐stranded RNA induced the release of TNF‐α by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8 h post‐challenge and expression of adhesion molecules E‐selectin and ICAM‐1. These responses were significantly reduced by neutralising TNF‐α. Conclusion By facilitating kinetic profiling, the microfluidic co‐culture system has enabled identification of a key signaling mechanism between the epithelial and endothelial barriers. Better understanding of cell–cell cross‐talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation. PMID:28250924

  12. Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis

    PubMed Central

    Campόdonico, Victoria L; Gadjeva, Mihaela; Paradis-Bleau, Catherine; Uluer, Ahmet; Pier, Gerald B

    2013-01-01

    Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism. PMID:18262467

  13. Airway Epithelial Expression Quantitative Trait Loci Reveal Genes Underlying Asthma and Other Airway Diseases

    PubMed Central

    Luo, Wei; Obeidat, Ma’en; Di Narzo, Antonio Fabio; Chen, Rong; Sin, Don D.; Paré, Peter D.

    2016-01-01

    Genome-wide association studies (GWASs) have identified loci that are robustly associated with asthma and related phenotypes; however, the molecular mechanisms underlying these associations need to be explored. The most relevant tissues to study the functional consequences of asthma are the airways. We used publically available data to derive expression quantitative trait loci (eQTLs) for human epithelial cells from small and large airways and applied the eQTLs in the interpretation of GWAS results of asthma and related phenotypes. For the small airways (n = 105), we discovered 660 eQTLs at a 10% false discovery rate (FDR), among which 315 eQTLs were not previously reported in a large-scale eQTL study of whole lung tissue. A large fraction of the identified eQTLs is supported by data from Encyclopedia of DNA Elements (ENCODE) showing that the eQTLs reside in regulatory elements (57.5 and 67.6% of cis- and trans-eQTLs, respectively). Published pulmonary GWAS hits were enriched as airway epithelial eQTLs (9.2-fold). Further, genes regulated by asthma GWAS loci in epithelium are significantly enriched in immune response pathways, such as IL-4 signaling (FDR, 5.2 × 10−4). The airway epithelial eQTLs described in this study are complementary to previously reported lung eQTLs and represent a powerful resource to link GWAS-associated variants to their regulatory function and thus elucidate the molecular mechanisms underlying asthma and airway-related conditions. PMID:26102239

  14. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    PubMed Central

    Yaghi, Asma; Dolovich, Myrna B.

    2016-01-01

    Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations. PMID:27845721

  15. 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.

  16. Cigarette smoke extract reduces VEGF in primary human airway epithelial cells.

    PubMed

    Thaikoottathil, J V; Martin, R J; Zdunek, J; Weinberger, A; Rino, J G; Chu, H W

    2009-04-01

    Reduced vascular endothelial growth factor (VEGF) has been reported in bronchoalveolar lavage fluid and lungs of severe emphysema patients. Airway epithelial cells (AEC) are exposed to various environmental insults like cigarette smoke and bacterial infections, but their direct effect on VEGF production in well-differentiated primary human AEC remains unclear. The current authors determined the effect of cigarette smoke extract (CSE) alone and in combination with Mycoplasma pneumoniae (Mp) on VEGF production in well-differentiated primary normal human bronchial epithelial (NHBE) and small airway epithelial cells (SAEC) in air-liquid interface cultures. Secretion and expression of VEGF were determined by ELISA and real-time RT-PCR, respectively. Cell growth, apoptosis, extracellular signal-regulated kinase (ERK)1/2 and protein kinase (PK)C signalling pathways were evaluated to further dissect VEGF regulation under CSE treatment. CSE significantly reduced VEGF secretion in NHBE and SAEC. In SAEC, Mp alone significantly increased the VEGF, while the presence of CSE attenuated Mp-induced VEGF production. While ERK inhibitor reduced VEGF secretion only in NHBE, a PKC inhibitor significantly decreased VEGF secretion in both NHBE and SAEC. In conclusion, direct cigarette smoke extract exposure significantly reduced vascular endothelial growth factor production in well-differentiated primary human airway epithelial cells, in part through modifying extracellular signal-regulated kinase 1/2 and protein kinase C signalling pathways.

  17. 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.

  18. 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.

  19. Nitric Oxide and Airway Epithelial Barrier Function: Regulation of Tight Junction Proteins and Epithelial Permeability

    PubMed Central

    Olson, Nels; Greul, Anne-Katrin; Hristova, Milena; Bove, Peter F.; Kasahara, David I.; van der Vliet, Albert

    2008-01-01

    Acute airway inflammation is associated with enhanced production of nitric oxide (NO•) and altered airway epithelial barrier function, suggesting a role of NO• or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o- cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2-/- mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H2O2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity. PMID:19100237

  20. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

    PubMed

    Allahverdian, Sima; Harada, Norihiro; Singhera, Gurpreet K; Knight, Darryl A; Dorscheid, Delbert R

    2008-02-01

    Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

  1. 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

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

    PubMed

    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; van der Vliet, Albert

    2016-11-03

    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.

  3. 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

  4. Metformin prevents the effects of Pseudomonas aeruginosa on airway epithelial tight junctions and restricts hyperglycaemia-induced bacterial growth.

    PubMed

    Patkee, Wishwanath R A; Carr, Georgina; Baker, Emma H; Baines, Deborah L; Garnett, James P

    2016-04-01

    Lung disease and elevation of blood glucose are associated with increased glucose concentration in the airway surface liquid (ASL). Raised ASL glucose is associated with increased susceptibility to infection by respiratory pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. We have previously shown that the anti-diabetes drug, metformin, reduces glucose-induced S. aureus growth across in vitro airway epithelial cultures. The aim of this study was to investigate whether metformin has the potential to reduce glucose-induced P. aeruginosa infections across airway epithelial (Calu-3) cultures by limiting glucose permeability. We also explored the effect of P. aeruginosa and metformin on airway epithelial barrier function by investigating changes in tight junction protein abundance. Apical P. aeruginosa growth increased with basolateral glucose concentration, reduced transepithelial electrical resistance (TEER) and increased paracellular glucose flux. Metformin pre-treatment of the epithelium inhibited the glucose-induced growth of P. aeruginosa, increased TEER and decreased glucose flux. Similar effects on bacterial growth and TEER were observed with the AMP activated protein kinase agonist, 5-aminoimidazole-4-carboxamide ribonucleotide. Interestingly, metformin was able to prevent the P. aeruginosa-induced reduction in the abundance of tight junction proteins, claudin-1 and occludin. Our study highlights the potential of metformin to reduce hyperglycaemia-induced P. aeruginosa growth through airway epithelial tight junction modulation, and that claudin-1 and occludin could be important targets to regulate glucose permeability across airway epithelia and supress bacterial growth. Further investigation into the mechanisms regulating metformin and P. aeruginosa action on airway epithelial tight junctions could yield new therapeutic targets to prevent/suppress hyperglycaemia-induced respiratory infections, avoiding the use of antibiotics.

  5. 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

  6. 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.

  7. High glucose induces dysfunction of airway epithelial barrier through down-regulation of connexin 43.

    PubMed

    Yu, Hongmei; Yang, Juan; Zhou, Xiangdong; Xiao, Qian; Lü, Yang; Xia, Li

    2016-03-01

    The airway epithelium is a barrier to the inhaled antigens and pathogens. Connexin 43 (Cx43) has been found to play critical role in maintaining the function of airway epithelial barrier and be involved in the pathogenesis of the diabetic retinal vasculature, diabetes nephropathy and diabetes skin. Hyperglycemia has been shown to be an independent risk factor for respiratory infections. We hypothesize that the down-regulation of Cx43 induced by HG alters the expression of tight junctions (zonula occludens-1 (ZO-1) and occludin) and contributes to dysfunction of airway epithelial barrier, and Cx43 plays a critical role in the process in human airway epithelial cells (16 HBE). We show that high glucose (HG) decreased the expression of ZO-1 and occludin, disassociated interaction between Cx43 and tight junctions, and then increased airway epithelial transepithelial electrical resistance (TER) and permeability by down-regulation of Cx43 in human airway epithelial cells. These observations demonstrate an important role for Cx43 in regulating HG-induced dysfunction of airway epithelial barrier. These findings may bring new insights into the molecular pathogenesis of pulmonary infection related to diabetes mellitus and lead to novel therapeutic intervention for the dysfunction of airway epithelial barrier in chronic inflammatory airway diseases.

  8. 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.

  9. 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.

  10. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  11. Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl.

    PubMed

    Foster, Matthew W; Gwinn, William M; Kelly, Francine L; Brass, David M; Valente, Ashlee M; Moseley, M Arthur; Thompson, J Will; Morgan, Daniel L; Palmer, Scott M

    2017-02-03

    Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.

  12. NDRG1 is important to maintain the integrity of airway epithelial barrier through claudin-9 expression.

    PubMed

    Gon, Yasuhiro; Maruoka, Shuichiro; Kishi, Hiroyuki; Kozu, Yutaka; Kazumichi, Kuroda; Nomura, Yasuyuki; Takeshita, Ikuko; Oshima, Takeshi; Hashimoto, Shu

    2017-02-13

    Impairment of epithelial barrier integrity caused by environmental triggers is associated with the pathogenesis of airway inflammation. Using human airway epithelial cells, we attempted to identify molecule(s) that promote airway epithelial barrier integrity. Microarray analyses were conducted using the Affimetrix human whole genome gene chip, and we identified the N-myc downstream-regulated gene 1 (NDRG1) gene, which was induced during the development of the epithelial cell barrier. Immunohistochemical analysis revealed strong NDRG1 expression in ciliated epithelial cells in nasal tissues sampled from patients with chronic rhinosinusitis (CRS), and the low expression of NDRG1 was observed in goblet cells or damaged epithelial cells. NDRG1 gene knockdown with its specific siRNA decreased the transepithelial electrical resistance and increased the dextran permeability. Immunocytochemistry revealed that NDRG1 knockdown disrupted tight junctions of airway epithelial cells. Next, we analyzed the effects of NDRG1 knockdown on the expression of tight and adhesion junction molecules. NDRG1 knockdown significantly decreased only claudin-9 expression, but did not decrease other claudin family molecules, such as E-cadherin, and ZO-1, -2, or -3. Knockdown of claudin-9 markedly impaired the barrier function in airway epithelial cells. These results suggest that NDRG1 is important for the barrier integrity in airway epithelial cells.

  13. Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces

    PubMed Central

    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

    2016-01-01

    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

  14. 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.

  15. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells

    EPA Science Inventory

    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. Th...

  16. AMPK agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells.

    PubMed

    Myerburg, Michael M; King, J Darwin; Oyster, Nicholas M; Fitch, Adam C; Magill, Amy; Baty, Catherine J; Watkins, Simon C; Kolls, Jay K; Pilewski, Joseph M; Hallows, Kenneth R

    2010-06-01

    The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel and epithelial Na(+) channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I(sc)), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I(sc) in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.

  17. AMPK Agonists Ameliorate Sodium and Fluid Transport and Inflammation in Cystic Fibrosis Airway Epithelial Cells

    PubMed Central

    Myerburg, Michael M.; King, J Darwin; Oyster, Nicholas M.; Fitch, Adam C.; Magill, Amy; Baty, Catherine J.; Watkins, Simon C.; Kolls, Jay K.; Pilewski, Joseph M.; Hallows, Kenneth R.

    2010-01-01

    The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl− channel and epithelial Na+ channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (Isc), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2–5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent Isc in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red–dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03–1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease. PMID:19617399

  18. The Anti-inflammatory Effect of Alpha-1 Antitrypsin in Rhinovirus-infected Human Airway Epithelial Cells

    PubMed Central

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

    2017-01-01

    Objective Excessive airway inflammation is seen in chronic obstructive pulmonary disease (COPD) patients experiencing acute exacerbations, which are often associated with human rhinovirus (HRV) infection. Alpha-1 antitrypsin (A1AT) has anti-inflammatory function in endothelial cells and monocytes, but its anti-inflammatory effect has not been investigated in COPD airway epithelial cells. We determined A1AT’s anti-inflammatory function in COPD airway epithelial cells and the underlying mechanisms such as the role of caspase-1. Methods Brushed bronchial epithelial cells from COPD and normal subjects were cultured at air-liquid interface and treated with A1AT or bovine serum albumin (BSA, control) two hours prior to whole cigarette smoke (WCS) or air exposure, followed by HRV-16 infection. After 24 hours of viral infection, cell supernatants were collected for measuring IL-8, and cells were examined for caspase-1. The in vivo anti-inflammatory function of A1AT was determined by infecting mice intranasally with HRV-1B followed by aerosolized A1AT or BSA. Results A1AT significantly reduced WCS and HRV-16-induced IL-8 production in normal and COPD airway epithelial cells. COPD cells are less sensitive to A1AT’s anti-inflammatory effect than normal cells. A1AT exerted the anti-inflammatory function in part via reducing caspase-1 in normal cells, but not in COPD cells. In mice, A1AT significantly reduced HRV-1B induced lung neutrophilic inflammation. Conclusions A1AT exerts an anti-inflammatory effect in cigarette smoke-exposed and HRV-infected human airway epithelial cells, which may be related to its inhibitory effect on caspase-1 activity. PMID:28191362

  19. Asthmatic airway epithelial cells differentially regulate fibroblast expression of extracellular matrix components

    PubMed Central

    Reeves, Stephen R.; Kolstad, Tessa; Lien, Tin-Yu; Elliott, Molly; Ziegler, Steven F.; Wight, Thomas N.; Debley, Jason S.

    2014-01-01

    Background Airway remodeling may explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling may regulate HLF ECM expression. Objectives Determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. Methods Primary AECs were obtained from well-characterized atopic-asthmatic (N=10) and healthy children (N=10) intubated under anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface (ALI) for 3 weeks, then co-cultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1), collagen III (COL3A1), hyaluronan synthase 2 (HAS2), and fibronectin (FNDC) expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs was assessed by RT-PCR. TGFb1&2 concentrations in media were measured by ELISA. Results COL1A1 and COL3A1 expression by HLFs co-cultured with asthmatic AECs was greater than HLFs co-cultured with healthy AECs (2.2 fold, p<0.02; 10.8 fold, p<0.02). HAS2 expression by HLFs co-cultured with asthmatic AECs was 2.5-fold higher than by HLFs co-cultured with healthy AECs (p<0.002). FNDC expression by HLFs co-cultured with asthmatic AECs was significantly greater than by HLFs alone. TGFb2 activity was elevated in asthmatic AEC-HLF co-cultures (p<0.05) while PGES2 was down regulated in AEC-HLF co-cultures (2.2 fold, p<0.006). Conclusions HLFs co-cultured with asthmatic AECs showed differential expression of ECM constituents COL1A1 & COL3A1, and HAS2 compared to HLFs co-cultured with healthy AECs. These findings support a role for altered ECM production in asthmatic airway remodeling, possibly regulated by unbalanced AEC signaling. PMID:24875618

  20. 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, ...

  1. Fluid and electrolyte transport by cultured human airway epithelia.

    PubMed Central

    Smith, J J; Welsh, M J

    1993-01-01

    An understanding of the fluid and electrolyte transport properties of any epithelium requires knowledge of the direction, rate, and regulation of fluid transport and the composition of the fluid. Although human airway epithelial likely play a key role in controlling the quantity and composition of the respiratory tract fluid, evidence for such a role is not available. To obtain such knowledge, we measured fluid and electrolyte transport by cultured human nasal epithelia. Under basal conditions we found that epithelia absorbed Na+ and fluid; both processes were inhibited by addition of amiloride to the mucosal surface. These data suggest that active Na+ absorption is responsible for fluid absorption. Interestingly, Na+ absorption was not accompanied by the net absorption of Cl-; some other anion accompanied Na+. The combination of cAMP agonists and mucosal amiloride stimulated the secretion of NaCl-rich fluid. But surprisingly, the response to cAMP agonists in the absence of amiloride showed substantial intersubject variability: cAMP stimulated fluid secretion across some epithelia, for others, cAMP stimulated fluid absorption. The explanation for the differences in response is uncertain, but we speculate that the magnitude of apical membrane Na+ conductance may modulate the direction of fluid transport in response to cAMP. We also found that airway epithelial secrete H+ and absorb K+ under basal conditions; both processes were inhibited by cAMP agonists. Because the H+/K(+)-ATPase inhibitor, SCH 28080, inhibited K+ absorption, an apical membrane H+/K(+)-ATPase may be at least partly responsible for K+ and H+ transport. However, H+/K+ exchange could not entirely account for the luminal acidification. The finding that cAMP agonists inhibited luminal acidification may be explained by the recent finding that cAMP increases apical HCO3- conductance. These results provide new insights into how the intact airway epithelium may modify the composition of the respiratory

  2. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

    NASA Astrophysics Data System (ADS)

    Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

    1990-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

  3. Allergic airway inflammation induces a pro-secretory epithelial ion transport phenotype in mice.

    PubMed

    Anagnostopoulou, P; Dai, L; Schatterny, J; Hirtz, S; Duerr, J; Mall, M A

    2010-12-01

    The airway epithelium is a central effector tissue in allergic inflammation and T-helper cell (Th) type 2-driven epithelial responses, such as mucus hypersecretion contribute to airflow obstruction in allergic airway disease. Previous in vitro studies demonstrated that Th2 cytokines also act as potent modulators of epithelial ion transport and fluid secretion, but the in vivo effect of allergic inflammation on airway ion transport remains unknown. We, therefore, induced allergic inflammation by intratracheal instillation of Aspergillus fumigatus extract or interleukin-13 in mice and determined effects on ion transport in native tracheal and bronchial tissues. We demonstrate that allergic inflammation enhanced basal Cl(-) secretion in both airway regions and inhibited epithelial Na(+) channel (ENaC)-mediated Na(+) absorption and increased Ca²(+)-dependent Cl(-) secretion in bronchi. Allergen-induced alterations in bronchial ion transport were associated with reduced transcript levels of α-, β- and γENaC, and were largely abrogated in signal transducer and activator of transcription (Stat)6(-/-) mice. Our studies demonstrate that Th2-dependent airway inflammation produced a pro-secretory ion transport phenotype in vivo, which was largely Stat6-dependent. These results suggest that Th2-mediated fluid secretion may improve airway surface hydration and clearance of mucus that is hypersecreted in allergic airway diseases such as asthma, and identify epithelial Stat6 signalling as a potential therapeutic target to promote mucus hydration and airway clearance.

  4. GENETIC INFLUENCES ON IN VTIRO PARTICULATE MATTER-INDUCED AIRWAY EPITHELIAL INJURY AND INFLAMMATORY MEDIATOR RELEASE

    EPA Science Inventory

    GENETIC INFLUENCES ON IN VITRO PARTICULATE MATTER-INDUCED AIRWAY EPITHELIAL INJURY AND INFLAMMATORY MEDIATOR RELEASE.
    JA Dye, JH Richards, DA Andrews, UP Kodavanti. US EPA, RTP, NC, USA.

    Particulate matter (PM) air pollution is capable of damaging the airway epitheli...

  5. Cortactin mediates elevated shear stress-induced mucin hypersecretion via actin polymerization in human airway epithelial cells.

    PubMed

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

    2013-12-01

    Mucus hypersecretion is a remarkable pathophysiological manifestation in airway obstructive diseases. These diseases are usually accompanied with elevated shear stress due to bronchoconstriction. Previous studies have reported that shear stress induces mucin5AC (MUC5AC) secretion via actin polymerization in cultured nasal epithelial cells. Furthermore, it is well known that cortactin, an actin binding protein, is a central mediator of actin polymerization. Therefore, we hypothesized that cortactin participates in MUC5AC hypersecretion induced by elevated shear stress via actin polymerization in cultured human airway epithelial cells. Compared with the relevant control groups, Src phosphorylation, cortactin phosphorylation, actin polymerization and MUC5AC secretion were significantly increased after exposure to elevated shear stress. Similar effects were found when pretreating the cells with jasplakinolide, and transfecting with wild-type cortactin. However, these effects were significantly attenuated by pretreating with Src inhibitor, cytochalasin D or transfecting cells with the specific small interfering RNA of cortactin. Collectively, these results suggest that elevated shear stress induces MUC5AC hypersecretion via tyrosine-phosphorylated cortactin-associated actin polymerization in cultured human airway epithelial cells.

  6. 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

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

    PubMed

    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 (5×10(4) 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.

  8. 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.

  9. Molecular modulation of airway epithelial ciliary response to sneezing.

    PubMed

    Zhao, Ke-Qing; Cowan, Andrew T; Lee, Robert J; Goldstein, Natalia; Droguett, Karla; Chen, Bei; Zheng, Chunquan; Villalon, Manuel; Palmer, James N; Kreindler, James L; Cohen, Noam A

    2012-08-01

    Our purpose was to evaluate the effect of the mechanical force of a sneeze on sinonasal cilia function and determine the molecular mechanism responsible for eliciting the ciliary response to a sneeze. A novel model was developed to deliver a stimulation simulating a sneeze (55 mmHg for 50 ms) at 26°C to the apical surface of mouse and human nasal epithelial cells. Ciliary beating was visualized, and changes in ciliary beat frequency (CBF) were determined. To interrogate the molecular cascades driving sneeze-induced changes of CBF, pharmacologic manipulation of intra- and extracellular calcium, purinergic, PKA, and nitric oxide (NO) signaling were performed. CBF rapidly increases by ≥150% in response to a sneeze, which is dependent on the release of adenosine triphosphate (ATP), calcium influx, and PKA activation. Furthermore, apical release of ATP is independent of calcium influx, but calcium influx and subsequent increase in CBF are dependent on the ATP release. Lastly, we observed a blunted ciliary response in surgical specimens derived from patients with chronic rhinosinusitis compared to control patients. Apical ATP release with subsequent calcium mobilization and PKA activation are involved in sinonasal ciliary response to sneezing, which is blunted in patients with upper-airway disease.

  10. Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease.

    PubMed

    Puchelle, Edith; Zahm, Jean-Marie; Tournier, Jean-Marie; Coraux, Christelle

    2006-11-01

    In chronic obstructive pulmonary disease (COPD), exacerbations are generally associated with several causes, including pollutants, viruses, bacteria that are responsible for an excess of inflammatory mediators, and proinflammatory cytokines released by activated epithelial and inflammatory cells. The normal response of the airway surface epithelium to injury includes a succession of cellular events, varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even complete denudation of the basement membrane. The epithelium then has to repair and regenerate to restore its functions, through several mechanisms, including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. In COPD, the remodeling of the airway epithelium, such as squamous metaplasia and mucous hyperplasia that occur during injury, may considerably disturb the innate immune functions of the airway epithelium. In vitro and in vivo models of airway epithelial wound repair and regeneration allow the study of the spatiotemporal modulation of cellular and molecular interaction factors-namely, the proinflammatory cytokines, the matrix metalloproteinases and their inhibitors, and the intercellular adhesion molecules. These factors may be markedly altered during exacerbation periods of COPD and their dysregulation may induce remodeling of the airway mucosa and a leakiness of the airway surface epithelium. More knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to cytoprotective and regenerative therapeutics, allowing the reconstitution of a functional, well-differentiated airway epithelium in COPD.

  11. The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types

    PubMed Central

    Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A.; Yu, Jing-yu; Lim, Dong Hyun; Rosania, Gus R.

    2013-01-01

    Purpose We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Methods Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Results Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Conclusion Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types. PMID:23708857

  12. 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.

  13. Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells

    PubMed Central

    Daniel, Nadia M.; van der Vlugt, Luciën E. P. M.; van Schadewijk, Annemarie; Taube, Christian; Hiemstra, Pieter S.

    2016-01-01

    Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression. PMID:27829065

  14. 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

  15. Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

    PubMed

    Lynch, Thomas J; Anderson, Preston J; Xie, Weiliang; Crooke, Adrianne K; Liu, Xiaoming; Tyler, Scott R; Luo, Meihui; Kusner, David M; Zhang, Yulong; Neff, Traci; Burnette, Daniel C; Walters, Katherine S; Goodheart, Michael J; Parekh, Kalpaj R; Engelhardt, John F

    2016-06-24

    Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016.

  16. Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

    PubMed Central

    Knox, Alan J.

    2015-01-01

    Human airway smooth muscle cells (HASMC) contribute to asthma pathophysiology through an increased smooth muscle mass and elevated cytokine/chemokine output. Little is known about how HASMC and the airway epithelium interact to regulate chronic airway inflammation and remodeling. Amphiregulin is a member of the family of epidermal growth factor receptor (EGFR) agonists with cell growth and proinflammatory roles and increased expression in the lungs of asthma patients. Here we show that bradykinin (BK) stimulation of HASMC increases amphiregulin secretion in a mechanism dependent on BK-induced COX-2 expression, increased PGE2 output, and the stimulation of HASMC EP2 and EP4 receptors. Conditioned medium from BK treated HASMC induced CXCL8, VEGF, and COX-2 mRNA and protein accumulation in airway epithelial cells, which were blocked by anti-amphiregulin antibodies and amphiregulin siRNA, suggesting a paracrine effect of HASMC-derived amphiregulin on airway epithelial cells. Consistent with this, recombinant amphiregulin induced CXCL8, VEGF, and COX-2 in airway epithelial cells. Finally, we found that conditioned media from amphiregulin-stimulated airway epithelial cells induced amphiregulin expression in HASMC and that this was dependent on airway epithelial cell COX-2 activity. Our study provides evidence of a dynamic axis of interaction between HASMC and epithelial cells that amplifies CXCL8, VEGF, COX-2, and amphiregulin production. PMID:26047642

  17. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  18. Highly Differentiated Human Airway Epithelial Cells: a Model to Study Host cell-parasite Interactions in Pertussis

    PubMed Central

    Guevara, Claudia; Zhang, Chengxian; Gaddy, Jennifer A.; Iqbal, Junaid; Guerra, Julio; Greenberg, David P.; Decker, Michael D.; Carbonetti, Nicholas; Starner, Timothy D.; McCray, Paul B.; Mooi, Frits R.

    2017-01-01

    Background Bordetella pertussis colonizes the human respiratory mucosa. Most studies on B. pertussis adherence have relied on cultured mammalian cells that lack key features present in differentiated human airway cells or on animal models that are not natural hosts of B. pertussis. The objectives of this work are to evaluate B. pertussis infection on highly differentiated human airway cells in vitro and to show the role of B. pertussis fimbriae in cell adherence. Methods Primary human airway epithelial (PHAE) cells from human bronchi and a human bronchial epithelial (HBE) cell line were grown in vitro under air-liquid interface conditions. Results PHAE and HBE cells infected with B. pertussis wild type strain revealed bacterial adherence to cell’s apical surface and bacterial induced cytoskeleton changes and cell detachment. Mutations in the major fimbrial subunits Fim2/3 or in the minor fimbrial adhesin subunit FimD affected B. pertussis adherence to predominantly HBE cells. This cell model recapitulates the morphologic features of the human airway infected by B. pertussis and confirms the role of fimbriae in B. pertussis adherence. Furthemore, HBE cells show that fimbrial subunits, and specifically FimD adhesin, are critical in B. pertussis adherence to airway cells. Conclusions The relevance of this model to study host-parasite interaction in pertussis lies in the striking physiologic and morphologic similarity between the PHAE and HBE cells and the human airway ciliated and goblet cells in vivo. These cells can proliferate in vitro, differentiate, and express the same genetic profile as human respiratory cells in vivo. PMID:26492208

  19. 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

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

    PubMed Central

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

    2014-01-01

    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 3–7 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. PMID:24880893

  1. 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.

  2. A CEACAM6-High Airway Neutrophil Phenotype and CEACAM6-High Epithelial Cells Are Features of Severe Asthma.

    PubMed

    Shikotra, Aarti; Choy, David F; Siddiqui, Salman; Arthur, Greer; Nagarkar, Deepti R; Jia, Guiquan; Wright, Adam K A; Ohri, Chandra M; Doran, Emma; Butler, Claire A; Hargadon, Beverley; Abbas, Alexander R; Jackman, Janet; Wu, Lawren C; Heaney, Liam G; Arron, Joseph R; Bradding, Peter

    2017-03-08

    Severe asthma represents a major unmet clinical need; understanding the pathophysiology is essential for the development of new therapies. Using microarray analysis, we previously found three immunological clusters in asthma: Th2-high, Th17-high, and Th2/17-low. Although new therapies are emerging for Th2-high disease, identifying molecular pathways in Th2-low disease remains an important goal. Further interrogation of our previously described microarray dataset revealed upregulation of gene expression for carcinoembryonic Ag cell adhesion molecule (CEACAM) family members in the bronchi of patients with severe asthma. Our aim was therefore to explore the distribution and cellular localization of CEACAM6 using immunohistochemistry on bronchial biopsy tissue obtained from patients with mild-to-severe asthma and healthy control subjects. Human bronchial epithelial cells were used to investigate cytokine and corticosteroid in vitro regulation of CEACAM6 gene expression. CEACAM6 protein expression in bronchial biopsies was increased in airway epithelial cells and lamina propria inflammatory cells in severe asthma compared with healthy control subjects. CEACAM6 in the lamina propria was localized to neutrophils predominantly. Neutrophil density in the bronchial mucosa was similar across health and the spectrum of asthma severity, but the percentage of neutrophils expressing CEACAM6 was significantly increased in severe asthma, suggesting the presence of an altered neutrophil phenotype. CEACAM6 gene expression in cultured epithelial cells was upregulated by wounding and neutrophil elastase. In summary, CEACAM6 expression is increased in severe asthma and primarily associated with airway epithelial cells and tissue neutrophils. CEACAM6 may contribute to the pathology of treatment-resistant asthma via neutrophil and airway epithelial cell-dependent pathways.

  3. Expression of IL-4/IL-13 receptors in differentiating human airway epithelial cells

    PubMed Central

    Martin, Linda D.; Stern, Randi; Laxman, Bharathi; Marroquin, Bertha A.

    2010-01-01

    IL-4 and IL-13 elicit several important responses in airway epithelium including chemokine secretion and mucous secretion that may contribute to airway inflammation, cell migration, and differentiation. These cytokines have overlapping but not identical effector profiles likely due to shared subunits in their receptor complexes. These receptors are variably described in epithelial cells, and the relative expression, localization, and function of these receptors in differentiated and repairing epithelial cells are not clear. We examined IL-4/IL-13 receptor expression and localization in primary airway epithelial cells collected from normal human lungs and grown under conditions yielding both undifferentiated and differentiated cells inclusive of basal, goblet, and ciliated cell phenotypes. Gene expression of the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2 receptor subunits increased with differentiation, but different patterns of localization and protein abundance were seen for each subunit based on both differentiation and the cell subtypes present. Increased expression of receptor subunits observed in more differentiated cells was associated with more substantial functional responses to IL-4 stimulation including increased eotaxin-3 expression and accelerated migration after injury. We demonstrate substantial differences in IL-4/IL-13 receptor subunit expression and responsiveness to IL-4 based on the extent of airway epithelial cell differentiation and suggest that these differences may have functional consequences in airway inflammation. PMID:20729386

  4. Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

    PubMed

    Cerri, Chiara; Chimenti, Daniele; Conti, Ilaria; Neri, Tommaso; Paggiaro, Pierluigi; Celi, Alessandro

    2006-08-01

    Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

  5. 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.

  6. deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

    PubMed

    Arason, Ari Jon; Jonsdottir, Hulda R; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K

    2014-01-01

    The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

  7. Grepafloxacin inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in human airway epithelial cells.

    PubMed

    Hashimoto, S; Matsumoto, K; Gon, Y; Maruoka, S; Hayashi, S; Asai, Y; Machino, T; Horie, T

    2000-01-01

    We examined the effect of grepafloxacin (GPFX), a new fluoroquinolone antimicrobial agent, on interleukin-8 (IL-8) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human airway epithelial cells (AEC). GPFX inhibited IL-8 protein production as well as mRNA expression in a concentration-dependent manner (2.5 - 25 micro g/ml), but the inhibition of IL-8 expression by corresponding concentrations of GPFX to serum and airway lining fluids was not complete. We discuss the modulatory effect of GPFX on IL-8 production in the context of its efficacy on controlling chronic airway inflammatory diseases.

  8. Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells.

    PubMed

    Liu, Chi; Liu, Hui-jun; Xiang, Yang; Tan, Yu-rong; Zhu, Xiao-lin; Qin, Xiao-qun

    2010-08-01

    Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.

  9. Cultured airway epithelium responses to mineral particles: role of the oxidative stress.

    PubMed

    Guilianelli, C; Baeza-Squiban, A; Lapart, E; Marano, F

    1996-11-01

    We tested the hypothesis according to which mineral particles containing iron would be able to produce cytotoxic-ROS. We approached this problem in vitro using primary cultures of rabbit tracheal epithelial cells. The oxidizing power of three mineral particles, i.e. nemalite, chrysotile and hematite, has been evaluated for their capacity to induce lipid peroxidation, and to activate intra-cellular anti-oxidant enzymes. The results show that nemalite and chrysotile which contain Fe2+ have a strong oxidizing power, inducing an oxidative stress on airway epithelial cells, whereas hematite, the Fe3+ containing particles, is without effect.

  10. Acid and organic aerosol coatings on magnetic nanoparticles increase iron concentrations in human airway epithelial cells.

    PubMed

    Ghio, Andrew J; Dailey, Lisa A; Richards, Judy H; Jang, Myoseon

    2009-07-01

    Numerous industrial applications for man-made nanoparticles have been proposed. Interactions of nanoparticles with agents in the atmosphere may impact human health. We tested the postulate that in vitro exposures of respiratory epithelial cells to airborne magnetic nanoparticles (MNP; Fe(3)O(4)) with and without a secondary organic aerosol (SOA) and an inorganic acid could affect iron homeostasis, oxidative stress, and interleukin (IL)-8 release. Cell iron concentrations were increased after exposures to MNP and values were further elevated with co-exposures to either SOA or inorganic acid. Increased expression of ferritin and elevated levels of RNA for DMT1, proteins for iron storage and transport respectively, followed MNP exposures, but values were significant for only those with co-exposures to inorganic acid and organic aerosols. Cell iron concentration corresponded to a measure of oxidative stress in the airway epithelial cells; MNP with co-exposures to SOA and inorganic acid increased both available metal and indices of oxidant generation. Finally, the release of a proinflammatory cytokine (i.e. IL-8) by the exposed cells similarly increased with cell iron concentration. We conclude that MNP can interact with a SOA and an inorganic acid to present metal in a catalytically reactive state to cultured respiratory cells. This produces an oxidative stress to affect a release of IL-8.

  11. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

    PubMed

    Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

    2013-04-11

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

  12. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

    PubMed Central

    2013-01-01

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells. PMID:23577829

  13. 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

  14. 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

  15. Mometasone Furoate Suppresses PMA-Induced MUC-5AC and MUC-2 Production in Human Airway Epithelial Cells

    PubMed Central

    Koontongkaew, Sittichai; Monthanapisut, Paopanga; Pattanacharoenchai, Napaporn

    2017-01-01

    Background Mucus hypersecretion from airway epithelium is a characteristic feature of airway inflammatory diseases. Tumor necrosis factor α (TNF-α) regulates mucin synthesis. Glucocorticoids including mometasone fuorate (MF) have been used to attenuate airway inflammation. However, effects of MF on mucin production have not been reported. Methods Effects of MF and budesonide (BUD) on the phorbol-12-myristate-13-acetate (PMA)–induction of mucin and TNF-α in human airway epithelial cells (NCI-H292) were investigated in the present study. Confluent NCI-H292 cells were pretreated with PMA (200 nM) for 2 hours. Subsequently, the cells were stimulated with MF (1–500 ng/mL) or BUD (21.5 ng/mL) for 8 hours. Dexamethasone (1 µg/mL) was used as the positive control. Real-time polymerase chain reaction was used to determine MUC2 and MUC5AC mRNA levels. The level of total mucin, MUC2, MUC5AC, and TNF-α in culture supernatants were measured using enzyme-linked immunosorbent assay. Results MF and BUD significantly suppressed MUC2 and MUC5AC gene expression in PMA-stimulated NCI-H292 cells. The inhibitory effects of the two steroid drugs were also observed in the production of total mucin, MUC2 and MUC5AC proteins, and TNF-α. Conclusion Our findings demonstrated that MF and BUD attenuated mucin and TNF-α production in PMA-induced human airway epithelial cells. PMID:28119748

  16. Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer.

    PubMed

    Watkins, D Neil; Berman, David M; Burkholder, Scott G; Wang, Baolin; Beachy, Philip A; Baylin, Stephen B

    2003-03-20

    Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.

  17. 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.

  18. Analysis of airway epithelial regeneration and repair following endobronchial brush biopsy in sheep.

    PubMed

    Yahaya, B; Baker, A; Tennant, P; Smith, S H; Shaw, D J; McLachlan, G; Collie, D D S

    2011-11-01

    Understanding the fundamental processes involved in repairing the airway wall following injury is fundamental to understanding the way in which these processes are perturbed during disease pathology. Indeed complex diseases such as asthma and chronic obstructive pulmonary disease (COPD) have at their core evidence of airway wall remodeling processes that play a crucial functional role in these diseases. The authors sought to understand the dynamic cellular events that occur during bronchial airway epithelial repair in sheep. The injury was induced by endobronchial brush biopsy (BBr), a process that causes epithelial débridement and induces a consequential repair process. In addition, the current experimental protocol allowed for the time-dependent changes in airway wall morphology to be studied both within and between animals. The initial débridement was followed by evidence of dedifferentiation in the intact epithelium at the wound margins, followed by proliferation of cells both within the epithelium and in the deeper wall structures, notably in association with the submucosal glands and smooth muscle bundles. Seven days after injury, although the airway wall was thickened at the site of damage, the epithelial layer was intact, with evidence of redifferentiation. These studies, in demonstrating broad agreement with previous studies in small animals, indicate the wider relevance of this system as a comparative model and should provide a solid basis upon which to further characterize the critical cellular and molecular interactions that underlie both effective restitution and pathological repair.

  19. 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...

  20. Organic electrochemical transistor array for recording transepithelial ion transport of human airway epithelial cells.

    PubMed

    Yao, Chunlei; Xie, Changyan; Lin, Peng; Yan, Feng; Huang, Pingbo; Hsing, I-Ming

    2013-12-03

    An organic electrochemical transistor array is integrated with human airway epithelial cells. This integration provides a novel method to couple transepithelial ion transport with electrical current. Activation and inhibition of transepithelial ion transport are readily detected with excellent time resolution. The organic electrochemical transistor array serves as a promising platform for physiological studies and drug testing.

  1. THE EFFECT OF SIZE FRACTIONED PARTICULATE MATTER ON HUMAN AIRWAY EPITHELIAL CELLS IN VITRO

    EPA Science Inventory

    THE EFFECT OF SIZE FRACTIONATED PARTICULATE MATTER ON HUMAN AIRWAY EPITHELIAL CELLS IN VITRO. LA Dailey1, C Sioutas2, JM Soukup1, S Becker1, RB Devlin1. 1National Health & Environmental Effects Research Laboratory, USEPA, RTP, NC,USA; 2USC, Civil & Environmental Engineering, LA, ...

  2. 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, ...

  3. 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

  4. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    NASA Astrophysics Data System (ADS)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-09-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO2 NPs (size range 4-33 nm), two preparations of CeO2 NPs (9-36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15-240 μg/cm2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm2, in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO2 and CeO2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured nanomaterials.

  5. 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

  6. Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases

    PubMed Central

    Aldhahrani, Adil; Verdon, Bernard; Pearson, Jeffery

    2017-01-01

    Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B). The immortalised human bronchial epithelial cell line (BEAS-2B) was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL)-8, IL-6 and granulocyte−macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L−1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L−1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo. This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury. PMID:28344983

  7. Staphylococcus aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line

    PubMed Central

    Gierok, Philipp; Harms, Manuela; Methling, Karen; Hochgräfe, Falko; Lalk, Michael

    2016-01-01

    The Gram positive opportunistic human pathogen Staphylococcus aureus induces a variety of diseases including pneumonia. S. aureus is the second most isolated pathogen in cystic fibrosis patients and accounts for a large proportion of nosocomial pneumonia. Inside the lung, the human airway epithelium is the first line in defence with regard to microbial recognition and clearance as well as regulation of the immune response. The metabolic host response is, however, yet unknown. To address the question of whether the infection alters the metabolome and metabolic activity of airway epithelial cells, we used a metabolomics approach. The nutrition uptake by the human airway epithelial cell line A549 was monitored over time by proton magnetic resonance spectroscopy (1H-NMR) and the intracellular metabolic fingerprints were investigated by gas chromatography and high performance liquid chromatography (GC-MS) and (HPLC-MS). To test the metabolic activity of the host cells, glutamine analogues and labelled precursors were applied after the infection. We found that A549 cells restrict uptake of essential nutrients from the medium after S. aureus infection. Moreover, the infection led to a shutdown of the purine and pyrimidine synthesis in the A549 host cell, whereas other metabolic routes such as the hexosamine biosynthesis pathway remained active. In summary, our data show that the infection with S. aureus negatively affects growth, alters the metabolic composition and specifically impacts the de novo nucleotide biosynthesis in this human airway epithelial cell model. PMID:27834866

  8. Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer.

    PubMed

    Spira, Avrum; Beane, Jennifer E; Shah, Vishal; Steiling, Katrina; Liu, Gang; Schembri, Frank; Gilman, Sean; Dumas, Yves-Martine; Calner, Paul; Sebastiani, Paola; Sridhar, Sriram; Beamis, John; Lamb, Carla; Anderson, Timothy; Gerry, Norman; Keane, Joseph; Lenburg, Marc E; Brody, Jerome S

    2007-03-01

    Lung cancer is the leading cause of death from cancer in the US and the world. The high mortality rate (80-85% within 5 years) results, in part, from a lack of effective tools to diagnose the disease at an early stage. Given that cigarette smoke creates a field of injury throughout the airway, we sought to determine if gene expression in histologically normal large-airway epithelial cells obtained at bronchoscopy from smokers with suspicion of lung cancer could be used as a lung cancer biomarker. Using a training set (n = 77) and gene-expression profiles from Affymetrix HG-U133A microarrays, we identified an 80-gene biomarker that distinguishes smokers with and without lung cancer. We tested the biomarker on an independent test set (n = 52), with an accuracy of 83% (80% sensitive, 84% specific), and on an additional validation set independently obtained from five medical centers (n = 35). Our biomarker had approximately 90% sensitivity for stage 1 cancer across all subjects. Combining cytopathology of lower airway cells obtained at bronchoscopy with the biomarker yielded 95% sensitivity and a 95% negative predictive value. These findings indicate that gene expression in cytologically normal large-airway epithelial cells can serve as a lung cancer biomarker, potentially owing to a cancer-specific airway-wide response to cigarette smoke.

  9. Receptor-promoted exocytosis of airway epithelial mucin granules containing a spectrum of adenine nucleotides.

    PubMed

    Kreda, Silvia M; Seminario-Vidal, Lucia; van Heusden, Catharina A; O'Neal, Wanda; Jones, Lisa; Boucher, Richard C; Lazarowski, Eduardo R

    2010-06-15

    Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. We now demonstrate that protease-activated-receptor (PAR) agonists also stimulate the simultaneous release of mucins and ATP from airway epithelial cells. PAR-mediated mucin and ATP release were dependent on intracellular Ca(2+) and actin cytoskeleton reorganization since BAPTA AM, cytochalasin D, and inhibitors of Rho and myosin light chain kinases blocked both responses. To test the hypothesis that ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of isolated mucin granules purified based on their MUC5AC and VAMP-8 content by density gradients. Mucin granules contained ATP, but the levels of ADP and AMP within granules exceeded by nearly 10-fold that of ATP. Consistent with this finding, apical secretions from PAR-stimulated cells contained relatively high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and also are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules is likely to provide a major source of airway surface adenosine to signal in a paracrine faction ciliated cell A(2b) receptors to activate ion/water secretion and appropriately hydrate goblet cell-released mucins.

  10. Receptor-promoted exocytosis of airway epithelial mucin granules containing a spectrum of adenine nucleotides

    PubMed Central

    Kreda, Silvia M; Seminario-Vidal, Lucia; van Heusden, Catharina A; O’Neal, Wanda; Jones, Lisa; Boucher, Richard C; Lazarowski, Eduardo R

    2010-01-01

    Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. We now demonstrate that protease-activated-receptor (PAR) agonists also stimulate the simultaneous release of mucins and ATP from airway epithelial cells. PAR-mediated mucin and ATP release were dependent on intracellular Ca2+ and actin cytoskeleton reorganization since BAPTA AM, cytochalasin D, and inhibitors of Rho and myosin light chain kinases blocked both responses. To test the hypothesis that ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of isolated mucin granules purified based on their MUC5AC and VAMP-8 content by density gradients. Mucin granules contained ATP, but the levels of ADP and AMP within granules exceeded by nearly 10-fold that of ATP. Consistent with this finding, apical secretions from PAR-stimulated cells contained relatively high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and also are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules is likely to provide a major source of airway surface adenosine to signal in a paracrine faction ciliated cell A2b receptors to activate ion/water secretion and appropriately hydrate goblet cell-released mucins. PMID:20421285

  11. 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.

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

    PubMed Central

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

    2015-01-01

    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

  13. 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.

  14. 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.

  15. Muscarinic receptor subtypes in cilia-driven transport and airway epithelial development

    PubMed Central

    Klein, Maike K.; Haberberger, Rainer V.; Hartmann, Petra; Faulhammer, Petra; Lips, Katrin S.; Krain, Benjamin; Wess, Jürgen; Kummer, Wolfgang; König, Peter

    2014-01-01

    Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1–M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect most likely through the M1 receptor and normalized the ATP response. M1, M4, and M5 receptor deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptor are missing. None of the receptors is necessary for epithelial development. PMID:19213795

  16. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

    PubMed

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-11-23

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

  17. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers

    PubMed Central

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-01-01

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions. PMID:27886077

  18. Staphylococcus aureus Inhibits IL-8 Responses Induced by Pseudomonas aeruginosa in Airway Epithelial Cells.

    PubMed

    Chekabab, Samuel M; Silverman, Richard J; Lafayette, Shantelle L; Luo, Yishan; Rousseau, Simon; Nguyen, Dao

    2015-01-01

    Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam3CysSK4 but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.

  19. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro.

    PubMed

    Fang, Shijing; Crews, Anne L; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong; Adler, Kenneth B

    2013-04-15

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

  20. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

    PubMed Central

    Fang, Shijing; Crews, Anne L.; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong

    2013-01-01

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation. PMID:23377348

  1. 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

  2. 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.

  3. Role of Allergen Source-Derived Proteases in Sensitization via Airway Epithelial Cells

    PubMed Central

    Matsumura, Yasuhiro

    2012-01-01

    Protease activity is a characteristic common to many allergens. Allergen source-derived proteases interact with lung epithelial cells, which are now thought to play vital roles in both innate and adaptive immune responses. Allergen source-derived proteases act on airway epithelial cells to induce disruption of the tight junctions between epithelial cells, activation of protease-activated receptor-2, and the production of thymic stromal lymphopoietin. These facilitate allergen delivery across epithelial layers and enhance allergenicity or directly activate the immune system through a nonallergic mechanism. Furthermore, they cleave regulatory cell surface molecules involved in allergic reactions. Thus, allergen source-derived proteases are a potentially critical factor in the development of allergic sensitization and appear to be strongly associated with heightened allergenicity. PMID:22523502

  4. 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

  5. Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

    PubMed

    Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

    2014-03-01

    Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

  6. "Epithelial Cell TRPV1-Mediated Airway Sensitivity as a Mechanism for Respiratory Symptoms Associated with Gulf War Illness?

    DTIC Science & Technology

    2010-06-01

    TITLE: “Epithelial Cell TRPV1 -Mediated Airway Sensitivity as a Mechanism for Respiratory Symptoms Associated with Gulf War Illness” PRINCIPAL...66,),&$7,212) E7(/(3+21(180%(5 ,QFOXGHDUHDFRGH 01-06-2010 Annual Report 1 JUN 2009 - 31 MAY 2010 Epithelial Cell TRPV1 -Mediated Airway...express functional TRPV1 . More recently we found that these cells also express another important irritant receptor, namely TRPA1. Activation of

  7. Hyaluronan and layilin mediate loss of airway epithelial barrier function induced by cigarette smoke by decreasing E-cadherin.

    PubMed

    Forteza, Rosanna Malbran; Casalino-Matsuda, S Marina; Falcon, Nieves S; Valencia Gattas, Monica; Monzon, Maria E

    2012-12-07

    Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.

  8. Hyaluronan and Layilin Mediate Loss of Airway Epithelial Barrier Function Induced by Cigarette Smoke by Decreasing E-cadherin*

    PubMed Central

    Forteza, Rosanna Malbran; Casalino-Matsuda, S. Marina; Falcon, Nieves S.; Valencia Gattas, Monica; Monzon, Maria E.

    2012-01-01

    Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS. PMID:23048036

  9. 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

  10. Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

    PubMed Central

    Sibley, Christopher D.; Grinwis, Margot E.; Field, Tyler R.; Eshaghurshan, Christina S.; Faria, Monica M.; Dowd, Scot E.; Parkins, Michael D.; Rabin, Harvey R.; Surette, Michael G.

    2011-01-01

    The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured. PMID:21829484

  11. 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

  12. Metals mimic airway epithelial injury induced by in vitro exposure to Utah Valley ambient particulate matter extracts.

    PubMed

    Pagan, Ines; Costa, Daniel L; McGee, John K; Richards, Judy H; Dye, Janice A

    2003-06-27

    Epidemiologic studies have shown positive associations between changes in ambient particulate matter (PM) levels in Utah Valley during 1986-1988, and the respiratory health of the local population. Ambient PM reductions coincided with closure of an open-hearth steel mill, the major industrial source of particulate emissions in the valley. In this report, water extracts of PM filters from steel mill operational (UE-86, UE-88) and closure (UE-87) periods were analyzed for their elemental composition. Their relative toxicity was determined by exposing primary rodent airway epithelial cultures to equal masses of extracted material. To elucidate extract subcomponents mediating the effects observed, cells were also exposed to surrogate metal mixtures. Potential interactions between the two predominant metals in the UE-86/88 samples, zinc (Zn) and copper (Cu), were further investigated. Data indicated that, relative to the UE-87 (plant closed) sample, UE-86/88 samples contained more sulfate, calcium, potassium,magnesium and, although present in much lower amounts, a variety of metals including Zn,Cu, iron, lead, strontium, nickel, manganese, and vanadium (V). Cell exposure to UE-86 and UE-88, but not UE-87, resulted in time- and concentration-dependent epithelial injury based on biochemical and light/electron microscopic changes. Cell injury induced by metal mixtures containing equivalent amounts of Zn + Cu + V was commensurate with that induced by the corresponding extract, although divergent antioxidant responses were observed. Exposure to Zn + Cu resulted in significantly greater epithelial toxicity and stress (c-Jun N-terminal protein kinase activation) responses than did exposure to Zn or Cu individually. The parallel epithelial injury induced by the extracts and their surrogate Zn + Cu + V mixtures suggests that these metals are mediating the acute airway epithelial effects observed; however, metal interactions appear to play a critical role in the overall cellular

  13. Role of mitochondrial hydrogen peroxide induced by intermittent hypoxia in airway epithelial wound repair in vitro.

    PubMed

    Hamada, Satoshi; Sato, Atsuyasu; Hara-Chikuma, Mariko; Satooka, Hiroki; Hasegawa, Koichi; Tanimura, Kazuya; Tanizawa, Kiminobu; Inouchi, Morito; Handa, Tomohiro; Oga, Toru; Muro, Shigeo; Mishima, Michiaki; Chin, Kazuo

    2016-05-15

    The airway epithelium acts as a frontline barrier against various environmental insults and its repair process after airway injury is critical for the lung homeostasis restoration. Recently, the role of intracellular reactive oxygen species (ROS) as transcription-independent damage signaling has been highlighted in the wound repair process. Both conditions of continuous hypoxia and intermittent hypoxia (IH) induce ROS. Although IH is important in clinical settings, the roles of IH-induced ROS in the airway repair process have not been investigated. In this study, we firstly showed that IH induced mitochondrial hydrogen peroxide (H2O2) production and significantly decreased bronchial epithelial cell migration, prevented by catalase treatment in a wound scratch assay. RhoA activity was higher during repair process in the IH condition compared to in the normoxic condition, resulting in the cellular morphological changes shown by immunofluorescence staining: round cells, reduced central stress fiber numbers, pronounced cortical actin filament distributions, and punctate focal adhesions. These phenotypes were replicated by exogenous H2O2 treatment under the normoxic condition. Our findings confirmed the transcription-independent role of IH-induced intracellular ROS in the bronchial epithelial cell repair process and might have significant implications for impaired bronchial epithelial cell regeneration.

  14. Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression

    PubMed Central

    Hays, L E; Zodrow, D M; Yates, J E; Deffebach, M E; Jacoby, D B; Olson, S B; Pankow, J F; Bagby, G C

    2008-01-01

    Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis. PMID:18475298

  15. 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

  16. Inhibitory effects of carbocisteine on type A seasonal influenza virus infection in human airway epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Shinya, Kyoko; Hatachi, Yukimasa; Sasaki, Takahiko; Yasuda, Hiroyasu; Yoshida, Motoki; Asada, Masanori; Fujino, Naoya; Suzuki, Takaya; Deng, Xue; Kubo, Hiroshi; Nagatomi, Ryoichi

    2010-08-01

    Type A human seasonal influenza (FluA) virus infection causes exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD). l-carbocisteine, a mucolytic agent, reduces the frequency of common colds and exacerbations in COPD. However, the inhibitory effects of l-carbocisteine on FluA virus infection are uncertain. We studied the effects of l-carbocisteine on FluA virus infection in airway epithelial cells. Human tracheal epithelial cells were pretreated with l-carbocisteine and infected with FluA virus (H(3)N(2)). Viral titers in supernatant fluids, RNA of FluA virus in the cells, and concentrations of proinflammatory cytokines in supernatant fluids, including IL-6, increased with time after infection. l-carbocisteine reduced viral titers in supernatant fluids, RNA of FluA virus in the cells, the susceptibility to FluA virus infection, and concentrations of cytokines induced by virus infection. The epithelial cells expressed sialic acid with an alpha2,6-linkage (SAalpha2,6Gal), a receptor for human influenza virus on the cells, and l-carbocisteine reduced the expression of SAalpha2,6Gal. l-carbocisteine reduced the number of acidic endosomes from which FluA viral RNA enters into the cytoplasm and reduced the fluorescence intensity from acidic endosomes. Furthermore, l-carbocisteine reduced NF-kappaB proteins including p50 and p65 in the nuclear extracts of the cells. These findings suggest that l-carbocisteine may inhibit FluA virus infection, partly through the reduced expression of the receptor for human influenza virus in the human airway epithelial cells via the inhibition of NF-kappaB and through increasing pH in endosomes. l-carbocisteine may reduce airway inflammation in influenza virus infection.

  17. Effect of eosinophil peroxidase on airway epithelial permeability in the guinea pig.

    PubMed

    Brottman, G M; Regelmann, W E; Slungaard, A; Wangensteen, O D

    1996-03-01

    Increased numbers of eosinophils and increased concentrations of plasma proteins have been found in the airways of patients with mild asthma. We used an intact guinea pig trachea model to investigate the role of eosinophil peroxidase (EPO) in altering the function of the airway epithelial barrier. EPO in the presence of hydrogen peroxide (H(2)O(2)) and bromide (Br(-)) catalyzes the production of hypobromous acid (HOBr), which is felt to have a toxic effect on airway epithelial cells. An intact guinea pig trachea was mounted on an apparatus in a way that would allow the tracheal epithelium to be exposed to different solutions. Following these exposures, a test solution containing (14)C-sucrose (S), (3)H-inulin (I), and FITC-dextran-20 (D) was placed in the tracheal lumen and positioned in the center of the segment for 90 minutes. Flux of these molecules across the epithelial barrier into a bath was measured, and the permeability (P) was calculated for each molecule to quantify epithelial barrier function. Light and electron micrographic studies were performed to assess cellular damage. We found that there was a dose response to EPO (in the presence of fixed amounts of H(2)(O)(2) and Br(-)). EPO at 7.3 x 10(-7) M caused no increase in P over controls (Ringer's solution alone) for S, I, or D (P> 0.05), whereas EPO at 2.7 x 10(-6) M caused a significant increase in P over controls (P = 0.008) for all test molecules. Light and electron micrographs of the latter tracheas showed no evidence of microscopic changes despite the increased P. Further testing verified that the increase in permeability was caused by the EPO catalyzed reaction and not the individual substrates themselves, and that the reaction was inhibited by a peroxidase inhibitor. We conclude that EPO can alter the barrier function of the airway epithelium before gross cellular damage becomes visible. We hypothesize that changes in the tight junctions are responsible for the alteration in the barrier function

  18. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

    PubMed Central

    Sesma, Juliana I.; Kreda, Silvia M.; Okada, Seiko F.; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C.; O'Neal, Wanda K.; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori

    2013-01-01

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins. PMID:23467297

  19. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

    PubMed

    Sesma, Juliana I; Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C; O'Neal, Wanda K; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori; Lazarowski, Eduardo R

    2013-05-15

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

  20. 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.

  1. 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

  2. Influence of cetirizine and levocetirizine on two cytokines secretion in human airway epithelial cells.

    PubMed

    Shih, Mei-Yin; Hsu, Jeng-Yuan; Weng, Yueh-Shan; Fu, Lin-Shien

    2008-01-01

    Recent studies suggest that several second-generation antihistamines can modulate various inflammatory reactions besides their H(1)-receptor antagonism. The antihistamine cetirizine is a racemic mixture of levocetirizine and dextrocetirizine. The aim of this study was to investigate the effects of these two antihistamines (cetirizine and levocetirizine) on granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8 secretion in A549 human airway epithelial cells. A549 cells were preincubated with cetirizine (0.1, 1, 2.5, 5, and 10 microM) or levocetirizine (0.1, 1, 2.5, 5, and 10 microM) individually for 16 hours and were then stimulated with IL-1beta for 8 hours. The levels of GM-CSF and IL-8 in cultured supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed that cetirizine (5 and 10 microM) and levocetirizine (2.5, 5, and 10 microM) significantly suppressed GM-CSF secretion from A549 cells stimulated with IL-1beta (p<0.05). Cetirizine (10 microM) and levocetirizine (5 and 10 microM) significantly suppressed IL-8 secretion after A549 was stimulated. The suppressive effect was comparable between levocetirizine, 2.5 microM, and cetirizine, 5 microM, as well as levocetirizine, 5 microM, and cetirizine, 10 microM. Moreover, levocetirizine, 5 microM, was better than cetirizine, 5 microM, on suppressing IL-8 secretion, but such a difference did not appear in other conditions. Our results suggest that cetirizine and levocetirizine at higher concentrations can reduce the release of GM-CSF and IL-8 from A549 cells stimulated with IL-1beta. These observations indicate that the two second-generation antihistamines may exert anti-inflammatory effects beyond histamine H(1)-receptor antagonist, and levocetirizine plays a major role in terms of this activity.

  3. Ca2+ signaling in airway epithelial cells facilitates leukocyte recruitment and transepithelial migration

    PubMed Central

    Chun, Jarin; Prince, Alice

    2009-01-01

    In airway cells, TLR2 stimulation by bacterial products activates Ca2+ fluxes that signal leukocyte recruitment to the lung and facilitates transepithelial migration into the airway lumen. TLR2 is apically displayed on airway cells, where it senses bacterial stimuli. Biochemical and genetic approaches demonstrate that TLR2 ligands stimulate release of Ca2+ from intracellular stores by activating TLR2 phosphorylation by c-Src and recruiting PI3K and PLCγ to affect Ca2+ release through IP3Rs. This Ca2+ release plays a pivotal role in signaling TLR2-dependent NF-κB activation and chemokine expression to recruit PMNs to the lung. In addition, TLR2-initiated Ca2+ release activates Ca2+-dependent proteases, calpains, which cleave the transmembrane proteins occludin and E-cadherin to promote PMN transmigration. This review highlights recent findings that demonstrate a central role for Ca2+ signaling in airway epithelial cells to induce proinflammatory gene transcription and to initiate junctional changes that accommodate transmigration of recruited PMNs. PMID:19605699

  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. 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

  6. Antibacterial Defense of Human Airway Epithelial Cells from Chronic Obstructive Pulmonary Disease Patients Induced by Acute Exposure to Nontypeable Haemophilus influenzae: Modulation by Cigarette Smoke.

    PubMed

    Amatngalim, Gimano D; Schrumpf, Jasmijn A; Henic, Almira; Dronkers, Esther; Verhoosel, Renate M; Ordonez, Soledad R; Haagsman, Henk P; Fuentes, Maria E; Sridhar, Sriram; Aarbiou, Jamil; Janssen, Richard A J; Lekkerkerker, Annemarie N; Hiemstra, Pieter S

    2017-02-08

    Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable Haemophilus influenzae (NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.

  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. 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

  9. Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery.

    PubMed

    Heijink, I H; Brandenburg, S M; Postma, D S; van Oosterhout, A J M

    2012-02-01

    Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from COPD patients, nonsmokers and healthy smokers. We demonstrate that CSE rapidly and transiently impairs 16HBE barrier function, largely due to disruption of cell-cell contacts. CSE induced a similar, but stronger and more sustained, defect in PBECs. Application of the specific epidermal growth factor receptor (EGFR) inhibitor AG1478 showed that EGFR activation contributes to the CSE-induced defects in both 16HBE cells and PBECs. Furthermore, our data indicate that the endogenous protease calpain mediates these defects through tight junction protein degradation. CSE also delayed the reconstitution of 16HBE intercellular contacts during wound healing and attenuated PBEC barrier function upon wound regeneration. These findings were comparable between PBECs from smokers, healthy smokers and COPD patients. In conclusion, we demonstrate for the first time that CSE reduces epithelial integrity, probably by EGFR and calpain-dependent disruption of intercellular contacts. This may increase susceptibility to environmental insults, e.g. inhaled pathogens. Thus, EGFR may be a promising target for therapeutic strategies to improve mucosal barrier function in cigarette smoking-related disease.

  10. Cadmium regulates the expression of the CFTR chloride channel in human airway epithelial cells.

    PubMed

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N; Davis, Ian C; Knoell, Daren L; Parinandi, Narasimham L; Cormet-Boyaka, Estelle

    2010-07-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.

  11. GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

    1992-11-01

    Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

  12. Pneumocystis jirovecii Can Be Productively Cultured in Differentiated CuFi-8 Airway Cells

    PubMed Central

    Schildgen, Verena; Mai, Stephanie; Khalfaoui, Soumaya; Lüsebrink, Jessica; Pieper, Monika; Tillmann, Ramona L.; Brockmann, Michael

    2014-01-01

    ABSTRACT Although Pneumocystis jirovecii is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether P. jirovecii could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with bronchoalveolar fluid that had been confirmed to be positive for P. jirovecii using PCR. Five days later, the cells and basal medium were harvested and tested for P. jirovecii using quantitative PCR (qPCR), commercially available immunofluorescence detection assays, and Grocott staining of formalin-fixed, paraffin-embedded thin sections of infected-cell cultures. We successfully productively cultivated and propagated P. jirovecii from these P. jirovecii-positive bronchoalveolar lavage fluid (BALF) samples. Furthermore, we provide evidence that P. jirovecii induced cytopathic effects on lung epithelial cells and was even invasive in cell culture. To the best of our knowledge, the cell culture system developed herein represents the first methodology to enable molecular analyses of this pathogen’s life cycle and further in vitro studies of P. jirovecii, such as assessments of drug sensitivity and resistance as well as investigations of the pathogen’s stability against environmental factors and disinfectants. PMID:24825015

  13. Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

    PubMed

    Zsembery, Akos; Boyce, Amanda T; Liang, Lihua; Peti-Peterdi, János; Bell, P Darwin; Schwiebert, Erik M

    2003-04-11

    Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

  14. 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

  15. Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy

    PubMed Central

    Spann, Kirsten M; Baturcam, Engin; Schagen, Johanna; Jones, Carmen; Straub, Claire P; Preston, F Maxine; Chen, Linping; Phipps, Simon; Sly, Peter D; Fantino, Emmanuelle

    2014-01-01

    Background Airway epithelial cells (AEC) from patients with asthma, appear to have an impaired interferon (IFN)-β and -λ response to infection with rhinovirus. Objectives To determine if impaired IFN responses can be identified in young children at risk of developing asthma due to atopy and/or early life wheeze, and if the site of infection or the infecting virus influence the antiviral response. Methods Nasal (N) and tracheal (T) epithelial cells (EC) were collected from children categorised with atopy and/or wheeze based on specific IgE to locally common aeroallergens and a questionnaire concerning respiratory health. Submerged primary cultures were infected with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV), and IFN production, inflammatory cytokine expression and viral replication quantified. Results Nasal epithelial cells (NEC), but not tracheal epithelial cells (TEC), from children with wheeze and/or atopy produced less IFN-β, but not IFN-λ, in response to RSV infection; this was associated with higher viral shedding. However, IFN-regulated factors IRF-7, Mx-1 and CXCL-10, and inflammatory cytokines were not differentially regulated. NECs and TECs from children with wheeze and/or atopy demonstrated no impairment of the IFN response (β or λ) to hMPV infection. Despite this, more hMPV was shed from these cells. Conclusions AECs from children with wheeze and/or atopy do not have an intrinsic defect in the production of IFN-β or -λ, however, this response is influenced by the infecting virus. Higher viral load is associated with atopy and wheeze suggesting an impaired antiviral response to RSV and hMPV that is not influenced by production of IFNs. PMID:24811725

  16. Local blockade of epithelial PDL-1 in the airways enhances T cell function and viral clearance during influenza virus infection.

    PubMed

    McNally, Beth; Ye, Fang; Willette, Meredith; Flaño, Emilio

    2013-12-01

    In order to maintain the gas exchange function of the lung following influenza virus infection, a delicate orchestration of positive and negative regulatory pathways must be maintained to attain viral eradication while minimizing local inflammation. The programmed death receptor 1 ligand/programmed death receptor 1 (PDL-1/PD-1) pathway plays an important immunoregulatory role, particularly in the context of T cell function. Here, we have shown that influenza virus infection of primary airway epithelial cells strongly enhances PDL-1 expression and does so in an alpha interferon receptor (IFNAR) signaling-dependent manner. PD-1 is expressed primarily on effector T cells in the lung, compared to effector memory and central memory cells, and shortly after influenza virus infection, an increased number of PD-1(+) T cells are recruited to the airways. Using in vitro cocultures of airway epithelial cells and T cells and in vivo models of influenza virus infection, we have demonstrated that blockade of airway epithelial PDL-1 improves CD8 T cell function, defined by increased production of gamma interferon (IFN-γ) and granzyme B and expression of CD107ab. Furthermore, PDL-1 blockade in the airways served to accelerate influenza virus clearance and enhance infection recovery. Our findings suggest that local manipulation of the PDL-1/PD-1 axis in the airways may represent a therapeutic alternative during acute influenza virus infection.

  17. IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17

    PubMed Central

    Booth, Brian W; Sandifer, Tracy; Martin, Erika L; Martin, Linda D

    2007-01-01

    Background The pleiotrophic cytokine interleukin (IL)-13 features prominently in allergic and inflammatory diseases. In allergic asthma, IL-13 is well established as an inducer of airway inflammation and tissue remodeling. We demonstrated previously that IL-13 induces release of transforming growth factor-α (TGFα) from human bronchial epithelial cells, with proliferation of these cells mediated by the autocrine/paracrine action of this growth factor. TGFα exists as an integral membrane protein and requires proteolytic processing to its mature form, with a disintegrin and metalloproteinase (ADAM)17 responsible for this processing in a variety of tissues. Methods In this study, normal human bronchial epithelial (NHBE) cells grown in air/liquid interface (ALI) culture were used to examine the mechanisms whereby IL-13 induces release of TGFα and cellular proliferation. Inhibitors and antisense RNA were used to examine the role of ADAM17 in these processes, while IL-13-induced changes in the intracellular expression of TGFα and ADAM17 were visualized by confocal microscopy. Results IL-13 was found to induce proliferation of NHBE cells, and release of TGFα, in an ADAM17-dependent manner; however, this IL-13-induced proliferation did not appear to result solely from ADAM17 activation. Rather, IL-13 induced a change in the location of TGFα expression from intracellular to apical regions of the NHBE cells. The apical region was also found to be a site of significant ADAM17 expression, even prior to IL-13 stimulation. Conclusion Results from this study indicate that ADAM17 mediates IL-13-induced proliferation and TGFα shedding in NHBE cells. Furthermore, they provide the first example wherein a cytokine (IL-13) induces a change in the intracellular expression pattern of a growth factor, apparently inducing redistribution of intracellular stores of TGFα to the apical region of NHBE cells where expression of ADAM17 is prominent. Thus, IL-13-induced, ADAM17-mediated

  18. Chlorzoxazone or 1-EBIO increases Na(+) absorption across cystic fibrosis airway epithelial cells.

    PubMed

    Gao, L; Yankaskas, J R; Fuller, C M; Sorscher, E J; Matalon, S; Forman, H J; Venglarik, C J

    2001-11-01

    Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl(-) secretion by increasing basolateral K(+) conductance (G(K)) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778-787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across Delta F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na(+) absorption. The addition of chlorzoxazone (400 microM) or 1-EBIO (2 mM) increased the amiloride-sensitive I(sc) approximately 2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G(K) with a K(+) gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an approximately 10-fold increase in clotrimazole-sensitive G(K). In contrast, chlorzoxazone did not alter epithelial Na(+) channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G(K) alone can modulate epithelial Na(+) transport.

  19. Burkholderia cenocepacia ET12 Strain Activates TNFR1 Signaling in Cystic Fibrosis Airway Epithelial Cells

    PubMed Central

    Umadevi Sajjan, S.; Hershenson, Marc B.; Forstner, Janet F.; LiPuma, John J.

    2011-01-01

    Burkholderia cenocepacia is an important pulmonary pathogen in individuals with cystic fibrosis. Infection is often associated with severe pulmonary inflammation and some patients develop a fatal necrotizing pneumonia and sepsis (‘cepacia syndrome’). The mechanisms by which this species causes severe pulmonary inflammation are poorly understood. Here, we demonstrate that B. cenocepacia BC7, a potentially virulent representative of the epidemic ET12 lineage binds to tumor necrosis factor receptor I (TNFR1) and activates TNFR1-related signaling pathway similar to TNF-α, a natural ligand for TNFR1. This interaction participates in stimulating a robust IL-8 production from CF airway epithelial cells. In contrast, BC45, a relatively less virulent ET12 representative, and ATCC 25416, an environmental B. cepacia strain do not bind to TNFR1 and stimulate only minimal IL-8 production from CF cells. Further, TNFR1 expression is increased in CF airway epithelial cells compared to non-CF cells. We also show that B. cenocepacia ET12 strain colocaizes with TNFR1 in vitro and in the lungs of CF patient who died due to infection with B. cenocepacia, ET12 strain. Together, these results suggest that interaction of B. cenocepacia, ET12 strain with TNFR1 may contribute to robust inflammatory responses elicited by this organism. PMID:17697131

  20. Dysfunction of mitochondria Ca2+ uptake in cystic fibrosis airway epithelial cells.

    PubMed

    Antigny, Fabrice; Girardin, Nathalie; Raveau, Dorothée; Frieden, Maud; Becq, Frédéric; Vandebrouck, Clarisse

    2009-07-01

    In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca(2+) mobilization is abnormally increased. Because the uptake of Ca(2+) by mitochondria during Ca(2+) influx or Ca(2+) release from ER stores may be crucial for maintaining a normal Ca(2+) homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (DeltaPsi(mit)) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2-AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca(2+). The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the DeltaPsi(mit) of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca(2+) uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca(2+) uptake.

  1. 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

  2. The role of reactive oxygen and nitrogen species in airway epithelial gene expression.

    PubMed Central

    Martin, L D; Krunkosky, T M; Voynow, J A; Adler, K B

    1998-01-01

    The body first encounters deleterious inhaled substances, such as allergens, industrial particles, pollutants, and infectious agents, at the airway epithelium. When this occurs, the epithelium and its resident inflammatory cells respond defensively by increasing production of cytokines, mucus, and reactive oxygen and nitrogen species (ROS/RNS). As inflammation in the airway increases, additional infiltrating cells increase the level of these products. Recent interest has focused on ROS/RNS as potential modulators of the expression of inflammation-associated genes important to the pathogenesis of various respiratory diseases. ROS/RNS appear to play a variety of roles that lead to changes in expression of genes such as interleukin-6 and intercellular adhesion molecule 1. By controlling this regulation, the reactive species can serve as exogenous stimuli, as intercellular signaling molecules, and as modulators of the redox state in epithelial cells. Unraveling the molecular mechanisms affected by ROS/RNS acting in these capacities should aid in the understanding of how stimulated defense mechanisms within the airway can lead to disease. Images Figure 1 PMID:9788898

  3. Epithelial-mesenchymal transition as a fundamental underlying pathogenic process in COPD airways: fibrosis, remodeling and cancer.

    PubMed

    Nowrin, Kaosia; Sohal, Sukhwinder Singh; Peterson, Gregory; Patel, Rahul; Walters, Eugene Haydn

    2014-10-01

    Chronic obstructive pulmonary disease (COPD) is a complex condition, frequently with a mix of airway and lung parenchymal damage. However, the earliest changes are in the small airways, where most of the airflow limitation occurs. The pathology of small airway damage seems to be wall fibrosis and obliteration, but the whole airway is involved in a 'field effect'. Our novel observations on active epithelial-mesenchymal transition (EMT) in the airways of smokers, particularly in those with COPD, are changing the understanding of this airway pathology and the aetiology of COPD. EMT involves a cascade of regulatory changes that destabilise the epithelium with a motile and mesenchymal epithelial cell phenotype emerging. One important manifestation of EMT activity involves up-regulation of specific key transcription factors (TFs), such as Smads, Twist, and β-catenin. Such TFs can be used as EMT biomarkers, in recognisable patterns reflecting the potential major drivers of the process; for example, TGFβ, Wnt, and integrin-linked kinase systems. Thus, understanding the relative changes in TF activity during EMT may provide rich information on the mechanisms driving this whole process, and how they may change over time and with therapy. We have sought to review the current literature on EMT and the relative expression of specific TF activity, to define the networks likely to be involved in a similar process in the airways of patients with smoking-related COPD.

  4. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  5. Nicotine-induced epithelial-mesenchymal transition via Wnt/β-catenin signaling in human airway epithelial cells.

    PubMed

    Zou, Weifeng; Zou, Yimin; Zhao, Zhuxiang; Li, Bing; Ran, Pixin

    2013-02-15

    Epithelial-mesenchymal transition (EMT) has been proposed to be a mechanism in airway remodeling, which is a characteristic of chronic obstructive pulmonary disease (COPD). Studies have shown that cigarette smoke and nicotine are factors that induce Wnt/β-catenin activation, which is a pathway that has also been implicated in EMT. The main aim of this study was to test whether human bronchial epithelial cells are able to undergo EMT in vitro following nicotine stimulation via the Wnt3a/β-catenin signaling pathway. We show that nicotine activates the Wnt3a signal pathway, which leads to the translocation of β-catenin into the nucleus and activation of β-catenin/Tcf-dependent transcription in the human bronchial epithelial cell (HBEC) line. This accumulation was accompanied by an increase in smooth muscle actin, vimentin, matrix metalloproteinases-9, and type I collagen expression as well as downregulation of E-cadherin, which are typical characteristics of EMT. We also noted that the release of TGF-β(1) from these cells was stimulated by nicotine. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented these effects, implying that β-catenin activation in these responses is Wnt3a dependent. Furthermore, specific knockdown of TGF-β(1) with TGF-β(1) siRNA partially prevented nicotine-induced EMT, suggesting that TGF-β(1) has a role in nicotine-mediated EMT in HBECs. These results suggest that HBECs are able to undergo EMT in vitro upon nicotine stimulation via the Wnt3a/β-catenin signaling pathway.

  6. 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

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

    PubMed

    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

    2007-10-01

    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 Ca(2+)-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca(2+)-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca(2+)-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 P2Y(2) receptor-stimulated increase of cytosolic Ca(2+) 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.

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

    PubMed

    Park, Sin-Hye; Gong, Ju-Hyun; 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.

  9. Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

    PubMed Central

    Zhang, Zhaolin; Leir, Shih-Hsing

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator gene (CFTR) expression in human airway epithelial cells involves the recruitment of distal cis-regulatory elements, which are associated with airway-selective DNase hypersensitive sites at −44 kb and −35 kb from the gene. The −35-kb site encompasses an enhancer that is regulated by the immune mediators interferon regulatory factor 1 and 2 and by nuclear factor Y. Here we investigate the −44-kb element, which also has enhancer activity in vitro in airway epithelial cells but is inactive in intestinal epithelial cells. This site contains an antioxidant response element (ARE) that plays a critical role in its function in airway cell lines and primary human bronchial epithelial cells. The natural antioxidant sulforaphane (SFN) induces nuclear translocation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcription factor that regulates genes with AREs in their promoters, many of which are involved in response to injury. Under normal conditions, the −44-kb ARE is occupied by the repressor BTB and CNC homology 1, basic leucine zipper transcription factor (Bach1), and v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) heterodimers. After 2 hours of SFN treatment, Nrf2 displaces these repressive factors and activates CFTR expression. Site-directed mutagenesis shows that both the ARE and an adjacent NF-κB binding site are required for activation of the –44-kb element in airway epithelial cells. Moreover, this element is functionally linked to the −35-kb enhancer in modulating CFTR expression in response to environmental stresses in the airway. PMID:25259561

  10. Overexpression of farnesoid X receptor in small airways contributes to epithelial to mesenchymal transition and COX-2 expression in chronic obstructive pulmonary disease

    PubMed Central

    Chen, Bi; You, Wen-Jie; Xue, Shan; Qin, Hui; Zhao, Xu-Ji; Zhang, Miao; Liu, Xue-Qing; Zhu, Shu-Yang

    2016-01-01

    Background Epithelial-mesenchymal transition (EMT) and cyclooxygenase-2 (COX-2) contribute to airway remodelling and inflammation in chronic obstructive pulmonary disease (COPD). Recent data suggest that the farnesoid X receptor (FXR), a nuclear receptor traditionally considered as bile acid-activated receptor, is also expressed in non-classical bile acids target tissues with novel functions beyond regulating bile acid homeostasis. This study aimed to investigate the potential role of FXR in the development of COPD, as well as factors that affect FXR expression. Methods Expression of FXR, EMT biomarkers and COX-2 was examined by immunohistochemistry in lung tissues from non-smokers, smokers, and smokers with COPD. The role of FXR in TGF-β1-induced EMT and COX-2 expression in human bronchial epithelial (HBE) cells was evaluated in vitro. Factors regulating FXR expression were assessed in cultured HBE cells and a cigarette smoke-induced rat model of COPD. Results Expression of FXR, EMT markers and COX-2 was significantly elevated in small airway epithelium of COPD patients compared with controls. The staining scores of FXR in small airway epithelium were negatively related with FEV1% of predicted of smokers without and with COPD. FXR agonist GW4064 remarkably enhanced and FXR antagonist Z-Guggulsterone significantly inhibited EMT changes in TGF-β1-treated HBE cells. Both chenodeoxycholic acid (CDCA) and GW4064 increased COX-2 expression in HBE cells, whereas Z-Guggulsterone dramatically restrained CDCA-induced COX-2 expression. Finally, FXR expression is induced by IL-4 and IL-13 in HBE cells, as well as by cigarette smoke exposure in a rat model of COPD. Conclusions Overexpression of FXR in small airway may contribute to airway remodelling and inflammation in COPD by regulating EMT and COX-2 expression. PMID:28066584

  11. Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration

    PubMed Central

    Moodley, Serisha; Derouet, Mathieu; Bai, Xiao Hui; Xu, Feng; Kapus, Andras; Yang, Burton B.; Liu, Mingyao

    2016-01-01

    Repair of airway epithelium after injury requires migration of neighboring epithelial cells to injured areas. However, the molecular mechanisms regulating airway epithelial cell migration is not well defined. We have previously shown that XB130, a scaffold protein, is required for airway epithelial repair and regeneration in vivo, and interaction between XB130 and another scaffold protein, Tks5, regulates cell proliferation and survival in human bronchial epithelial cells. The objective of the present study was to determine the role of XB130 and Tks5 interaction in airway epithelial cell migration. Interestingly, we found that XB130 only promotes lateral cell migration, whereas, Tks5 promotes cell migration/invasion via proteolysis of extracellular matrix. Upon stimulation with EGF, PKC activator phorbol 12, 13-dibutyrate or a nicotinic acetylcholine receptor ligand, XB130 and Tks5 translocated to the cell membrane in a stimulus-dependent manner. The translocation and distribution of XB130 is similar to lamellipodial marker, WAVE2; whereas Tks5 is similar to podosome marker, N-WASP. Over-expression of XB130 or Tks5 alone enhances cell migration, whereas co-expression of both XB130 and Tks5 inhibits cell migration processes and signaling. Furthermore, XB130 interacts with Rac1 whereas Tks5 interacts with Cdc42 to promote Rho GTPase activity. Our results suggest that dissociation between XB130 and Tks5 may facilitate lateral cell migration via XB130/Rac1, and vertical cell migration via Tks5/Cdc42. These molecular mechanisms will help our understanding of airway epithelial repair and regeneration. PMID:27835612

  12. Activation of eicosanoid metabolism in human airway epithelial cells by ozonolysis products of membrane fatty acids.

    PubMed

    Leikauf, G D; Zhao, Q; Zhou, S; Santrock, J

    1995-09-01

    Inhaled ozone can react with a variety of cellular macromolecules within the lung. Recent analyses of the chemistry of ozone reactions with unsaturated fatty acids, which are present in all membranes and in mucus in the airways, indicate that ozonolysis yields one aldehyde and one hydroxyhydroperoxide molecule for each molecule of ozone. The hydroxyhydroperoxide molecule is unstable in aqueous environments, and subsequently yields a second aldehyde and hydrogen peroxide. The structure of common unsaturated fatty acids is such that attack by ozone at the carbon-carbon double bonds will yield 3-, 6-, and 9-carbon saturated and unsaturated aldehydes and hydroxyhydroperoxide. This study examines the effects of ozonolysis products on eicosanoid metabolism in human airway epithelial cells. Eicosanoid biosynthesis is important in a wide array of pathophysiological responses in the airway, and the release of eicosanoids by the epithelial barrier is likely to be significant in diseases induced by environmental factors. Previously, we demonstrated that ozone can increase eicosanoid synthesis from airway epithelial cells exposed in vitro. Human exposures to concentrations of ozone below the current National Ambient Air Quality Standard (0.12 ppm, not to be exceeded for more than one hour once per year) also resulted in increased eicosanoids in bronchoalveolar lavage fluid. To determine whether ozonolysis products could activate eicosanoid release, we exposed human airway epithelial cells to 3-, 6-, and 9-carbon aldehydes, hydroxyhydroperoxides, and hydrogen peroxide. We measured (1) eicosanoid metabolism using high-performance liquid chromatography and radioimmunoassays, and (2) the effects of the aldehydes, hydroxyhydroperoxides, and hydrogen peroxide on cell lysis. Eicosanoid release increased after exposure to aldehyde; release induced by 9-carbon (nonanal) aldehyde was greater than that induced by the 6-carbon (hexanal) or 3-carbon (propanal) aldehydes

  13. 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.

  14. Airway Epithelial Orchestration of Innate Immune Function in Response to Virus Infection. A Focus on Asthma.

    PubMed

    Ritchie, Andrew I; Jackson, David J; Edwards, Michael R; Johnston, Sebastian L

    2016-03-01

    Asthma is a very common respiratory condition with a worldwide prevalence predicted to increase. There are significant differences in airway epithelial responses in asthma that are of particular interest during exacerbations. Preventing exacerbations is a primary aim when treating asthma because they often necessitate unscheduled healthcare visits and hospitalizations and are a significant cause of morbidity and mortality. The most common cause of asthma exacerbations is a respiratory virus infection, of which the most likely type is rhinovirus infection. This article focuses on the role played by the epithelium in orchestrating the innate immune responses to respiratory virus infection. Recent studies show impaired bronchial epithelial cell innate antiviral immune responses, as well as augmentation of a pro-Th2 response characterized by the epithelial-derived cytokines IL-25 and IL-33, crucial in maintaining the Th2 cytokine response to virus infection in asthma. A better understanding of the mechanisms of these abnormal immune responses has the potential to lead to the development of novel therapeutic targets for virus-induced exacerbations. The aim of this article is to highlight current knowledge regarding the role of viruses and immune modulation in the asthmatic epithelium and to discuss exciting areas for future research and novel treatments.

  15. 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...

  16. 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...

  17. CO-EXPOSURE OF HUMAN AIRWAY EPITHELIAL CELLS TO OZONE AND PARTICULATE MATTER: EFFECTS ON ARACHIDONIC ACID METABOLISM

    EPA Science Inventory

    Co-exposure of human airway epithelial cells to ozone and particulate matter: effects on arachidonic acid metabolism.

    D. Stamm1, L. Dailey2, M.C. Madden2
    1 University of North Carolina-Chapel Hill, School of Medicine
    2 U.S. EPA, ORD, NHEERL, HSD, Chapel Hill, NC, USA...

  18. Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells

    PubMed Central

    Singh, Brajesh K.; Li, Ni; Mark, Anna C.; Mateo, Mathieu; Cattaneo, Roberto

    2016-01-01

    ABSTRACT Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-μm) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-μm) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE. IMPORTANCE Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell

  19. Human metapneumovirus glycoprotein G disrupts mitochondrial signaling in airway epithelial cells.

    PubMed

    Bao, Xiaoyong; Kolli, Deepthi; Ren, Junping; Liu, Tianshuang; Garofalo, Roberto P; Casola, Antonella

    2013-01-01

    Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.

  20. 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.

  1. 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

  2. Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

    PubMed Central

    Maestre-Batlle, Danay; Pena, Olga M.; Hirota, Jeremy A.; Gunawan, Evelyn; Rider, Christopher F.; Sutherland, Darren; Alexis, Neil E.; Carlsten, Chris

    2017-01-01

    Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. PMID:28165060

  3. A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence.

    PubMed

    Lam, E; Ramke, M; Groos, S; Warnecke, G; Heim, A

    2011-12-01

    The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established. These primary cells proved to be fully permissive for HAdV-C5 infection in submerged culture, demonstrating efficient HAdV genome replication, infectious viral particle release (1.07×10(8) TCID(50)/ml±6.63×10(7)) and development of cytopathic effect (CPE). Differentiation of porcine bronchial epithelial cells was achieved at the air-liquid interface on collagen I coated 0.4μm polyester membranes. Morphology, expression of tubulin and occludin, the development of tight-junctions and cilia were similar to human bronchial epithelial cells. Infection with HAdV-C5 from the basolateral side resulted in release of infectious virus progeny (2.05×10(7) TCID(50)/ml±2.39×10(7)) to the apical surface as described recently in human bronchial epithelial cells, although complete CPE was not observed. Differentiated porcine bronchial epithelial cells hold promise as a novel method for studying the virulence and pathophysiology of pneumonia associated HAdV types.

  4. 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

    2010-11-03

    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.

  5. Short Palate, Lung, and Nasal Epithelial Clone–1 Is a Tightly Regulated Airway Sensor in Innate and Adaptive Immunity

    PubMed Central

    Britto, Clemente J.; Liu, Qing; Curran, David R.; Patham, Bhargavi; Dela Cruz, Charles S.

    2013-01-01

    Short palate, lung, and nasal epithelial clone–1 (SPLUNC1) is a protein abundantly expressed by the respiratory epithelium of the proximal lower respiratory tract, a site of great environmental exposure. Previous studies showed that SPLUNC1 exerts antimicrobial effects, regulates airway surface liquid and mucociliary clearance, and suppresses allergic airway inflammation. We studied SPLUNC1 to gain insights into its role in host defense. In the lower respiratory tract, concentrations of SPLUNC1 are high under basal conditions. In models of pneumonia caused by common respiratory pathogens, and in Th1-induced and Th2-induced airway inflammation, SPLUNC1 secretion is markedly reduced. Pathogen-associated molecular patterns and IFN-γ act directly on airway epithelial cells to inhibit SPLUNC1 mRNA expression. Thus, SPLUNC1 is quickly suppressed during infection, in response to an insult on the epithelial surface. These experiments highlight the finely tuned fluctuations of SPLUNC1 in response to exposures in the respiratory tract, and suggest that the loss of SPLUNC1 is a crucial feature of host defense across air-breathing animal species. PMID:23470624

  6. 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

  7. 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.

  8. 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.

  9. Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation

    PubMed Central

    Kale, Sagar L.; Agrawal, Komal; Gaur, Shailendra Nath; Arora, Naveen

    2017-01-01

    Protease allergens are known to enhance allergic inflammation but their exact role in initiation of allergic reactions at mucosal surfaces still remains elusive. This study was aimed at deciphering the role of serine protease activity of Per a 10, a major cockroach allergen in initiation of allergic inflammation at mucosal surfaces. We demonstrate that Per a 10 increases epithelial permeability by disruption of tight junction proteins, ZO-1 and occludin, and enhances the migration of Monocyte derived dendritic cell precursors towards epithelial layer as exhibited by trans-well studies. Per a 10 exposure also leads to secretion of IL-33, TSLP and intracellular Ca2+ dependent increase in ATP levels. Further, in vivo experiments revealed that Per a 10 administration in mice elevated allergic inflammatory parameters along with high levels of IL-33, TSLP, IL-1α and uric acid in the mice lungs. We next demonstrated that Per a 10 cleaves CD23 (low affinity IgE receptor) from the surface of PBMCs and purified B cells and CD25 (IL-2 receptor) from the surface of PBMCs and purified T cells in an activity dependent manner, which might favour Th2 responses. In conclusion, protease activity of Per a 10 plays a significant role in initiation of allergic airway inflammation at the mucosal surfaces. PMID:28198394

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

    PubMed Central

    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

  11. Cigarette smoke alters IL-33 expression and release in airway epithelial cells.

    PubMed

    Pace, Elisabetta; Di Sano, Caterina; Sciarrino, Serafina; Scafidi, Valeria; Ferraro, Maria; Chiappara, Giuseppina; Siena, Liboria; Gangemi, Sebastiano; Vitulo, Patrizio; Giarratano, Antonino; Gjomarkaj, Mark

    2014-09-01

    Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored. Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.

  12. 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

  13. Toluene diisocyanate exposure induces airway inflammation of bronchial epithelial cells via the activation of transient receptor potential melastatin 8

    PubMed Central

    Kim, Joo-Hee; Jang, Young-Sook; Jang, Seung-Hun; Jung, Ki-Suck; Kim, Seung-Hyun; Ye, Young-Min; Park, Hae-Sim

    2017-01-01

    Toluene diisocyanate (TDI) is the most important cause of occupational asthma (OA), and various pathogenic mechanisms have been suggested. Of these mechanisms, neurogenic inflammation is an important inducer of airway inflammation. Transient receptor potential melastatin 8 (TRPM8) is a well-established cold-sensing cation channel that is expressed in both neuronal cells and bronchial epithelial cells. A recent genome-wide association study of TDI-exposed workers found a significant association between the phenotype of TDI-induced OA and the single-nucleotide polymorphism rs10803666, which has been mapped to the TRPM8 gene. We hypothesized that TRPM8 located in airway epithelial cells may be involved in the pathogenic mechanisms of TDI-induced OA and investigated its role. Bronchial epithelial cells were treated with TDI in a dose- and time-dependent manner. The expression levels of TRPM8 mRNA and protein were determined by quantitative real-time polymerase chain reaction and western blotting. TDI-induced morphological changes in the cells were evaluated by immunocytochemistry. Alterations in the transcripts of inflammatory cytokines were examined in accordance with TRPM8 activation by TDI. TRPM8 expression at both the mRNA and protein levels was enhanced by TDI in airway epithelial cells. TRPM8 activation by TDI led to significant increases in the mRNA of interleukin (IL)-4, IL-13, IL-25 and IL-33. The increased expression of the cytokine genes by TDI was partly attenuated after treatment with a TRPM8 antagonist. TDI exposure induces increased expression of TRPM8 mRNA in airway epithelial cells coupled with enhanced expression of inflammatory cytokines, suggesting a novel role of TRPM8 in the pathogenesis of TDI-induced OA. PMID:28255167

  14. 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

  15. Ozone-induced airway epithelial cell death, the neurokinin-1 receptor pathway, and the postnatal developing lung

    PubMed Central

    Murphy, Shannon R.; Oslund, Karen L.; Hyde, Dallas M.; Miller, Lisa A.; Van Winkle, Laura S.

    2014-01-01

    Children are uniquely susceptible to ozone because airway and lung growth continue for an extensive period after birth. Early-life exposure of the rhesus monkey to repeated ozone cycles results in region-specific disrupted airway/lung growth, but the mediators and mechanisms are poorly understood. Substance P (SP), neurokinin-1 receptor (NK-1R); and nuclear receptor Nur77 (NR4A1) are signaling pathway components involved in ozone-induced cell death. We hypothesize that acute ozone (AO) exposure during postnatal airway development disrupts SP/NK-1R/Nur77 pathway expression and that these changes correlate with increased ozone-induced cell death. Our objectives were to 1) spatially define the normal development of the SP/NK-1R/Nur77 pathway in conducting airways; 2) compare how postnatal age modulates responses to AO exposure; and 3) determine how concomitant, episodic ozone exposure modifies age-specific acute responses. Male infant rhesus monkeys were assigned at age 1 mo to two age groups, 2 or 6 mo, and then to one of three exposure subgroups: filtered air (FA), FA+AO (AO: 8 h/day × 2 days), or episodic biweekly ozone exposure cycles (EAO: 8 h/day × 5 days/14-day cycle+AO). O3 = 0.5 ppm. We found that 1) ozone increases SP/NK-1R/Nur77 pathway expression in conducting airways, 2) an ozone exposure cycle (5 days/cycle) delivered early at age 2 mo resulted in an airway that was hypersensitive to AO exposure at the end of 2 mo, and 3) continued episodic exposure (11 cycles) resulted in an airway that was hyposensitive to AO exposure at 6 mo. These observations collectively associate with greater overall inflammation and epithelial cell death, particularly in early postnatal (2 mo), distal airways. PMID:25063800

  16. 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...

  17. 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

  18. 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.

  19. Synergism between rhinovirus infection and oxidant pollutant exposure enhances airway epithelial cell cytokine production.

    PubMed Central

    Spannhake, E William; Reddy, Sekhar P M; Jacoby, David B; Yu, Xiao-Ying; Saatian, Bahman; Tian, Jingyan

    2002-01-01

    Of the several factors believed to exacerbate asthmatic symptoms, air pollution and viral infections are considered to be particularly important. Although evidence indicates that each of these respiratory insults individually can increase asthma severity in susceptible individuals, we know little about the extent to which exposure to environmental oxidant pollutants can influence the course of respiratory viral infection and its associated inflammation. To investigate the interaction of these two stimuli within their common epithelial cell targets in the upper and lower respiratory tracks, we infected primary human nasal epithelial cells and cells of the BEAS-2B line grown at the air-liquid interface with human rhinovirus type 16 (RV16) and exposed them to NO2 (2.0 ppm) or O3 (0.2 ppm) for 3 hr. Independently, RV16, NO2, and O3 rapidly increased release of the inflammatory cytokine interleukin-8 through oxidant-dependent mechanisms. The combined effect of RV16 and oxidant ranged from 42% to 250% greater than additive for NO2 and from 41% to 67% for O3. We abrogated these effects by treating the cells with the antioxidant N-acetylcysteine. Surface expression of intercellular adhesion molecule 1 (ICAM-1) underwent additive enhancement in response to combined stimulation. These data indicate that oxidant pollutants can amplify the generation of proinflammatory cytokines by RV16-infected cells and suggest that virus-induced inflammation in upper and lower airways may be exacerbated by concurrent exposure to ambient levels of oxidants commonly encountered the indoor and outdoor environments. PMID:12117643

  20. 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

  1. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

    PubMed Central

    Baaske, Romina; Richter, Mandy; Möller, Nils; Ziesemer, Sabine; Eiffler, Ina; Müller, Christian; Hildebrandt, Jan-Peter

    2016-01-01

    Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla). This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins) activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L), which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin. PMID:27929417

  2. 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

  3. Dung biomass smoke activates inflammatory signaling pathways in human small airway epithelial cells.

    PubMed

    McCarthy, Claire E; Duffney, Parker F; Gelein, Robert; Thatcher, Thomas H; Elder, Alison; Phipps, Richard P; Sime, Patricia J

    2016-12-01

    Animal dung is a biomass fuel burned by vulnerable populations who cannot afford cleaner sources of energy, such as wood and gas, for cooking and heating their homes. Exposure to biomass smoke is the leading environmental risk for mortality, with over 4,000,000 deaths each year worldwide attributed to indoor air pollution from biomass smoke. Biomass smoke inhalation is epidemiologically associated with pulmonary diseases, including chronic obstructive pulmonary disease (COPD), lung cancer, and respiratory infections, especially in low and middle-income countries. Yet, few studies have examined the mechanisms of dung biomass smoke-induced inflammatory responses in human lung cells. Here, we tested the hypothesis that dung biomass smoke causes inflammatory responses in human lung cells through signaling pathways involved in acute and chronic lung inflammation. Primary human small airway epithelial cells (SAECs) were exposed to dung smoke at the air-liquid interface using a newly developed, automated, and reproducible dung biomass smoke generation system. The examination of inflammatory signaling showed that dung biomass smoke increased the production of several proinflammatory cytokines and enzymes in SAECs through activation of the activator protein (AP)-1 and arylhydrocarbon receptor (AhR) but not nuclear factor-κB (NF-κB) pathways. We propose that the inflammatory responses of lung cells exposed to dung biomass smoke contribute to the development of respiratory diseases.

  4. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

  5. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  6. 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.

  7. 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.

  8. Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α*S⃞

    PubMed Central

    Bove, Peter F.; Hristova, Milena; Wesley, Umadevi V.; Olson, Nels; Lounsbury, Karen M.; van der Vliet, Albert

    2008-01-01

    Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1α and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1α stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1α activation. Activation of HIF-1α by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1α mutant construct inhibited epithelial wound repair, implicating HIF-1α in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1α. Finally, NO-mediated inhibition of cell migration was associated with HIF-1α-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1α and p53, with potential consequences for epithelial repair and remodeling during airway inflammation. PMID

  9. Association of epithelial damage and signs of neutrophil mobilization in the airways during acute exacerbations of paediatric asthma

    PubMed Central

    Yoshihara, S; Yamada, Y; Abe, T; Lindén, A; Arisaka, O

    2006-01-01

    We examined whether epithelial damage is associated with mobilization of neutrophils or eosinophils in the airway lumen during acute exacerbations of paediatric asthma. Aspirated sputum samples were harvested from 65 paediatric patients (mean age 3·4 ± 0·4 years) during acute exacerbations of asthma. Patients with signs of infection were excluded. The presence of conglomerates of epithelial cells (i.e. ‘Creola bodies) in the aspirated sputum was utilized as a marker of epithelial damage. Among the paediatric asthma patients, 60% displayed Creola bodies (CrB+: n = 39) in their sputum samples whereas the remaining patients did not (CrB–: n = 26). CrB+ patients displayed more than a 20-fold increase in the concentration of the neutrophil-mobilizing cytokine interleukin (IL)-8 (pg/ml) and of the neutrophil product neutrophil elastase (NE, g/l), respectively, compared with CrB– patients (IL-8: 7468·2 ± 1953·6 versus 347·9 ± 72·6, P < 0·01; NE: 2072·4 ± 419·0 versus 438·5 ± 125·7, P < 0·01). Even though not statistically significant, a corresponding trend was observed for the relative number of sputum neutrophils. In contrast, the concentration of the eosinophil-mobilizing cytokine IL-5 and the esoinophil product ECP tended to be lower in CrB+ than in CrB– patients (P > 0·05). In conclusion, as indicated by the analysis of aspirated sputum, epithelial damage is associated with a locally enhanced chemotactic signal for and activity of neutrophils, but not eosinophils, during acute exacerbations of paediatric asthma. It remains to be determined whether these indirect signs of neutrophil mobilization in the airway lumen mirror an increased number of neutrophils in the surrounding airway tissue. PMID:16634793

  10. Inhibition of beta-defensin gene expression in airway epithelial cells by low doses of residual oil fly ash is mediated by vanadium.

    PubMed

    Klein-Patel, Marcia E; Diamond, Gill; Boniotto, Michele; Saad, Sherif; Ryan, Lisa K

    2006-07-01

    Poor ambient air quality is associated with increased morbidity and mortality, including respiratory infections. However, its effects on various host-defense mechanisms are poorly understood. This study utilized an in vitro model to study the effect of particulate matter (PM(2.5)) on one antimicrobial mechanism of host defense in the airway, beta-defensin-2 and its bovine homologue, tracheal antimicrobial peptide (TAP) induction in response to lipopolysaccharide (LPS) and IL-1beta. Our model utilized cultured primary bovine tracheal epithelial (BTE) cells and the human alveolar type II epithelial cell line, A549, treated with 0-20 microg/cm(2) residual oil fly ash (ROFA) for 6 h. The cells were then washed and stimulated for 18 h with 100 ng/ml LPS or for 6 h with 100 ng/ml IL-1beta. ROFA inhibited the LPS-induced increase in TAP mRNA and protein without inducing significant cytotoxicity. As little as 2.5 microg/cm(2) of ROFA inhibited LPS-induced TAP gene expression by 30%. The inhibitory activity was associated with the soluble fraction and not the washed particle. The activity in the leachate was attributed to vanadium, but not nickel or iron. SiO(2) and TiO(2) were utilized as controls and did not inhibit LPS induction of TAP gene expression in BTE. ROFA also inhibited the increase of IL-1beta-induced human beta-defensin-2, a homologue of TAP, in A549 cells. The results show that ROFA, V(2)O(5), and VOSO(4) inhibit the ability of airway epithelial cells to respond to inflammatory stimuli at low, physiologically relevant doses and suggest that exposure to these agents could result in an impairment of defense against airborne pathogens.

  11. Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis.

    PubMed Central

    Cheng, P W; Boat, T F; Cranfill, K; Yankaskas, J R; Boucher, R C

    1989-01-01

    Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface. Images PMID:2738159

  12. Capsular Polysaccharide of Mycoplasma ovipneumoniae Induces Sheep Airway Epithelial Cell Apoptosis via ROS-Dependent JNK/P38 MAPK Pathways

    PubMed Central

    Jiang, Zhongjia; Song, Fuyang; Li, Yanan; Xue, Di; Zhao, Ning; Zhang, Jiamei; Deng, Guangcun; Li, Min

    2017-01-01

    In an attempt to better understand the pathogen-host interaction between invading Mycoplasma ovipneumoniae (M. ovipneumoniae) and sheep airway epithelial cells, biological effects and possible molecular mechanism of capsular polysaccharide of M. ovipneumoniae (CPS) in the induction of cell apoptosis were explored using sheep bronchial epithelial cells cultured in air-liquid interface (ALI). The CPS of M. ovipneumoniae was first isolated and purified. Results showed that CPS had a cytotoxic effect by disrupting the integrity of mitochondrial membrane, accompanied with an increase of reactive oxygen species and decrease of mitochondrial membrane potential (ΔΨm). Of importance, the CPS exhibited an ability to induce caspase-dependent cell apoptosis via both intrinsic and extrinsic apoptotic pathways. Mechanistically, the CPS induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways. These findings provide the first evidence that CPS of M. ovipneumoniae induces a caspase-dependent apoptosis via both intrinsic and extrinsic apoptotic pathways in sheep bronchial epithelial cells, which may be mainly attributed by a ROS-dependent JNK and p38 MAPK signaling pathways. PMID:28367270

  13. Effects of transforming growth factor-[beta] and budesonide on mitogen-activated protein kinase activation and apoptosis in airway epithelial cells.

    PubMed

    Pelaia, Girolamo; Cuda, Giovanni; Vatrella, Alessandro; Fratto, Donatella; Grembiale, Rosa D; Tagliaferri, Pierosandro; Maselli, Rosario; Costanzo, Francesco S; Marsico, Serafino A

    2003-07-01

    Airway epithelial cells play a central role in the inflammatory, apoptotic, and remodeling processes associated with asthma. Within this context, a key function is exerted by transforming growth factor-beta (TGF-beta), whose biological effects are mediated at least in part by mitogen-activated protein kinases (MAPKs). The aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the effects of TGF-beta (10 ng/ml) on both MAPK activation and apoptosis, in the presence or absence of a pretreatment with budesonide (10-8 M). MAPK activation was detected by Western blotting, using anti-phospho-MAPK monoclonal antibodies, which specifically recognize the phosphorylated, active forms of these enzymes. Apoptosis was assayed by caspase-3 activation and fluorescence microscopy, using annexin-V (An-V) and propidium iodide (PI) as markers of cell death. Our results show that TGF-beta induced a marked ( reverse similar 9-fold) increase in p38 MAPK phosphorylation, and also dramatically enhanced cell death, which was completely prevented by specific MAPK inhibitors. Both MAPK activation and apoptosis were effectively inhibited by budesonide (BUD), thereby suggesting that the powerful antiapoptotic action of inhaled glucocorticoids may be very important for their protective role against epithelial injury, which represents a key pathogenic event in asthma.

  14. 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.

  15. 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.

  16. Functional activity of L-carnitine transporters in human airway epithelial cells.

    PubMed

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

    2016-02-01

    Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects.

  17. 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.

  18. MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis

    PubMed Central

    Perdomo, Catalina; Campbell, Joshua D.; Gerrein, Joseph; Tellez, Carmen S.; Garrison, Carly B.; Walser, Tonya C.; Drizik, Eduard; Si, Huiqing; Gower, Adam C.; Vick, Jessica; Anderlind, Christina; Jackson, George R.; Mankus, Courtney; Schembri, Frank; O’Hara, Carl; Gomperts, Brigitte N.; Dubinett, Steven M.; Hayden, Patrick; Belinsky, Steven A.; Lenburg, Marc E.; Spira, Avrum

    2013-01-01

    Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis. PMID:24158479

  19. 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.

  20. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  1. Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed

    PubMed Central

    Leonard, Vincent H.J.; Sinn, Patrick L.; Hodge, Gregory; Miest, Tanner; Devaux, Patricia; Oezguen, Numan; Braun, Werner; McCray, Paul B.; McChesney, Michael B.; Cattaneo, Roberto

    2008-01-01

    The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV. PMID:18568079

  2. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  3. Human airway epithelial cell responses to Neisseria lactamica and purified porin via Toll-like receptor 2-dependent signaling.

    PubMed

    Liu, Xiuping; Wetzler, Lee M; Nascimento, Laura Oliveira; Massari, Paola

    2010-12-01

    The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

  4. Interferon-γ promotes double-stranded RNA-induced TLR3-dependent apoptosis via upregulation of transcription factor Runx3 in airway epithelial cells.

    PubMed

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

    2016-12-01

    Viral respiratory tract infections are the most common illness in humans. Infection of the respiratory viruses results in accumulation of viral replicative double-stranded RNA (dsRNA), which is one of the important components of infecting viruses for the induction of lung epithelial cell apoptosis and innate immune response, including the production of interferon (IFN). In the present study, we have investigated the regulation of dsRNA-induced airway epithelial cell apoptosis by IFN. We found that transcription factor Runx3 was strongly induced by type-II IFNγ, slightly by type-III IFNλ, but essentially not by type-I IFNα in airway epithelial cells. IFNγ-induced expression of Runx3 was predominantly mediated by JAK-STAT1 pathway and partially by NF-κB pathway. Interestingly, Runx3 can be synergistically induced by IFNγ with a synthetic analog of viral dsRNA polyinosinic-polycytidylic acid [poly(I:C)] or tumor necrosis factor-α (TNFα) through both JAK-STAT1 and NF-κB pathways. We further found that dsRNA poly(I:C)-induced apoptosis of airway epithelial cells was mediated by dsRNA receptor toll-like receptor 3 (TLR3) and was markedly augmented by IFNγ through the enhanced expression of TLR3 and subsequent activation of both extrinsic and intrinsic apoptosis pathways. Last, we demonstrated that upregulation of Runx3 by IFNγ promoted TLR3 expression, thus amplifying the dsRNA-induced apoptosis in airway epithelial cells. These novel findings indicate that IFNγ promotes dsRNA-induced TLR3-dependent apoptosis via upregulation of transcription factor Runx3 in airway epithelial cells. Findings from our study may provide new insights into the regulation of airway epithelial cell apoptosis by IFNγ during viral respiratory tract infection.

  5. Epithelial mesenchymal transition (EMT) and non-small cell lung cancer (NSCLC): a mutual association with airway disease.

    PubMed

    Mahmood, Malik Quasir; Ward, Chris; Muller, Hans Konrad; Sohal, Sukhwinder Singh; Walters, Eugene Haydn

    2017-03-01

    NSCLC is a leading cause of morbidity and mortality worldwide. It includes adeno- and squamous cell carcinoma. In the background, COPD and smoking play a vital role in development of NSCLC. Local progression and metastasis of NSCLC has been associated with various mechanisms, but in particular by a process called epithelial mesenchymal transition (EMT), which is implicated in COPD pathogenesis. In this study, we have investigated whether expression of EGFR (activation marker) and S100A4, vimentin and N-cadherin (as EMT) is different both in central and leading edge of NSCLC and to what extent related to EMT activity of both small and large airways, stage and differentiation of NSCLC. We have investigated EMT biomarkers (S100A4, vimentin, and N-cadherin), an epithelial activation marker (EGFR) and a vascularity marker (Type-IV collagen) in surgically resected tissue from patients with NSCLC (adeno- and squamous cell carcinoma), and compared them with expression in the corresponding non-tumorous airways. EGFR, S100A4, vimentin, N-cadherin expression was higher in tumor cells located at the peripheral leading edge of NSCLC when compared with centrally located tumor cells of same subjects (P < 0.01). Type-IV collagen-expressing blood vessels were also more at the leading edge in comparison with central parts of NSCLC. EGFR and S100A4 expression was related to differentiation status (P < 0.05) and TNM stage (P < 0.05) of NSCLC. Moreover, EMT markers in the leading edge were significantly related to airway EMT activity, while peripheral edge vascularity of squamous cell carcinoma only was significantly related to large airway Rbm vascularity (P < 0.05). EGFR- and EMT-related protein expression was markedly high in the peripheral leading edge of NSCLCs and related to tumor characteristics associated with poor prognosis. The relationships between EMT-related tumor biomarker expression and those in the airway epithelium and Rbm provide a background for utility of

  6. Long-term exposure to diesel exhaust enhances antigen-induced eosinophilic inflammation and epithelial damage in the murine airway.

    PubMed

    Ichinose, T; Takano, H; Miyabara, Y; Sagai, M

    1998-07-01

    The histopathologic changes in the murine airway induced by long-term exposure to diesel exhaust (DE), ovalbumin (OA), or both were investigated. The relationship between the histopathologic appearances in the airway and immunoglobulin production or local cytokine levels in the lungs was also studied. ICR mice were exposed to clean air or DE at a soot concentrations of 0.3, 1.0, or 3.0 mg/m3 for 34 weeks. Fifteen weeks after exposure to DE, mice were sensitized intraperitoneally with 10 micrograms of OA and challenged by an aerosol of 1% OA six times at 3-week intervals during the last 18 weeks of the exposure. DE exposure caused a dose-dependent increase of nonciliated cell proliferation and epithelial cell hypertrophy in the airway, but showed no effect on goblet cell proliferation in the bronchial epithelium and eosinophil recruitment in the submucosa of the airway. OA treatment induced very slight changes in goblet cell proliferation and eosinophil recruitment. The combination of OA and DE exposure produced dose-dependent increases of goblet cells and eosinophils, in addition to further increases of the typical changes induced by DE. OA treatment induced OA-specific IgG1 and IgE production in plasma, whereas the adjuvant effects of DE exposure on immunoglobulin production were not observed. Inhalation of DE led to increased levels of IL-5 protein in the lung at a soot concentration of 1.0 and 3.0 mg/m3 with OA, although these increases did not reach statistical significance. We conclude that the combination of antigen and chronic exposure to DE produces increased eosinophilic inflammation, and cell damage to the epithelium may depend on the degree of eosinophilic inflammation in the airway.

  7. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  8. Glucocorticoid Insensitivity in Virally Infected Airway Epithelial Cells Is Dependent on Transforming Growth Factor-β Activity

    PubMed Central

    Radwan, Asmaa; Keenan, Christine R.; Langenbach, Shenna Y.; Li, Meina; Londrigan, Sarah L.; Gualano, Rosa C.; Stewart, Alastair G.

    2017-01-01

    Asthma and chronic obstructive pulmonary disease (COPD) exacerbations are commonly associated with respiratory syncytial virus (RSV), rhinovirus (RV) and influenza A virus (IAV) infection. The ensuing airway inflammation is resistant to the anti-inflammatory actions of glucocorticoids (GCs). Viral infection elicits transforming growth factor-β (TGF-β) activity, a growth factor we have previously shown to impair GC action in human airway epithelial cells through the activation of activin-like kinase 5 (ALK5), the type 1 receptor of TGF-β. In the current study, we examine the contribution of TGF-β activity to the GC-resistance caused by viral infection. We demonstrate that viral infection of human bronchial epithelial cells with RSV, RV or IAV impairs GC anti-inflammatory action. Poly(I:C), a synthetic analog of double-stranded RNA, also impairs GC activity. Both viral infection and poly(I:C) increase TGF-β expression and activity. Importantly, the GC impairment was attenuated by the selective ALK5 (TGFβRI) inhibitor, SB431542 and prevented by the therapeutic agent, tranilast, which reduced TGF-β activity associated with viral infection. This study shows for the first time that viral-induced glucocorticoid-insensitivity is partially mediated by activation of endogenous TGF-β. PMID:28046097

  9. 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

  10. Rhinovirus-Induced Barrier Dysfunction in Polarized Airway Epithelial Cells Is Mediated by NADPH Oxidase 1▿

    PubMed Central

    Comstock, Adam T.; Ganesan, Shyamala; Chattoraj, Asamanja; Faris, Andrea N.; Margolis, Benjamin L.; Hershenson, Marc B.; Sajjan, Umadevi S.

    2011-01-01

    Previously, we showed that rhinovirus (RV), which is responsible for the majority of common colds, disrupts airway epithelial barrier function, as evidenced by reduced transepithelial resistance (RT), dissociation of zona occludins 1 (ZO-1) from the tight junction complex, and bacterial transmigration across polarized cells. We also showed that RV replication is required for barrier function disruption. However, the underlying biochemical mechanisms are not known. In the present study, we found that a double-stranded RNA (dsRNA) mimetic, poly(I:C), induced tight junction breakdown and facilitated bacterial transmigration across polarized airway epithelial cells, similar to the case with RV. We also found that RV and poly(I:C) each stimulated Rac1 activation, reactive oxygen species (ROS) generation, and Rac1-dependent NADPH oxidase 1 (NOX1) activity. Inhibitors of Rac1 (NSC23766), NOX (diphenylene iodonium), and NOX1 (small interfering RNA [siRNA]) each blocked the disruptive effects of RV and poly(I:C) on RT, as well as the dissociation of ZO-1 and occludin from the tight junction complex. Finally, we found that Toll-like receptor 3 (TLR3) is not required for either poly(I:C)- or RV-induced reductions in RT. Based on these results, we concluded that Rac1-dependent NOX1 activity is required for RV- or poly(I:C)-induced ROS generation, which in turn disrupts the barrier function of polarized airway epithelia. Furthermore, these data suggest that dsRNA generated during RV replication is sufficient to disrupt barrier function. PMID:21507984

  11. Type II alveolar epithelial cell in vitro culture in aerobiosis.

    PubMed

    Aerts, C; Voisin, C; Wallaert, B

    1988-08-01

    A method of Type II alveolar epithelial cell culture in aerobiosis has been developed. Isolation of Type II cells was performed by digesting guinea-pig lung tissue with crude trypsin and elastase and using discontinuous Percoll density gradients. The Type II cells, as identified by light and electron microscopy, were cultured in aerobiosis for up to six days, in direct contact with the atmosphere in conditions mimicking those present in the lower respiratory tract. Significant activities of cellular superoxide dismutase (SOD), manganese dependent superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GSH-Px) were found at the time of isolation. In contrast, cell glutathione content varied widely from one experiment to another. Changes of antioxidant enzymes were evaluated during cell culture in aerobiosis. SOD, Mn-SOD and catalase were significantly decreased after three days but were not significantly different between a three day and six day culture. Antioxidant changes did not influence the cell culture. In marked contrast, decrease in cell glutathione was associated with rapid cell death, whereas good cell survival was obtained at high levels of cell glutathione. Cell culture in aerobiosis will permit a precise evaluation of the effects of gases, particularly oxidant gases, on a primary culture of Type II alveolar epithelial cells.

  12. 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

  13. 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.

  14. 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}.

  15. 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...

  16. 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...

  17. 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...

  18. Fetal Exposure of Rhesus Macaques to Bisphenol A Alters Cellular Development of the Conducting Airway by Changing Epithelial Secretory Product Expression

    PubMed Central

    Murphy, Shannon R.; Boetticher, Miriam V.; VandeVoort, Catherine A.

    2013-01-01

    Background: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium. Objective: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque. Methods: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection. Results: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased. Conclusions: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure. PMID:23757601

  19. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    PubMed

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  20. 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...

  1. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  2. 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.

  3. Wood smoke enhances cigarette smoke-induced inflammation by inducing the aryl hydrocarbon receptor repressor in airway epithelial cells.

    PubMed

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

    2015-03-01

    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/m(3) WS for 2 h/d, to 250 mg/m(3) 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.

  4. SIGIRR inhibits toll-like receptor 4, 5, 9-mediated immune responses in human airway epithelial cells.

    PubMed

    Zhang, Chun; Wu, Xueling; Zhao, Yunfeng; Deng, Zhaoxia; Qian, Guisheng

    2011-01-01

    Human airway epithelial cells (HAEC) may contribute to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) through toll-like receptors (TLRs)-mediated molecular mechanisms. TLRs exist on the surface of HAEC where binding to their cognate ligands initiates airway inflammation. Single immunoglobulin interleukin-1 receptor-related protein (SIGIRR) is a member of the toll-interleukin-1 receptor (TIR) family that can negatively modulate the immune response. We carried out studies to characterize SIGIRR modulation of TLR-mediated immune response in HAEC and to define its mechanisms of action. Following treatment with various concentrations of LPS, flagellin and CpG DNA, the levels of cognate TLRs 4, 5, and 9 were measured in the supernatants of HAEC over-expressing the SIGIRR molecule. Moreover, the interaction of the TLR adaptor myeloid differentiation factor 88 (MyD88) with SIGIRR in response to LPS-, flagellin- and CpG DNA-stimulation was examined by co-immunoprecipitation. The findings from this study revealed that overexpression of SIGIRR in HAEC stimulated by LPS, flagellin or CpG DNA resulted in attenuated production of the inflammatory mediators IL-6 and TNF-α. This attenuation was not the result of decreased expression of TLR4, 5 or 9, but rather a sequestration of MyD88 to the TLRs. In conclusion, SIGIRR can inhibit TLR4, 5, and 9-mediated immune responses in HAEC and may be a valuable therapeutic target for the prevention of ALI/ARDS.

  5. T cell-derived IL-17 mediates epithelial changes in the airway and drives pulmonary neutrophilia1

    PubMed Central

    Fogli, Laura K.; Sundrud, Mark S.; Goel, Swati; Bajwa, Sofia; Jensen, Kari; Derudder, Emmanuel; Sun, Amy; Coffre, Maryaline; Uyttenhove, Catherine; van Snick, Jacques; Schmidt-Supprian, Marc; Rao, Anjana; Grunig, Gabriele; Durbin, Joan; Casola, Stefano S.; Rajewsky, Klaus; Koralov, Sergei B.

    2013-01-01

    Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. Here we describe a novel mouse model of spontaneous IL-17-driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We utilized this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases. PMID:23966625

  6. 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.

  7. 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

  8. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

  9. 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

  10. In vivo imaging of tracheal epithelial cells in mice during airway regeneration.

    PubMed

    Kim, Jun Ki; Vinarsky, Vladimir; Wain, John; Zhao, Rui; Jung, Keehoon; Choi, Jinwoo; Lam, Adam; Pardo-Saganta, Ana; Breton, Sylvie; Rajagopal, Jayaraj; Yun, Seok Hyun

    2012-12-01

    Many human lung diseases, such as asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans, and cystic fibrosis, are characterized by changes in the cellular composition and architecture of the airway epithelium. Intravital fluorescence microscopy has emerged as a powerful approach in mechanistic studies of diseases, but it has been difficult to apply this tool for in vivo respiratory cell biology in animals in a minimally invasive manner. Here, we describe a novel miniature side-view confocal probe capable of visualizing the epithelium in the mouse trachea in vivo at a single-cell resolution. We performed serial real-time endotracheal fluorescence microscopy in live transgenic reporter mice to view the three major cell types of the large airways, namely, basal cells, Clara cells, and ciliated cells. As a proof-of-concept demonstration, we monitored the regeneration of Clara cells over 18 days after a sulfur dioxide injury. Our results show that in vivo tracheal microscopy offers a new approach in the study of altered, regenerating, or metaplastic airways in animal models of lung diseases.

  11. Phenotypic and physiologic variability in nasal epithelium cultured from smokers and non-smokers exposed to secondhand tobacco smoke

    EPA Science Inventory

    The emergence of air-liquid interface (ALI) culturing of mammalian airway epithelium is a recent innovation for experimental modeling of airway epithelial development, function, and pathogenic mechanisms associated with infectious agent and irritant exposure. This construct provi...

  12. Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.

    PubMed

    Beaudoin, Trevor; Lafayette, Shantelle; Nguyen, Dao; Rousseau, Simon

    2012-11-09

    The presence of the mucoid phenotype of Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis. As CF lung disease results from chronic infection leading to airway inflammation, we determined whether the switch to a mucoid phenotype by P. aeruginosa has an impact on the inflammatory response of airway epithelial cells. Exposure of airway epithelial cells to non-mucoid and mucoid P. aeruginosa-derived material leads to p38α MAPK activation, a key protein kinase involved in transmitting inflammatory signals. However, while the non-mucoid strain PAO1 activates p38α MAPK pathway solely via TLR5, the mucoid strain PACF508 activates p38α MAPK via both TLR5 and TLR2. Inactivation of mucA (the gene responsible for the mucoid phenotype) in PAO1 leads to p38α MAPK activation by both TLR2 and TLR5, as observed in the clinical mucoid isolate PACF508. Therefore, the switch to mucoid phenotype may contribute to more inflammation via TLR2 activation in addition to TLR5. Our findings highlight an important and under recognized role for TLR2 in the response of airway epithelial cells to infection.

  13. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    PubMed

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

  14. Characterising the mechanism of airway smooth muscle β2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells.

    PubMed

    Van Ly, David; Faiz, Alen; Jenkins, Christine; Crossett, Ben; Black, Judith L; McParland, Brent; Burgess, Janette K; Oliver, Brian G G

    2013-01-01

    Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2

  15. Characterising the Mechanism of Airway Smooth Muscle β2 Adrenoceptor Desensitization by Rhinovirus Infected Bronchial Epithelial Cells

    PubMed Central

    Van Ly, David; Faiz, Alen; Jenkins, Christine; Crossett, Ben; Black, Judith L.; McParland, Brent; Burgess, Janette K.; Oliver, Brian G. G.

    2013-01-01

    Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2

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

    PubMed Central

    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

  17. 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...

  18. 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.

  19. Regulation of cytokine production in human alveolar macrophages and airway epithelial cells in response to ambient air pollution particles: Further mechanistic studies

    SciTech Connect

    Becker, Susanne; Mundandhara, Sailaja; Devlin, Robert B.; Madden, Michael . E-mail: madden.michael@epa.gov

    2005-09-01

    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 endpoints of inflammation, and oxidant stress. Separation of Chapel Hill PM 10 into fine and coarse size particles revealed that the main proinflammatory response (TNF, IL-6, COX-2) in AM was driven by material present in the coarse PM, containing 90-95% of the stimulatory material in PM10. The particles did not affect expression of hemoxygenase-1 (HO-1), a sensitive marker of oxidant stress. Primary cultures of normal human bronchial epithelial cells (NHBE) also responded to the coarse fraction with higher levels of IL-8 and COX-2, than induced by fine or ultrafine PM. All size PM induced oxidant stress in NHBE, while fine PM induced the highest levels of HO-1 expression. The production of cytokines in AM by both coarse and fine particles was blocked by the toll like receptor 4 (TLR4) antagonist E5531 involved in the recognition of LPS and Gram negative bacteria. The NHBE were found to recognize coarse and fine PM through TLR2, a receptor with preference for recognition of Gram positive bacteria. Compared to ambient PM, diesel PM induced only a minimal cytokine response in both AM and NHBE. Instead, diesel suppressed LPS-induced TNF and IL-8 release in AM. Both coarse and fine ambient air PM were also found to inhibit LPS-induced TNF release while silica, volcanic ash or carbon black had no inhibitory effect. Diesel particles did not affect cytokine mRNA induction nor protein accumulation but interfered with the release of cytokine from the cells. Ambient coarse and fine PM, on the other hand, inhibited both mRNA induction and protein production. Exposure to coarse and fine PM decreased the expression of TLR4 in the macrophages. Particle-induced decrease in TLR4 and hyporesponsiveness to LPS

  20. Electrical properties of dog tracheal epithelial cells grown in monolayer culture.

    PubMed

    Coleman, D L; Tuet, I K; Widdicombe, J H

    1984-03-01

    Epithelial cells from dog trachea, when grown in tissue culture, formed confluent monolayers 5-6 days after plating. After 8-10 days, domes [mean diam 356 +/- (SE) 53 micron] appeared in monolayers grown in collagen-coated flasks. When grown on polycarbonate filters coated with collagen, a detectable resistance (greater than 5 omega X cm2) and transepithelial potential difference (PD) (greater than 0.1 mV) developed 6 days after plating and increased to approximately 15 omega X cm2 and 15 mV at 10 days. Serosal ouabain (10(-4) M) abolished PD and short-circuit current (Isc). Luminal ouabain had no effect. Luminal amiloride (10(-4) M) and serosal bumetanide (10(-4) M) each decreased PD and Isc. However, a combination of both of these drugs did not abolish Isc. Isoproterenol (10(-5) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), vasoactive intestinal peptide (10(-7) M), prostaglandin (PG) E2 (10(-5) M), PGF2 alpha (10(-5) M), and bradykinin (10(-5) M) each increased PD and Isc. Thus these monolayer cultures maintain electrical properties resembling those of the original tissue. This preparation may prove useful for the study of water and ion transport by airway epithelia.

  1. Differentiation of cultured epithelial cells: response to toxic agents.

    PubMed Central

    Rice, R H; LaMontagne, A D; Petito, C T; Rong, X H

    1989-01-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents. Images FIGURE 1. FIGURE 4. PMID:2466642

  2. Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells.

    PubMed

    Yasukawa, Atsushi; Hosoki, Koa; Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C

    2013-01-01

    Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

  3. 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.

  4. Distinct roles of short and long thymic stromal lymphopoietin isoforms in house dust mite-induced asthmatic airway epithelial barrier disruption

    PubMed Central

    Dong, Hangming; Hu, Yahui; Liu, Laiyu; Zou, Mengchen; Huang, Chaowen; Luo, Lishan; Yu, Changhui; Wan, Xuan; Zhao, Haijin; Chen, JiaLong; Xie, Zhefan; Le, Yanqing; Zou, Fei; Cai, Shaoxi

    2016-01-01

    Loss of airway epithelial integrity contributes significantly to asthma pathogenesis. Thymic stromal lymphopoietin (TSLP) may have dual immunoregulatory roles. In inflammatory disorders of the bowel, the long isoform of TSLP (lfTSLP) promotes inflammation while the short isoform (sfTSLP) inhibits inflammation. We hypothesize that lfTSLP contributes to house dust mite (HDM)-induced airway epithelial barrier dysfunction and that synthetic sfTSLP can prevent these effects. In vitro, airway epithelial barrier function was assessed by monitoring transepithelial electrical resistance, fluorescent-dextran permeability, and distribution of E-cadherin and β-catenin. In vivo, BALB/c mice were exposed to HDM by nasal inhalation for 5 consecutive days per week to establish an asthma model. sfTSLP and 1α,25-Dihydroxyvitamin D3 (1,25D3) were administered 1 h before HDM exposure. After 8 weeks, animal lung function tests and pathological staining were performed to evaluate asthma progression. We found that HDM and lfTSLP impaired barrier function. Treatment with sfTSLP and 1,25D3 prevented HDM-induced airway epithelial barrier disruption. Moreover, sfTSLP and 1,25D3 treatment ameliorated HDM-induced asthma in mice. Our data emphasize the importance of the different expression patterns and biological properties of sfTSLP and lfTSLP. Moreover, our results indicate that sfTSLP and 1,25D3 may serve as novel therapeutic agents for individualized treatment of asthma. PMID:27996052

  5. Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion

    PubMed Central

    Garnett, James Peter; Kalsi, Kameljit K.; Sobotta, Mirko; Bearham, Jade; Carr, Georgina; Powell, Jason; Brodlie, Malcolm; Ward, Christopher; Tarran, Robert; Baines, Deborah L.

    2016-01-01

    The cystic fibrosis (CF) airway surface liquid (ASL) provides a nutrient rich environment for bacterial growth including elevated glucose, which together with defective bacterial killing due to aberrant HCO3− transport and acidic ASL, make the CF airways susceptible to colonisation by respiratory pathogens such as Pseudomonas aeruginosa. Approximately half of adults with CF have CF related diabetes (CFRD) and this is associated with increased respiratory decline. CF ASL contains elevated lactate concentrations and hyperglycaemia can also increase ASL lactate. We show that primary human bronchial epithelial (HBE) cells secrete lactate into ASL, which is elevated in hyperglycaemia. This leads to ASL acidification in CFHBE, which could only be mimicked in non-CF HBE following HCO3− removal. Hyperglycaemia-induced changes in ASL lactate and pH were exacerbated by the presence of P. aeruginosa and were attenuated by inhibition of monocarboxylate lactate-H+ co-transporters (MCTs) with AR-C155858. We conclude that hyperglycaemia and P. aeruginosa induce a metabolic shift which increases lactate generation and efflux into ASL via epithelial MCT2 transporters. Normal airways compensate for MCT-driven H+ secretion by secreting HCO3−, a process which is dysfunctional in CF airway epithelium leading to ASL acidification and that these processes may contribute to worsening respiratory disease in CFRD. PMID:27897253

  6. Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H(+) secretion.

    PubMed

    Garnett, James Peter; Kalsi, Kameljit K; Sobotta, Mirko; Bearham, Jade; Carr, Georgina; Powell, Jason; Brodlie, Malcolm; Ward, Christopher; Tarran, Robert; Baines, Deborah L

    2016-11-29

    The cystic fibrosis (CF) airway surface liquid (ASL) provides a nutrient rich environment for bacterial growth including elevated glucose, which together with defective bacterial killing due to aberrant HCO3(-) transport and acidic ASL, make the CF airways susceptible to colonisation by respiratory pathogens such as Pseudomonas aeruginosa. Approximately half of adults with CF have CF related diabetes (CFRD) and this is associated with increased respiratory decline. CF ASL contains elevated lactate concentrations and hyperglycaemia can also increase ASL lactate. We show that primary human bronchial epithelial (HBE) cells secrete lactate into ASL, which is elevated in hyperglycaemia. This leads to ASL acidification in CFHBE, which could only be mimicked in non-CF HBE following HCO3(-) removal. Hyperglycaemia-induced changes in ASL lactate and pH were exacerbated by the presence of P. aeruginosa and were attenuated by inhibition of monocarboxylate lactate-H(+) co-transporters (MCTs) with AR-C155858. We conclude that hyperglycaemia and P. aeruginosa induce a metabolic shift which increases lactate generation and efflux into ASL via epithelial MCT2 transporters. Normal airways compensate for MCT-driven H(+) secretion by secreting HCO3(-), a process which is dysfunctional in CF airway epithelium leading to ASL acidification and that these processes may contribute to worsening respiratory disease in CFRD.

  7. Use of primary cultures of human bronchial epithelial cells isolated from cystic fibrosis patients for the pre-clinical testing of CFTR modulators.

    PubMed

    Neuberger, Timothy; Burton, Bill; Clark, Heather; Van Goor, Fredrick

    2011-01-01

    The use of human bronchial epithelial (HBE) cell cultures derived from the bronchi of CF patients offers the opportunity to study the effects of CFTR correctors and potentiators on CFTR function and epithelial cell biology in the native pathological environment. Cultured HBE cells derived from CF patients exhibit many of the morphological and functional characteristics believed to be associated with CF airway disease in vivo, including abnormal ion and fluid transport leading to dehydration of the airway surface and the loss of cilia beating. In addition, they can be generated in sufficient quantities to support routine lab testing of compound potency and efficacy and retain reproducible levels of CFTR function over time. Here we describe the development and validation of the CF HBE pharmacology model and its use to characterize, optimize, and select clinical candidates. It is expected that the pre-clinical testing of CFTR potentiators and correctors using epithelial cell cultures derived from CF patients will help to increase their likelihood of clinical efficacy.

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

    PubMed

    Ferguson, Timothy E G; Reihill, James A; 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.

  9. 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

  10. 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.

  11. Store-operated CRAC channels regulate PAR2-activated Ca2+ signaling and cytokine production in airway epithelial cells

    PubMed Central

    Jairaman, Amit; Yamashita, Megumi; Schleimer, Robert P.; Prakriya, Murali

    2015-01-01

    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 Ca2+ is likely a critical but poorly understood event. Here, we show that Ca2+ release-activated Ca2+ (CRAC) channels encoded by STIM1 and Orai1 are a major route of Ca2+ entry in primary human AECs and drive the Ca2+ elevations seen in response to PAR2 activation. Activation of CRAC channels induces the production of several key inflammatory mediators from AECs including TSLP, IL-6 and PGE2, in part through stimulation of gene expression via NFAT (nuclear factor of activated T-cells). 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 Ca2+ influx in AECs and a vital checkpoint for the induction of PAR2-induced proinflammatory cytokines. PMID:26238490

  12. In vitro methods to culture primary human breast epithelial cells.

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  13. Interaction of platelet-activating factor with cultured guinea pig tracheal epithelial cells.

    PubMed Central

    Churchill, L; Chilton, F H; Proud, D

    1991-01-01

    The present study has examined the interaction of platelet-activating factor (PAF) with cultured guinea pig tracheal epithelial cells (GTE). PAF stimulated GTE to release endogenous arachidonic acid and metabolize it to prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Prostanoid production by GTE in response to PAF was dose-dependent (0.1-100 nM) and was maximal within 5 min. PGE2 and PGF2 alpha levels increased by 3.3 +/- 0.8 and 3.2 +/- 0.6 ng/10(6) cells respectively over basal levels in response to 100 nM-PAF. The ability of GTE to synthesize and/or catabolize PAF was also examined. GTE readily incorporated [3H]acetate into a product which migrated on t.l.c. with PAF. However, further characterization of this product suggested that label had not been incorporated into PAF, but rather that it was incorporated into another lipid product with chromatographic characteristics similar to those of PAF. In contrast, GTE readily metabolized PAF to inactive products. When [3H]PAF was incubated with GTE, 50% of the total [3H]PAF added was catabolized in approx. 15 min. The major route of catabolism of PAF by GTE was the deacetylation-reacylation pathway, which yielded 1-O-[3H]alkyl-2-acyl-sn-glycerophosphocholine. Determination of the nature of the long-chain acyl group incorporated into the sn-2 position of the newly synthesized products revealed that oleic and linoleic acids were the major fatty acids present. Taken together, these results suggest that respiratory epithelial cells respond to stimulation by PAF with enhanced production of PGE2 and PGF2 alpha, and also have the capacity to modulate inflammatory reactions in the airways by their ability to degrade this potent inflammatory mediator. Images Fig. 1. PMID:2064601

  14. Porcine Nasal Epithelial Cultures for Studies of Cystic Fibrosis Sinusitis

    PubMed Central

    Dean, Nichole; Ranganath, Neel K.; Jones, Brandon; Zhang, Shaoyan; Skinner, Daniel; Rowe, Steven M.; Sorscher, Eric J.; Woodworth, Bradford A.

    2014-01-01

    Background Transgenic cystic fibrosis (CF) murine models do not develop spontaneous lung or sinus disease, two major causes of morbidity in human CF patients. Because of these limitations, transgenic CFTR−/− pigs have been developed and are currently being characterized. These CF animal models have phenotypes closely resembling that of human CF subjects. The objectives of the current study were to develop primary porcine nasal epithelial (PNE) cultures and evaluate their usefulness as a means to investigate sinonasal transepithelial transport and CFTR function. Methods PNE derived from the septum or turbinates of CFTR+/+ and CFTR−/− pigs were cultured at an air-liquid interface to confluence and full differentiation. Epithelial monolayers were mounted in Ussing chambers to investigate pharmacologic manipulation of ion transport. Ciliary beat frequency (CBF) and scanning electron microscopy of monolayers were used to indicate degree of ciliation and cell differentiation. Results Stimulation of CFTR-mediated anion transport(ΔIsc in μA/cm2) was significantly greater in epithelia derived from the septum when compared to turbinates(33.04+/−1.17 vs. 18.9+/−0.73;p<0.05). cAMP-activated Cl− secretion was absent in CFTR−/− and present in CFTR+/+ epithelia. Calcium-activated Cl− (CaCC) secretion was increased in CF, however, overall Cl− transport through CaCCs was very low. Degree of ciliation (90%) and CBF were similar between groups. Discussion Septal PNE exhibit a robust ion transport phenotype and indicate CFTR−/− sinus disease could be attributable to diminished alternative pathways for Cl− transport. Overall, PNE have similarities to human respiratory epithelia not demonstrated in murine cells and represent useful in vitro models for studying CF sinus disease. PMID:24733748

  15. Ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Nadine, Lusamba Kalonji; Ota, Chiharu; Kubo, Hiroshi; Nagatomi, Ryoichi

    2014-04-01

    The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.

  16. Mechanosensitive ATP Release Maintains Proper Mucus Hydration of Airways

    PubMed Central

    Button, Brian; Okada, Seiko F.; Frederick, Charles Brandon; Thelin, William R.; Boucher, Richard C.

    2013-01-01

    The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal auto-crine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis. PMID:23757023

  17. Mechanosensitive ATP release maintains proper mucus hydration of airways.

    PubMed

    Button, Brian; Okada, Seiko F; Frederick, Charles Brandon; Thelin, William R; Boucher, Richard C

    2013-06-11

    The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal autocrine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis.

  18. Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

    PubMed

    Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

    2013-07-01

    This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

  19. An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen

    PubMed Central

    Temelkovski, J.; Hogan, S.; Shepherd, D.; Foster, P.; Kumar, R.

    1998-01-01

    BACKGROUND—Existing murine models of asthma lack many of the inflammatory and epithelial changes that are typical of the human disease. Moreover, these models are frequently complicated by allergic alveolitis.
METHODS—High IgE responder BALB/c mice were systemically sensitised to ovalbumin and chronically challenged with low particle mass concentrations of aerosolised ovalbumin. Titres of anti-ovalbumin IgE in serum were measured at two weekly intervals by enzyme immunoassay, accumulation of inflammatory cells and histopathological abnormalities of the epithelium were quantified morphometrically in the trachea and the lungs, and airway reactivity was assessed by measuring bronchoconstriction following intravenous administration of methacholine.
RESULTS—Mice sensitised by two intraperitoneal injections of ovalbumin developed high titres of IgE antibodies to ovalbumin. Following exposure to low concentrations of aerosolised antigen for up to eight weeks these animals developed a progressive inflammatory response in the airways, characterised by the presence of intraepithelial eosinophils and by infiltration of the lamina propria with lymphoid/mononuclear cells, without associated alveolitis. Goblet cell hyperplasia/metaplasia was induced in the intrapulmonary airways, while epithelial thickening and subepithelial fibrosis were evident following chronic exposure. In parallel, the mice developed increased sensitivity to induction of bronchospasm, as well as increased maximal reactivity. Non-immunised mice exposed to aerosolised ovalbumin had low or absent anti-ovalbumin IgE and did not exhibit inflammatory or epithelial changes, but developed airway hyperreactivity.
CONCLUSIONS—This experimental model replicates many of the features of human asthma and should facilitate studies of pathogenetic mechanisms and of potential therapeutic agents. 

 PMID:10193371

  20. Macrophage/epithelial cell CCL2 contributes to rhinovirus-induced hyperresponsiveness and inflammation in a mouse model of allergic airways disease.

    PubMed

    Schneider, Dina; Hong, Jun Young; Bowman, Emily R; Chung, Yutein; Nagarkar, Deepti R; McHenry, Christina L; Goldsmith, Adam M; Bentley, J Kelley; Lewis, Toby C; Hershenson, Marc B

    2013-02-01

    Human rhinovirus (HRV) infections lead to exacerbations of lower airways disease in asthmatic patients but not in healthy individuals. However, underlying mechanisms remain to be completely elucidated. We hypothesized that the Th2-driven allergic environment enhances HRV-induced CC chemokine production, leading to asthma exacerbations. Ovalbumin (OVA)-sensitized and -challenged mice inoculated with HRV showed significant increases in the expression of lung CC chemokine ligand (CCL)-2/monocyte chemotactic protein (MCP)-1, CCL4/macrophage inflammatory protein (MIP)-1β, CCL7/MCP-3, CCL19/MIP-3β, and CCL20/MIP3α compared with mice treated with OVA alone. Inhibition of CCL2 with neutralizing antibody significantly attenuated HRV-induced airways inflammation and hyperresponsiveness in OVA-treated mice. Immunohistochemical stains showed colocalization of CCL2 with HRV in epithelial cells and CD68-positive macrophages, and flow cytometry showed increased CCL2(+), CD11b(+) cells in the lungs of OVA-treated, HRV-infected mice. Compared with lung macrophages from naïve mice, macrophages from OVA-exposed mice expressed significantly more CCL2 in response to HRV infection ex vivo. Pretreatment of mouse lung macrophages and BEAS-2B human bronchial epithelial cells with interleukin (IL)-4 and IL-13 increased HRV-induced CCL2 expression, and mouse lung macrophages from IL-4 receptor knockout mice showed reduced CCL2 expression in response to HRV, suggesting that exposure to these Th2 cytokines plays a role in the altered HRV response. Finally, bronchoalveolar macrophages from children with asthma elaborated more CCL2 upon ex vivo exposure to HRV than cells from nonasthmatic patients. We conclude that CCL2 production by epithelial cells and macrophages contributes to HRV-induced airway hyperresponsiveness and inflammation in a mouse model of allergic airways disease and may play a role in HRV-induced asthma exacerbations.

  1. Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells.

    PubMed

    March, Catalina; Moranta, David; Regueiro, Verónica; Llobet, Enrique; Tomás, Anna; Garmendia, Junkal; Bengoechea, José A

    2011-03-25

    Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.

  2. 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

  3. Newborn pig trachea cell line cultured in air-liquid interface conditions allows a partial in vitro representation of the porcine upper airway tissue

    PubMed Central

    2014-01-01

    Background The domestic pig is an excellent animal model to study human microbial diseases due to its similarity to humans in terms of anatomy, physiology, and genetics. We assessed the suitability of an in vitro air-liquid interface (ALI) culture system for newborn pig trachea (NPTr) cells as a practical tool for analyzing the immune response of respiratory epithelial cells to aggressors. This cell line offers a wide microbial susceptibility spectrum to both viruses and bacteria. The purpose of our study was to evaluate and characterize diverse aspects of cell differentiation using different culture media. After the NPTr cells reached confluence, the apical medium was removed and the cells were fed by medium from the basal side. Results We assessed the cellular layer’s capacity to polarize and differentiate in ALI conditions. Using immunofluorescence and electronic microscopy we evaluated the presence of goblet and ciliated cells, the epithelial junction organization, and the transepithelial electrical resistance. We found that the cellular layer develops a variable density of mucus producing cells and acquires a transepithelial resistance. We also identified increased development of cellular junctions over the culture period. Finally, we observed variable expression of transcripts associated to proteins such as keratin 8, mucins (MUC1, MUC2, and MUC4), occludin, and villin 1. Conclusions The culture of NPTr cells in ALI conditions allows a partial in vitro representation of porcine upper airway tissue that could be used to investigate some aspects of host/respiratory pathogen interactions. PMID:24885012

  4. Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells.

    PubMed

    Mitchell, Gabriel; Gattuso, Mariza; Grondin, Gilles; Marsault, Éric; Bouarab, Kamal; Malouin, François

    2011-05-01

    Small-colony variants (SCVs) often are associated with chronic Staphylococcus aureus infections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 μg/ml), whereas the growth of normal S. aureus strains was not significantly altered by tomatidine (MIC, >16 μg/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normal S. aureus strains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treated S. aureus at a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms of S. aureus.

  5. Roflumilast N-oxide prevents cytokine secretion induced by cigarette smoke combined with LPS through JAK/STAT and ERK1/2 inhibition in airway epithelial cells.

    PubMed

    Victoni, Tatiana; Gleonnec, Florence; Lanzetti, Manuella; Tenor, Hermann; Valença, Samuel; Porto, Luis Cristovão; Lagente, Vincent; Boichot, Elisabeth

    2014-01-01

    Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Airway epithelial cells and macrophages are the first defense cells against cigarette smoke and these cells are an important source of pro-inflammatory cytokines. These cytokines play a role in progressive airflow limitation and chronic airways inflammation. Furthermore, the chronic colonization of airways by Gram-negative bacteria, contributes to the persistent airways inflammation and progression of COPD. The current study addressed the effects of cigarette smoke along with lipolysaccharide (LPS) in airway epithelial cells as a representative in vitro model of COPD exacerbations. Furthermore, we evaluated the effects of PDE4 inhibitor, the roflumilast N-oxide (RNO), in this experimental model. A549 cells were stimulated with cigarette smoke extract (CSE) alone (0.4% to 10%) or in combination with a low concentration of LPS (0.1 µg/ml) for 2 h or 24 h for measurement of chemokine protein and mRNAs and 5-120 min for protein phosphorylation. Cells were also pre-incubated with MAP kinases inhibitors and Prostaglandin E2 alone or combined with RNO, before the addition of CSE+LPS. Production of cytokines was determined by ELISA and protein phosphorylation by western blotting and phospho-kinase array. CSE did not induce production of IL-8/CXCL8 and Gro-α/CXCL1 from A549 cells, but increase production of CCL2/MCP-1. However the combination of LPS 0.1 µg/ml with CSE 2% or 4% induced an important production of these chemokines, that appears to be dependent of ERK1/2 and JAK/STAT pathways but did not require JNK and p38 pathways. Moreover, RNO associated with PGE2 reduced CSE+LPS-induced cytokine release, which can happen by occur through of ERK1/2 and JAK/STAT pathways. We report here an in vitro model that can reflect what happen in airway epithelial cells in COPD exacerbation. We also showed a new pathway where CSE+LPS can induce cytokine release from A549 cells, which is reduced by RNO.

  6. Characterization of miRNAs involved in response to poly(I:C) in porcine airway epithelial cells.

    PubMed

    Wang, L; Wang, J K; Han, L X; Zhuo, J S; Du, X; Liu, D; Yang, X Q

    2017-04-01

    MicroRNAs (miRNA) have been implicated in a variety of pathological conditions including infectious diseases. Knowledge of the miRNAs affected by poly(I:C), a synthetic analog of viral double-stranded RNA, in porcine airway epithelial cells (PAECs) contributes to understanding the mechanisms of swine viral respiratory diseases, which bring enormous economic loss worldwide every year. In this study, we used high throughput sequencing to profile miRNA expression in PAECs treated with poly(I:C) as compared to the untreated control. This approach revealed 23 differentially expressed miRNAs (DEMs), five of which have not been implicated in viral infection before. Nineteen of the 23 miRNAs were down-regulated including members of the miR-17-92 cluster, a well-known polycistronic oncomir and extensively involved in viral infection in humans. Target genes of DEMs, predicted using bioinformatic methods and validated by luciferase reporter analysis on two representative DEMs, were significantly enriched in several pathways including transforming growth factor-β signaling. A large quantity of sequence variations (isomiRs) were found including a substitution at position 5, which was verified to redirect miRNAs to a new spectrum of targets by luciferase reporter assay together with bioinformatics analysis. Twelve novel porcine miRNAs conserved in other species were identified by homology analysis together with cloning verification. Furthermore, the expression analysis revealed the potential importance of three novel miRNAs in porcine immune response to viruses. Overall, our data contribute to clarifying the mechanisms underlying the host immune response against respiratory viruses in pigs, and enriches the repertoire of porcine miRNAs.

  7. Effects of 10 cigarette smoke condensates on primary human airway epithelial cells by comparative gene and cytokine expression studies.

    PubMed

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

    2010-03-01

    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 approximately 4 microg/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.

  8. Cistrome-based Cooperation between Airway Epithelial Glucocorticoid Receptor and NF-κB Orchestrates Anti-inflammatory Effects.

    PubMed

    Kadiyala, Vineela; Sasse, Sarah K; Altonsy, Mohammed O; Berman, Reena; Chu, Hong W; Phang, Tzu L; Gerber, Anthony N

    2016-06-10

    Antagonism of pro-inflammatory transcription factors by monomeric glucocorticoid receptor (GR) has long been viewed as central to glucocorticoid (GC) efficacy. However, the mechanisms and targets through which GCs exert therapeutic effects in diseases such as asthma remain incompletely understood. We previously defined a surprising cooperative interaction between GR and NF-κB that enhanced expression of A20 (TNFAIP3), a potent inhibitor of NF-κB. Here we extend this observation to establish that A20 is required for maximal cytokine repression by GCs. To ascertain the global extent of GR and NF-κB cooperation, we determined genome-wide occupancy of GR, the p65 subunit of NF-κB, and RNA polymerase II in airway epithelial cells treated with dexamethasone, TNF, or both using chromatin immunoprecipitation followed by deep sequencing. We found that GR recruits p65 to dimeric GR binding sites across the genome and discovered additional regulatory elements in which GR-p65 cooperation augments gene expression. GR targets regulated by this mechanism include key anti-inflammatory and injury response genes such as SERPINA1, which encodes α1 antitrypsin, and FOXP4, an inhibitor of mucus production. Although dexamethasone treatment reduced RNA polymerase II occupancy of TNF targets such as IL8 and TNFAIP2, we were unable to correlate specific binding sequences for GR or occupancy patterns with repressive effects on transcription. Our results suggest that cooperative anti-inflammatory gene regulation by GR and p65 contributes to GC efficacy, whereas tethering interactions between GR and p65 are not universally required for GC-based gene repression.

  9. Cytoplasmic Irradiation Induces Metabolic Shift in Human Small Airway Epithelial Cells via Activation of Pim-1 Kinase.

    PubMed

    Wu, Jinhua; Zhang, Qin; Wuu, Yen-Ruh; Zou, Sirui; Hei, Tom K

    2017-02-07

    The unique cellular and molecular consequences of cytoplasmic damage caused by ionizing radiation were studied using a precision microbeam irradiator. Our results indicated that targeted cytoplasmic irradiation induced metabolic shift from an oxidative to glycolytic phenotype in human small airway epithelial cells (SAE). At 24 h postirradiation, there was an increase in the mRNA expression level of key glycolytic enzymes as well as lactate secretion in SAE cells. Using RNA-sequencing analysis to compare genes that were responsive to cytoplasmic versus nuclear irradiation, we found a glycolysis related gene, Pim-1, was significantly upregulated only in cytoplasmic irradiated SAE cells. Inhibition of Pim-1 activity using the selective pharmaceutic inhibitor Smi-4a significantly reduced the level of lactate production and glucose uptake after cytoplasmic irradiation. In addition, Pim-1 also inhibited AMPK activity, which is a well-characterized negative regulator of glycolysis. Distinct from the glycolysis induced by cytoplasmic irradiation, targeted nuclear irradiation also induced a transient and minimal increase in glycolysis that correlated with increased expression of Hif-1α. In an effort to explore the underline mechanism, we found that inhibition of mitochondria fission using the cell-permeable inhibitor mdivi-1 suppressed the induction of Pim-1, thus confirming Pim-1 upregulation as a downstream effect of mitochondrial dysfunction. Our data show and, for the first time, that cytoplasmic irradiation mediate expression level of Pim-1, which lead to glycolytic shift in SAE cells. Additionally, since glycolysis is frequently linked to cancer cell metabolism, our findings further suggest a role of cytoplasmic damage in promoting neoplastic changes.

  10. 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.

  11. Carcinogenic effects of oil dispersants: A KEGG pathway-based RNA-seq study of human airway epithelial cells.

    PubMed

    Liu, Yao-Zhong; Zhang, Lei; Roy-Engel, Astrid M; Saito, Shigeki; Lasky, Joseph A; Wang, Guangdi; Wang, He

    2017-02-20

    The health impacts of the BP oil spill are yet to be further revealed as the toxicological effects of oil products and dispersants on human respiratory system may be latent and complex, and hence difficult to study and follow up. Here we performed RNA-seq analyses of a system of human airway epithelial cells treated with the BP crude oil and/or dispersants Corexit 9500 and Corexit 9527 that were used to help break up the oil spill. Based on the RNA-seq data, we then systemically analyzed the transcriptomic perturbations of the cells at the KEGG pathway level using two pathway-based analysis tools, GAGE (generally applicable gene set enrichment) and GSNCA (Gene Sets Net Correlations Analysis). Our results suggested a pattern of change towards carcinogenesis for the treated cells marked by upregulation of ribosomal biosynthesis (hsa03008) (p=1.97E-13), protein processing (hsa04141) (p=4.09E-7), Wnt signaling (hsa04310) (p=6.76E-3), neurotrophin signaling (hsa04722) (p=7.73E-3) and insulin signaling (hsa04910) (p=1.16E-2) pathways under the dispersant Corexit 9527 treatment, as identified by GAGE analysis. Furthermore, through GSNCA analysis, we identified gene co-expression changes for several KEGG cancer pathways, including small cell lung cancer pathway (hsa05222, p=9.99E-5), under various treatments of oil/dispersant, especially the mixture of oil and Corexit 9527. Overall, our results suggested carcinogenic effects of dispersants (in particular Corexit 9527) and their mixtures with the BP crude oil, and provided further support for more stringent safety precautions and regulations for operations involving long-term respiratory exposure to oil and dispersants.

  12. 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

  13. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    SciTech Connect

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  14. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  15. Human eccrine sweat gland epithelial cultures express ductal characteristics.

    PubMed Central

    Brayden, D J; Cuthbert, A W; Lee, C M

    1988-01-01

    1. Isolated human eccrine sweat glands were cultured in vitro. Cells were harvested and plated onto permeable supports to form confluent cell sheets, area 0.2 cm2. These were used to study the electrogenic transepithelial transport of ions by measurement of short-circuit current (SCC). Epithelial sheets had a basal SCC of 5.89 +/- 0.62 microA cm-2 (n = 33) and a transepithelial resistance of 74.1 +/- 5.6 omega cm2 (n = 33). The transepithelial potential difference varied between -0.2 and -1.8 mV with a mean value of -0.71 +/- 0.09 mV (n = 33). 2. The basal current was abolished by addition of 10 microM-amiloride to the apical bathing solution. The concentration of amiloride which inhibited basal SCC by 50% (EC50) was 0.4 microM. Cultures prepared from the secretory coil of sweat glands, rather than from whole glands, were similarly sensitive to amiloride (EC50 = 0.8 microM). 3. Lysylbradykinin (LBK), carbachol, isoprenaline, prostaglandin E2 (PGE2) and A23187 all increased SCC in cultures from whole glands. LBK responses were obtained with basolateral and not with apical application. Furthermore LBK actions were not substantially altered by cyclo-oxygenase inhibition but showed marked desensitization upon repeated application. Sheet cultures prepared from sweat gland coils also showed SCC responses to both carbachol and LBK. Forskolin, an activator of adenylate cyclase, did not alter SCC in either type of preparation. 4. Replacement of chloride and of chloride and bicarbonate in the bathing solution did not cause attenuation of the responses to LBK or carbachol in whole-gland sheet cultures. Furthermore responses were unaffected by piretanide or acetazolamide. These results were taken to indicate that anion secretion was not the basis for the SCC responses. 5. Responses to LBK and carbachol were significantly reduced by amiloride (10 microM), this effect being reversible. No responses to LBK or carbachol were seen when N-methyl-D-glucamine (NMDG) was used to

  16. Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cells and Skewing the Cells toward a T-helper 2 Bias

    PubMed Central

    Homma, Tetsuya; Kato, Atsushi; Bhushan, Bharat; Norton, James E.; Suh, Lydia A.; Carter, Roderick G.; Gupta, Dave S.

    2016-01-01

    Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1–biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C– and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho–IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C–induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF

  17. Exposure to PM2.5 induces aberrant activation of NF-κB in human airway epithelial cells by downregulating miR-331 expression.

    PubMed

    Song, Lei; Li, Dan; Li, Xiaoping; Ma, Lianjun; Bai, Xiaoxue; Wen, Zhongmei; Zhang, Xiufang; Chen, Dong; Peng, Liping

    2017-03-01

    Exposure to particulate matter (PM) with an aerodynamic diameter≤2.5μm (PM2.5) induces reactive oxygen species (ROS) and pro-inflammatory cytokine production, leading to airway epithelial injury. However, the mechanisms underlying the toxicity of PM2.5 have not been clarified. Here, we show that exposure to PM2.5 induces sustained activation of the nuclear factor kappa B (NF-κB) signaling in human airway epithelial Beas-2B (B2B) cells. In addition, PM2.5 exposure significantly decreased miR-331 expression in B2B cells, which was abrogated by inhibition of ROS or phosphoinositide 3-kinase (PI3K)/Akt pathway. Induction of miR-331 overexpression attenuated the PM2.5 exposure-induced NF-kBp65 nuclear translocation, IL-6 and IL-8 expression in B2B cells. Furthermore, miR-331 targeted the inhibitor of NF-κB kinase beta (IKK-β) by down-regulating the IKK-β-regulated luciferase activity in HEK293 cells. Moreover, induction of miR-331 over-expression inhibited IKK-β expression while induction of IKK-β over-expression prevented the inhibition of miR-331 on the PM2.5 exposure-induced NF-kBp65 nuclear translocation, IL-6 and IL-8 expression in B2B cells. Therefore, PM2.5 exposure decreased miR-331 expression via the ROS/PI3K/Akt pathway, resulting in an increase in the IKK-β expression and sustained NF-κB activation in human airway epithelial cells. Our findings may provide new insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure and aid in design of new therapeutic strategies to prevent PM2.5-induced toxicity.

  18. Epithelial-mesenchymal transitions during cell culture of primary thyroid tumors?

    PubMed

    Herrmann, M E; Trevor, K T

    1993-04-01

    Fibroblast contamination of epithelial tumor cell cultures is of great concern when examining tumor cells in vitro for specific biochemical and cytogenetic changes. The observations of normal karyotypes in thyroid tumor cell cultures have raised the concern of whether residual tissue fibroblasts might obscure the cytogenetic analysis of transformed epithelial cells. We have characterized early passaged thyroid tumor cells to examine the proportions of epithelial and fibroblastic cell types. Cells were analyzed by immunocytology using antibodies recognizing the thyroid prohormone thyroglobulin, epithelial cytokeratins, and vimentin, a mesenchyme marker. Tumors consisted of one follicular adenoma and five papillary carcinomas. When examined by day 15 in culture, all cells contained filaments composed of vimentin, which most likely represents an adaptation to culture conditions. Double immunofluorescence staining for thyroglobulin and cytokeratin revealed the presence of not only epithelial but also spindle-like fibroblastoid cells possessing thyroid epithelial cell markers. The results suggest that in thyroid tumor cultures there is a unique cell type intermediate between epithelial and mesenchyme phenotypes that must be considered when performing cytogenetic analysis.

  19. The effects on bronchial epithelial mucociliary cultures of coarse, fine, and ultrafine particulate matter from an underground railway station.

    PubMed

    Loxham, Matthew; Morgan-Walsh, Rebecca J; Cooper, Matthew J; Blume, Cornelia; Swindle, Emily J; Dennison, Patrick W; Howarth, Peter H; Cassee, Flemming R; Teagle, Damon A H; Palmer, Martin R; Davies, Donna E

    2015-05-01

    We have previously shown that underground railway particulate matter (PM) is rich in iron and other transition metals across coarse (PM10-2.5), fine (PM2.5), and quasi-ultrafine (PM0.18) fractions and is able to generate reactive oxygen species (ROS). However, there is little knowledge of whether the metal-rich nature of such particles exerts toxic effects in mucus-covered airway epithelial cell cultures or whether there is an increased risk posed by the ultrafine fraction. Monolayer and mucociliary air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) were exposed to size-fractionated underground railway PM (1.1-11.1 µg/cm(2)) and release of lactate dehydrogenase and IL-8 was assayed. ROS generation was measured, and the mechanism of generation studied using desferrioxamine (DFX) and N-acetylcysteine (NAC). Expression of heme oxygenase-1 (HO-1) was determined by RT-qPCR. Particle uptake was studied by transmission electron microscopy. Underground PM increased IL-8 release from PBECs, but this was diminished in mucus-secreting ALI cultures. Fine and ultrafine PM generated a greater level of ROS than coarse PM. ROS generation by ultrafine PM was ameliorated by DFX and NAC, suggesting an iron-dependent mechanism. Despite the presence of mucus, ALI cultures displayed increased HO-1 expression. Intracellular PM was observed within vesicles, mitochondria, and free in the cytosol. The results indicate that, although the mucous layer appears to confer some protection against underground PM, ALI PBECs nonetheless detect PM and mount an antioxidant response. The combination of increased ROS-generating ability of the metal-rich ultrafine fraction and ability of PM to penetrate the mucous layer merits further research.

  20. Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

    PubMed

    Kim, Ha Ryong; Cho, Han Soo; Shin, Da Young; Chung, Kyu Hyuck

    2017-02-01

    In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases.

  1. Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.

    PubMed

    Jiao, Jian; Meng, Na; Wang, Hong; Zhang, Luo

    2013-12-01

    The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

  2. Toxicological Assessment of CoO and La2O3 Metal Oxide Nanoparticles in Human Small Airway Epithelial Cells.

    PubMed

    Sisler, Jennifer D; Pirela, Sandra V; Shaffer, Justine; Mihalchik, Amy L; Chisholm, William P; Andrew, Michael E; Schwegler-Berry, Diane; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2016-04-01

    Cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles are 2 metal oxide nanoparticles with different redox potentials according to their semiconductor properties. By utilizing these two nanoparticles, this study sought to determine how metal oxide nanoparticle's mode of toxicological action is related to their physio-chemical properties in human small airway epithelial cells (SAEC). We investigated cellular toxicity, production of superoxide radicals and alterations in gene expression related to oxidative stress, and cellular death at 6 and 24 h following exposure to CoO and La2O3(administered doses: 0, 5, 25, and 50 µg/ml) nanoparticles. CoO nanoparticles induced gene expression related to oxidative stress at 6 h. After characterizing the nanoparticles, transmission electron microscope analysis showed SAEC engulfed CoO and La2O3nanoparticles. CoO nanoparticles were toxic after 6 and 24 h of exposure to 25.0 and 50.0 µg/ml administered doses, whereas, La2O3nanoparticles were toxic only after 24 h using the same administered doses. Based upon the Volumetric Centrifugation Methodin vivoSedimentation, Diffusion, and Dosimetry, the dose of CoO and La2O3nanoparticles delivered at 6 and 24 h were determined to be: CoO: 1.25, 6.25, and 12.5 µg/ml; La2O3: 5, 25, and 50 µg/ml and CoO: 4, 20, and 40 µg/ml; and La2O3: 5, 25, 50 µg/ml, respectively. CoO nanoparticles produced more superoxide radicals and caused greater stimulation of total tyrosine and threonine phosphorylation at both 6 and 24 h when compared with La2O3nanoparticles. Taken together, these data provide evidence that different toxicological modes of action were involved in CoO and La2O3metal oxide nanoparticle-induced cellular toxicity.

  3. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells.

    PubMed

    Economou, E C; Marinelli, S; Smith, M C; Routt, A A; Kravets, V V; Chu, H W; Spendier, K; Celinski, Z J

    2016-09-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.

  4. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells

    PubMed Central

    Economou, E. C.; Marinelli, S.; Smith, M. C.; Routt, A. A.; Kravets, V. V.; Chu, H. W.; Spendier, K.; Celinski, Z. J.

    2016-01-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles. PMID:27774374

  5. Human airway organoid engineering as a step toward lung regeneration and disease modeling.

    PubMed

    Tan, Qi; Choi, Kyoung Moo; Sicard, Delphine; Tschumperlin, Daniel J

    2017-01-01

    Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-β1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues.

  6. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    PubMed

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  7. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  8. Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle

    PubMed Central

    Zaman, Nishat; Cole, Darren J.; Walker, Matthew J.; Legant, Wesley R.; Boudou, Thomas; Chen, Christopher S.; Favreau, John T.; Gaudette, Glenn R.; Cowley, Elizabeth A.; Maksym, Geoffrey N.

    2013-01-01

    Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell “microtissues” capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma. PMID:23125251

  9. 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

  10. [Use of transport medium in sputum bacterial culture examination of lower airway infection].

    PubMed

    Muraki, Masato; Kitaguchi, Sayako; Ichihashi, Hideo; Tsuji, Fumio; Ohmori, Takashi; Haraguchi, Ryuta; Tohda, Yuji

    2006-06-01

    Our medical institution does not have a bacterial culture facility, requiring outsourcing of bacterial culture tests. Due to the time elapsed from the time of specimen collection to culturing, the identification of causative bacteria in respiratory tract infections tends to be difficult. We therefore used transport medium for sputum bacteria examinations. Expectorated purulent or purulent-mucous sputum specimens were collected from 32 patients with lower respiratory tract infection. We divided each of the sputum specimens into the two treatment groups: transport medium (Seedswab gamma2) ndar and stad disinfection container. Paired samples prepared from each patient were sent out for bacterial culture together. The time elapsed from collection to delivery to the lab were as follows: day 0 (same day, n = 14 patients), day 1 (n = 15), day 2 (n = 2), and day 3 (n = 1). The identified causative bacteria were Streptococcus pneumoniae (n = 6 patients), Haemophilus influenzae (n =5), Pseudomonas aeruginosa (n = 4), Staphylococcus aureus (n = 2), Moraxella catarrhalis (n = 2), Klebsiella pneumoniae (n = 1), and Streptococcus agalactiae (n = 1). Samples prepared by each of the two methods gave similar results. The utility of transport medium for examination of general bacteria for lower airway infection from sputum samples was not demonstrated. The rate of detection of bacteria decreased, when the transport of samples was delayed. Therefore, we need to send the sputum specimens as quickly as possible.

  11. 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

  12. Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity.

    PubMed

    Jang, Jun-Ho; Bruse, Shannon; Liu, Yushi; Duffy, Veronica; Zhang, Chunyu; Oyamada, Nathaniel; Randell, Scott; Matsumoto, Akiko; Thompson, David C; Lin, Yong; Vasiliou, Vasilis; Tesfaigzi, Yohannes; Nyunoya, Toru

    2014-03-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs.

  13. Airway epithelium stimulates smooth muscle proliferation.

    PubMed

    Malavia, Nikita K; Raub, Christopher B; Mahon, Sari B; Brenner, Matthew; Panettieri, Reynold A; George, Steven C

    2009-09-01

    Communication between the airway epithelium and stroma is evident during embryogenesis, and both epithelial shedding and increased smooth muscle proliferation are features of airway remodeling. Hence, we hypothesized that after injury the airway epithelium could modulate airway smooth muscle proliferation. Fully differentiated primary normal human bronchial epithelial (NHBE) cells at an air-liquid interface were co-cultured with serum-deprived normal primary human airway smooth muscle cells (HASM) using commercially available Transwells. In some co-cultures, the NHBE were repeatedly (x4) scrape-injured. An in vivo model of tracheal injury consisted of gently denuding the tracheal epithelium (x3) of a rabbit over 5 days and then examining the trachea by histology 3 days after the last injury. Our results show that HASM cell number increases 2.5-fold in the presence of NHBE, and 4.3-fold in the presence of injured NHBE compared with HASM alone after 8 days of in vitro co-culture. In addition, IL-6, IL-8, monocyte chemotactic protein (MCP)-1 and, more markedly, matrix metalloproteinase (MMP)-9 concentration increased in co-culture correlating with enhanced HASM growth. Inhibiting MMP-9 release significantly attenuated the NHBE-dependent HASM proliferation in co-culture. In vivo, the injured rabbit trachea demonstrated proliferation in the smooth muscle (trachealis) region and significant MMP-9 staining, which was absent in the uninjured control. The airway epithelium modulates smooth muscle cell proliferation via a mechanism that involves secretion of soluble mediators including potential smooth muscle mitogens such as IL-6, IL-8, and MCP-1, but also through a novel MMP-9-dependent mechanism.

  14. Epithelial monolayer culture system for real‐time single‐cell analyses

    PubMed Central

    Seo, Jong Bae; Moody, Mark; Koh, Duk‐Su

    2014-01-01

    Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single‐cell and subcellular levels, and can be extended to other cell types with minor modifications. PMID:24771696

  15. Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells.

    PubMed

    Pelaia, Girolamo; Cuda, Giovanni; Vatrella, Alessandro; Gallelli, Luca; Fratto, Donatella; Gioffrè, Vincenza; D'Agostino, Bruno; Caputi, Mario; Maselli, Rosario; Rossi, Francesco; Costanzo, Francesco S; Marsico, Serafino A

    2004-09-01

    The airway epithelium is continuously exposed to inhaled oxidants, including airborne pollutants and cigarette smoke, which can exert harmful proinflammatory and cytotoxic effects. Therefore, the aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the signal transduction pathways activated by increasing concentrations (0.25, 0.5, and 1 mM) of hydrogen peroxide (H(2)O(2)), as well as their effects on IL-8 production and cell viability. The reported results show that H(2)O(2) elicited, in a concentration-dependent fashion, a remarkable increase in phosphorylation-dependent activation of mitogen-activated protein kinases (MAPKs), associated with a significant induction of IL-8 synthesis and a dramatically enhanced cell death. Pre-treatment of HBEC with MAPK inhibitors was able to significantly inhibit the effects of H(2)O(2) on IL-8 secretion, and to effectively prevent cell death. Therefore, these findings suggest that MAPKs play a key role as molecular transducers of the airway epithelial injury triggered by oxidative stress, as well as potential pharmacologic targets for indirect antioxidant intervention.

  16. Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture.

    PubMed

    Leighton, J

    1994-09-01

    The biology of animal cells in culture is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and of morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial tissues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin permeable transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.

  17. Use of a feline respiratory epithelial cell culture system grown at the air-liquid interface to characterize the innate immune response following feline herpesvirus 1 infection.

    PubMed

    Nelli, Rahul K; Maes, Roger; Kiupel, Matti; Hussey, Gisela Soboll

    2016-03-02

    Infection with feline herpesvirus-1 (FHV-1) accounts for 50% of viral upper respiratory diseases in domestic cats and is a significant cause of ocular diseases. Despite the clinical significance and high prevalence of FHV-1 infection, currently available vaccines cannot completely protect cats from infection and lifelong latency. FHV-1 infects via the mucous membranes and replicates in respiratory epithelial cells, but very little is known about the early innate immunity at this site. To address questions about immunity to FHV-1, feline respiratory epithelial cells cultured at air-liquid interface (ALI-FRECs) were established by collecting respiratory tracts from 6 healthy cats after euthanasia. Cells were isolated, cultured and characterized histologically and immunologically before infection with FHV-1. The expression of Toll-like receptors (TLRs), cytokine and chemokine responses were measured by real time PCR. ALI-FRECs morphologically resembled the natural airways of cats with multilayered columnar epithelial cells and cilia. Immunological properties of the natural airways were maintained in ALI-FRECs, as evidenced by the expression of TLRs, cytokines, chemokines, interferons, beta-defensins, and other regulatory genes. Furthermore, ALI-FRECs were able to support infection and replication of FHV-1, as well as modulate transcriptional regulation of various immune genes in response to infection. IL-1β and TNFα were increased in ALI-FRECs by 24hpi, whereas expression levels of IFN-α and TLR9 were not increased until 36hpi. In contrast, TLR3, GM-CSF and TGF-1β expression was down-regulated at 36hpi. The data presented show the development of a system ideal for investigating the molecular pathogenesis and immunity of FHV-1 or other respiratory pathogens.

  18. Neurons Self-Organize Around Salivary Epithelial Cells in Novel Co-Culture Model

    PubMed Central

    Sommakia, Salah; Baker, Olga J.

    2016-01-01

    Salivary gland bioengineering requires understanding the interaction between salivary epithelium and surrounding tissues. An important component of salivary glands is the presence of neurons. No previous studies have investigated how neurons and salivary epithelial cells interact in an in vitro co-culture model. In this study, we describe the self-organization of neurons around salivary epithelial cells in co-culture, in a similar fashion to what occurs in native tissue. We cultured primary mouse cortical neurons (m-CN) with a salivary epithelial cell line (Par-C10) on growth factor-reduced Matrigel (GFR-MG) for 4 days. After this time, co-cultures were compared with native salivary glands using confocal microscopy. Our findings indicate that m-CN were able to self-organize basolaterally to salivary epithelial cell clusters in a similar manner to what occurs in native tissue. These results indicate that this model can be developed as a potential platform for studying neuron-salivary epithelial cell interactions for bioengineering purposes. PMID:27833941

  19. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

    PubMed Central

    Gidfar, Sanaz; Milani, Farnoud Y.; Milani, Behrad Y.; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J.; Sagha, Hossein; Djalilian, Ali R.

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells. PMID:28054657

  20. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    PubMed

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

  1. 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...

  2. Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

    PubMed

    Wong, J Z; Woodcock-Mitchell, J; Mitchell, J; Rippetoe, P; White, S; Absher, M; Baldor, L; Evans, J; McHugh, K M; Low, R B

    1998-05-01

    In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

  3. Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells

    PubMed Central

    Seo, Hyo-Seok; Sikder, Mohamed Asaduzzaman; Lee, Hyun Jae; Ryu, Jiho; Lee, Choong Jae

    2014-01-01

    In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells. PMID:25489420

  4. 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

  5. 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

  6. 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

  7. 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

    2016-09-16

    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.

  8. The impact of oil spill to lung health – insights from an RNA-seq study of human airway epithelial cells

    PubMed Central

    Liu, Yao-Zhong; Roy-Engel, Astrid M; Baddoo, Melody C; Flemington, Erik K; Wang, Guangdi; Wang, He

    2015-01-01

    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 three 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

  9. Expression and regulation of immune-modulatory enzyme indoleamine 2,3-dioxygenase (IDO) by human airway epithelial cells and its effect on T cell activation

    PubMed Central

    Sewell, Herb F.; Knox, Alan; Ghaemmaghami, Amir M.

    2016-01-01

    Indoleamine 2,3-dioxygenase (IDO) catalyzes the degradation of tryptophan, which plays a critical role in immune suppression through regulating the production of a series of metabolites that are generally referred to as kynurenines. It has become increasingly clear that epithelial cells (ECs) play an active role in maintaining lung homeostasis by modulating the function of immune cells via producing cytokines, chemokines, and anti-microbial mediators. In this study we assessed the regulation of IDO activity and expression in human primary ECs and EC lines under steady state conditions and in response to bacterial and allergenic stimuli. We also investigated the potential immune modulatory functions of IDO expression in human airway ECs. Our data clearly show that airway ECs produce IDO, which is down-regulated in response to allergens and TLR ligands while up-regulated in response to IFN-γ. Using gene silencing, we further demonstrate that IDO plays a key role in the EC-mediated suppression of antigen-specific and polyclonal proliferation of T cells. Interestingly, our data also show that ECs lose their inhibitory effect on T cell activation in response to different TLR agonists mimicking bacterial or viral infections. In conclusion, our work provides an understanding of how IDO is regulated in ECs as well as demonstrates that “resting” ECs can suppress T cell activation in an IDO dependent manner. These data provide new insight into how ECs, through the production of IDO, can influence downstream innate and adaptive responses as part of their function in maintaining immune homeostasis in the airways. PMID:27613847

  10. TMEM16A Inhibitors Reveal TMEM16A as a Minor Component of Calcium-activated Chloride Channel Conductance in Airway and Intestinal Epithelial Cells*

    PubMed Central

    Namkung, Wan; Phuan, Puay-Wah; Verkman, A. S.

    2011-01-01

    TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC50 < 10 μm, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16Ainh-A01), had an IC50 of ∼1 μm. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCCinh-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16Ainh-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production. PMID:21084298

  11. Preliminary Study on Gene Expression of Chitinase-Like Cytokines in Human Airway Epithelial Cell Under Chitin and Chitosan Microparticles Treatment.

    PubMed

    Alimohammadi, Masumeh; Yeganeh, Farshid; Haji Molla Hoseini, Mostafa

    2016-06-01

    Small-sized chitin and chitosan microparticles (MPs) reduce allergic inflammation. We examined the capacity of these glycans to stimulate A549 human airway epithelial cells to determine the feasibility of using of these glycans as allergic therapeutic modality. A549 cells were treated with MPs and then expressions levels of chitinase domain-containing 1 (CHID1) and chitinase 3-like 1 (CHI3L1) genes were determined by quantitative real-time PCR. IL-6 production was measured by ELISA. Chitin MPs resulted in upregulation of CHI3L1 expression by 35.7-fold while mRNA expression did not change with chitosan MPs. Compared to the untreated group, production of IL-6 was significantly decreased in the chitosan MPs-treated group, but chitin MPs treatment cause elevation of IL-6 level. This study demonstrates that chitin potently induces CHI3L1 expression, but chitosan is relatively inert. This effect and inhibition of pro-inflammatory cytokine (IL-6) suggest that chitosan MPs may possess more potential for therapeutic uses in human airway allergic inflammation.

  12. Organotypic culture of fetal lung type II alveolar epithelial cells: applications to pulmonary toxicology.

    PubMed Central

    Shami, S G; Aghajanian, J D; Sanders, R L

    1984-01-01

    Techniques for isolation and culture of fetal Type II alveolar epithelial cells, as well as the morphologic and biochemical characteristics of these histotypic cultures, are described. Type II alveolar epithelial cells can be isolated from fetal rat lungs and grown in an organotypic culture system as described in this review. The fetal Type II cells resemble differentiated rat Type II cells in morphology, biochemistry, and karyotype as they grow in culture for up to 5 weeks. The cells of the mature organotypic cultures form alveolarlike structures while growing on a gelatin sponge matrix. The Type II cells also synthesize and secrete pulmonary surfactant similar in biochemical composition to that produced in vivo. This system has been used to study the effects of hormones on surfactant production and composition. The organotypic model has many potential applications to the study of pulmonary toxicology. Images FIGURE 1. FIGURE 2. PMID:6548184

  13. The Three-Dimensional Culture of Epithelial Organoids Derived from Embryonic Chicken Intestine.

    PubMed

    Pierzchalska, Malgorzata; Panek, Malgorzata; Czyrnek, Malgorzata; Grabacka, Maja

    2016-10-28

    The intestinal epithelium isolated from chicken embryos in last 3 days of development can be used to establish the 3D culture of intestinal organoids. When fragments of epithelial tissue released by incubation with EGTA (2.5 mM, 2 h) are embedded in Matrigel matrix on cell culture inserts the formation of empty spheres covered by epithelial cells is observed in first 24 h of culture. The growth and survival of organoids are supported by the addition of R-spondin 1, Noggin, and prostaglandin E2 to the culture medium. The organoids are accompanied by myofibroblasts which become visible in the next 2 days of culture. The intestinal enteroids (free of myofibroblasts) can be obtained from adult chicken intestine.

  14. Putting the Squeeze on Airway Epithelia

    PubMed Central

    Park, Jin-Ah; Fredberg, Jeffrey J.

    2015-01-01

    Asthma is characterized by chronic inflammation, airway hyperresponsiveness, and progressive airway remodeling. The airway epithelium is known to play a critical role in the initiation and perpetuation of these processes. Here, we review how excessive epithelial stress generated by bronchoconstriction is sufficient to induce airway remodeling, even in the absence of inflammatory cells. PMID:26136543

  15. Air pollution particles mediated oxidative DNA base damage in a cell free system and in human airway epithelial cells in relation to particulate metal content and bioreactivity.

    PubMed

    Prahalad, A K; Inmon, J; Dailey, L A; Madden, M C; Ghio, A J; Gallagher, J E

    2001-07-01

    chelator before reacting with DNA or addition of catalase in the incubation mixture, suppressed 8-oxo-dG formation significantly (p < 0.05) in oil-derived fly ash particles only. To determine whether the oxidative responses of these particulates as shown in cell-free systems were consistent with responses using a more biologically relevant environment, human airway epithelial cells were treated with the particulates and induction of 8-oxo-dG was determined. All particles induced 8-oxo-dG in the DNA of cells above culture control, except CFA. Cells exposed to 10-400 mg/mL of ROFA for 2 h induced a dose-dependent increase in 8-oxo-dG formation. Treatment of ROFA with metal ion chelator attenuated these effects. Overall, damage enhancement by particulates in dG, calf thymus, and cellular DNA as determined by 8-oxo-dG formation under aerobic conditions is consistent with the concentration of water-soluble, not the total metal content of the particle.

  16. Phosphodiesterase 4 inhibitors augment the ability of formoterol to enhance glucocorticoid-dependent gene transcription in human airway epithelial cells: a novel mechanism for the clinical efficacy of roflumilast in severe chronic obstructive pulmonary disease.

    PubMed

    Moodley, Thunicia; Wilson, Sylvia M; Joshi, Taruna; Rider, Christopher F; Sharma, Pawan; Yan, Dong; Newton, Robert; Giembycz, Mark A

    2013-04-01

    Post-hoc analysis of two phase III clinical studies found that the phosphodiesterase 4 (PDE4) inhibitor, roflumilast, reduced exacerbation frequency in patients with severe chronic obstructive pulmonary disease (COPD) who were taking inhaled corticosteroids (ICS) concomitantly, whereas patients not taking ICS derived no such benefit. In contrast, in two different trials also performed in patients with severe COPD, roflumilast reduced exacerbation rates in the absence of ICS, indicating that PDE4 inhibition alone is sufficient for therapeutic activity to be realized. Given that roflumilast is recommended as an "add-on" medication to patients with severe disease who will inevitably be taking a long-acting β2-adrenoceptor agonist (LABA)/ICS combination therapy, we tested the hypothesis that roflumilast augments the ability of glucocorticoids to induce genes with anti-inflammatory activity. Using a glucocorticoid response element (GRE) luciferase reporter transfected into human airway epithelial cells [both bronchial epithelium + adenovirus 12 - SV40 hybrid (BEAS-2B) cells and primary cultures], roflumilast enhanced fluticasone propionate-induced GRE-dependent transcription. Roflumilast also produced a sinistral displacement of the concentration-response curves that described the augmentation of GRE-dependent transcription by the LABA formoterol. In BEAS-2B cells and primary airway epithelia, roflumilast interacted with formoterol in a positive cooperative manner to enhance the expression of several glucocorticoid-inducible genes that have anti-inflammatory potential. We suggest that the ability of roflumilast and formoterol to interact in this way supports the concept that these drugs together may impart clinical benefit beyond that achievable by an ICS alone, a PDE4 inhibitor alone, or an ICS/LABA combination therapy. Roflumilast may, therefore, be especially effective in patients with severe COPD.

  17. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid.

    PubMed

    Basso, Fernanda Gonçalves; Soares, Diana Gabriela; Pansani, Taisa Nogueira; Turrioni, Ana Paula Silveira; Scheffel, Débora Lopes; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-11-28

    Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

  18. Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense

    PubMed Central

    Tang, Xiao Xiao; Vargas Buonfiglio, Luis G.; Comellas, Alejandro P.; Thornell, Ian M.; Ramachandran, Shyam; Karp, Philip H.; Taft, Peter J.; Sheets, Kelsey; Abou Alaiwa, Mahmoud H.; Welsh, Michael J.; Stoltz, David A.; Zabner, Joseph

    2016-01-01

    While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl− and bicarbonate (HCO3−) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl− and HCO3− transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization. PMID:26801568

  19. Effect of cord blood serum on ex vivo human limbal epithelial cell culture.

    PubMed

    Chakraborty, Anindita; Dutta, Jayanta; Das, Sumantra; Datta, Himadri

    2012-12-01

    Limbal cell transplantation is an efficacious procedure for rehabilitation of visual acuity in patients with severe ocular surface disorders. Cultivation of limbal epithelial stem cell with fetal bovine serum for transplantation has been a promising treatment for reconstructing the ocular surface in severe limbal stem cell deficiency caused by Steven Johnson syndrome, chemical or thermal injury. This technique of "cell therapy" has been accepted worldwide but the cost of cultivating the cells for transplantation is high. The objective of this study was to investigate the effect of cord blood serum in place of fetal bovine serum on the growth of human limbal epithelial cell culture. Our group has experimented with human cord blood serum which was obtained free of cost from willing donors. The use of human cord blood serum in place of fetal bovine serum for ex vivo culture of limbal stem cell has helped us in reducing the cost of culture. Fresh human limbal tissues from donor cadavers were cultured on intact and denuded amniotic membrane. Cells were proliferated in vitro with cell culture media containing human cord blood serum. Reverse transcription-polymerase chain reaction and immunofluorescence cytochemistry of cultured human limbal epithelial stem cell was done for characterization of the cells.

  20. Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia.

    PubMed

    Seminario-Vidal, Lucia; Okada, Seiko F; Sesma, Juliana I; Kreda, Silvia M; van Heusden, Catharina A; Zhu, Yunxiang; Jones, Lisa C; O'Neal, Wanda K; Penuela, Silvia; Laird, Dale W; Boucher, Richard C; Lazarowski, Eduardo R

    2011-07-29

    ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.

  1. Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia*

    PubMed Central

    Seminario-Vidal, Lucia; Okada, Seiko F.; Sesma, Juliana I.; Kreda, Silvia M.; van Heusden, Catharina A.; Zhu, Yunxiang; Jones, Lisa C.; O'Neal, Wanda K.; Penuela, Silvia; Laird, Dale W.; Boucher, Richard C.; Lazarowski, Eduardo R.

    2011-01-01

    ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia. PMID:21606493

  2. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

    PubMed

    Schlage, Walter K; Iskandar, Anita R; Kostadinova, Radina; Xiang, Yang; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Talikka, Marja; Geertz, Marcel; Mathis, Carole; Ivanov, Nikolai; Hoeng, Julia; Peitsch, Manuel C

    2014-10-01

    Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

  3. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures

    PubMed Central

    Schlage, Walter K.; Kostadinova, Radina; Xiang, Yang; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Talikka, Marja; Geertz, Marcel; Mathis, Carole; Ivanov, Nikolai; Hoeng, Julia; Peitsch, Manuel C.

    2014-01-01

    Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air–liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products. PMID:25046638

  4. 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.

  5. In vivo-in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems.

    PubMed

    Sauer, Ursula G; Vogel, Sandra; Hess, Annemarie; Kolle, Susanne N; Ma-Hock, Lan; van Ravenzwaay, Bennard; Landsiedel, Robert

    2013-02-01

    The usefulness of in vitro systems to predict acute inhalation toxicity was investigated. Nineteen substances were tested in three-dimensional human airway epithelial models, EpiAirway™ and MucilAir™, and in A549 and 3T3 monolayer cell cultures. IC(50) values were compared to rat four-hour LC(50) values classified according to EPA and GHS hazard categories. Best results were achieved with a prediction model distinguishing toxic from non-toxic substances, with satisfactory specificities and sensitivities. Using a self-made four-level prediction model to classify substances into four in vitro hazard categories, in vivo-in vitro concordance was mediocre, but could be improved by excluding substances causing pulmonary edema and emphysema in vivo. None of the test systems was outstanding, and there was no evidence that tissue or monolayer systems using respiratory tract cells provide an added value. However, the test systems only reflected bronchiole epithelia and alveolar cells and investigated cytotoxicity. Effects occurring in other cells by other mechanisms could not be recognised. Further work should optimise test protocols and expand the set of substances tested to define applicability domains. In vivo respiratory toxicity data for in vitro comparisons should distinguish different modes of action, and their relevance for human health effects should be ensured.

  6. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus.

    PubMed

    Vilela, Ricardo Chaves; Benchimol, Marlene

    2013-02-01

    Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

  7. Decreasing SMPD1 activity in BEAS-2B bronchial airway epithelial cells results in increased NRF2 activity, cytokine synthesis and neutrophil recruitment.

    PubMed

    MacFadden-Murphy, Elyse; Roussel, Lucie; Martel, Guy; Bérubé, Julie; Rousseau, Simon

    2017-01-22

    Niemann-Pick disease (NPD) type B is a rare autosomal recessive disease characterized by variable levels of impairment in sphingomyelin phosphodiesterase 1 (SMPD1) activity. Lung involvement is the most important prognostic factor in NPD-B, with recurrent respiratory infections starting in infancy being the major cause of morbidity and mortality. We hypothesized that decreased SMPD1 activity impaired airway epithelium host defense response. SMPD1 activity was reduced using inducible shRNA. Surprisingly, decreasing SMPD1 activity by 50%, resulted in increased neutrophil recruitment, both at baseline and in response to bacterial stimulation. This correlated with elevated levels of cytokine mRNA shown to contribute to neutrophil recruitment in unstimulated (e.g. IL-8 and GRO-α) and infected cells (e.g. IL-8, GRO-α, GM-CSF and CCL20). Instead of preventing the host defence responses, decreased SMPD1 activity results in an inflammatory response even in the absence of infection. Moreover, decreasing SMPD1 activity resulted in a pro-oxidative shift. Accordingly, expression of an inactive mutant, SMPD1[L225P] but not the WT enzyme increased activation of the antioxidant transcription factor NRF2. Therefore, decreasing SMPD1 activity by 50% in airway epithelial cells, the equivalent of the loss of one allele, results in the accumulation of oxidants that activates NRF2 and a concomitant increased cytokine production as well as neutrophil recruitment. This can result in a chronic inflammatory state that impairs host defence similar to scenarios observe in other chronic inflammatory lung disease such as Chronic Obstructive Pulmonary Disease or Cystic Fibrosis.

  8. The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

    PubMed Central

    Jordan, R.; Mawhorter, M. E.; Noton, S. L.; Powers, J. G.; Fearns, R.; Cihlar, T.; Perron, M.

    2015-01-01

    ABSTRACT Human respiratory syncytial virus (RSV) is a single-stranded RNA virus that causes acute, and occasionally fatal, lower respiratory illness in young infants, the elderly, and immunocompromised patients. Therapeutic interventions able to cut short viral replication and quickly return the airways to normal function are needed. An understanding of antiviral activities and their effects on host defense mechanisms is important for the design of safe and effective therapy. We targeted functionally and temporally distinct steps within the viral life cycle using small-molecule RSV inhibitors and studied their antiviral activities and their effects on innate interferon responses of airway epithelial cells in vitro. Antivirals acting upstream of RSV polymerase activity (i.e., compounds targeting the fusion protein or the nucleoprotein) reduced viral load immediately postinfection and partially attenuated interferon responses. In contrast, antivirals directed to the RSV polymerase demonstrated activity throughout the viral replication cycle and specifically modulated the RIG-I/mitochondrial antiviral signaling protein (MAVS)/TBK1/IRF3/interferon-stimulated gene (ISG) axis, causing either an upregulation or a downregulation of interferon responses, depending on the mechanism of polymerase inhibition. Notably, polymerase inhibition leading to the accumulation of abortive RNA products correlated with the amplification of interferon-stimulated genes to up to 10 times above normal infection levels. Understanding how antiviral activities and their modulation of innate immunity may affect recovery from RSV infection will help guide the development of safe and effective therapies. IMPORTANCE RSV circulates seasonally, causing acute lower respiratory disease. Therapeutic interventions with efficacy throughout the viral replication cycle, rapid viral clearance, and prevention of potentially harmful inflammatory responses are desirable. Compounds targeting the RSV polymerase

  9. 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

  10. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

    PubMed

    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  11. An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

    PubMed Central

    Birch, Nigel P.; Suresh, Vinod

    2016-01-01

    Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI

  12. A Systems Biology Approach Reveals the Dose- and Time-Dependent Effect of Primary Human Airway Epithelium Tissue Culture After Exposure to Cigarette Smoke In Vitro

    PubMed Central

    Mathis, Carole; Gebel, Stephan; Poussin, Carine; Belcastro, Vincenzo; Sewer, Alain; Weisensee, Dirk; Hengstermann, Arnd; Ansari, Sam; Wagner, Sandra; Peitsch, Manuel C; Hoeng, Julia

    2015-01-01

    To establish a relevant in vitro model for systems toxicology-based mechanistic assessment of environmental stressors such as cigarette smoke (CS), we exposed human organotypic bronchial epithelial tissue cultures at the air liquid interface (ALI) to various CS doses. Previously, we compared in vitro gene expression changes with published human airway epithelia in vivo data to assess their similarities. Here, we present a follow-up evaluation of these in vitro transcriptomics data, using complementary computational approaches and an integrated mRNA–microRNA (miRNA) analysis. The main cellular pathways perturbed by CS exposure were related to stress responses (oxidative stress and xenobiotic metabolism), inflammation (inhibition of nuclear factor-κB and the interferon gamma-dependent pathway), and proliferation/differentiation. Within post-exposure periods up to 48 hours, a transient kinetic response was observed at lower CS doses, whereas higher doses resulted in more sustained responses. In conclusion, this systems toxicology approach has potential for product testing according to “21st Century Toxicology”. PMID:25788831

  13. A systems biology approach reveals the dose- and time-dependent effect of primary human airway epithelium tissue culture after exposure to cigarette smoke in vitro.

    PubMed

    Mathis, Carole; Gebel, Stephan; Poussin, Carine; Belcastro, Vincenzo; Sewer, Alain; Weisensee, Dirk; Hengstermann, Arnd; Ansari, Sam; Wagner, Sandra; Peitsch, Manuel C; Hoeng, Julia

    2015-01-01

    To establish a relevant in vitro model for systems toxicology-based mechanistic assessment of environmental stressors such as cigarette smoke (CS), we exposed human organotypic bronchial epithelial tissue cultures at the air liquid interface (ALI) to various CS doses. Previously, we compared in vitro gene expression changes with published human airway epithelia in vivo data to assess their similarities. Here, we present a follow-up evaluation of these in vitro transcriptomics data, using complementary computational approaches and an integrated mRNA-microRNA (miRNA) analysis. The main cellular pathways perturbed by CS exposure were related to stress responses (oxidative stress and xenobiotic metabolism), inflammation (inhibition of nuclear factor-κB and the interferon gamma-dependent pathway), and proliferation/differentiation. Within post-exposure periods up to 48 hours, a transient kinetic response was observed at lower CS doses, whereas higher doses resulted in more sustained responses. In conclusion, this systems toxicology approach has potential for product testing according to "21st Century Toxicology".

  14. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-12-25

    Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/{beta}-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/{beta}-catenin signaling were determined, suggested down-regulation of Wnt/{beta}-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3{alpha} can inhibit the epithelial differentiation of MSCs. A loss of {beta}-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated {beta}-catenin expression and subsequently decreased {beta}-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/{beta}-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

  15. Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

    PubMed Central

    Rother, Jan; Büchsenschütz-Göbeler, Matthias; Nöding, Helen; Steltenkamp, Siegfried; Samwer, Konrad; Janshoff, Andreas

    2015-01-01

    The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m−1, and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells. PMID:25566882

  16. Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

    PubMed Central

    Amemiya, Takeshi; Nakamura, Takahiro; Yamamoto, Toshiro; Kinoshita, Shigeru; Kanamura, Narisato

    2015-01-01

    The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects. PMID:25915046

  17. Splitting culture medium by air-jet and rewetting for the assessment of the wettability of cultured epithelial cell surfaces.

    PubMed

    Tanaka, Nobuyuki; Kondo, Makoto; Uchida, Ryohei; Kaneko, Makoto; Sugiyama, Hiroaki; Yamato, Masayuki; Okano, Teruo

    2013-12-01

    This study found that the phenomenon of rewetting after squeezing culture medium varied in different culture conditions for rat oral mucosal epithelial cells. When culture medium covering over cultured cells was squeezed by an air-jet application, the motion of squeezed culture medium was able to be observed by using a commercially available movie camera. Squeezed width on cells cultured in keratinocyte culture medium (KCM), which contained with fetal bovine serum, was one-sixth of that in FBS-free KCM. This result corresponded to the mucous layer staining statuses of cultured cells in both cases; positive in KCM and negative in FBS-free medium. Furthermore, the gene expression of mucous glycoprotein MUC4 in KCM was 100 times higher than that in FBS-free medium, and the expression of MUC4 protein only showed on the apical surface of cells cultured in KCM. The relative gene expression levels of MUC1, 13, 15, and 16 in both the normal and FBS-free medium were found to be no more than one-thirtieth of that of MUC4 in KCM. The main factor of the wettability difference between KCM and FBS-free medium was speculated to be the difference of MUC4 expression between both media. This method can be a simple technique for testing not only the surface wettability but also the mucous formation of cultured cells.

  18. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    PubMed Central

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

  19. Long-term culture and partial characterization of dog gallbladder epithelial cells

    SciTech Connect

    Oda, D.; Lee, S.P.; Hayashi, A. )

    1991-05-01

    We describe the successful isolation and maintenance of primary cultures of dog gallbladder epithelial cells. The surgically removed gallbladder was treated with trypsin/EDTA for 45 minutes and epithelial cells were collected and resuspended in Eagle's minimum essential medium with 10% fetal calf serum, and plated on Vitrogen-coated culture dishes. Each gallbladder yielded approximately 12 to 15 x 10{sup 6} columnar epithelial cells, greater than 95% of which were viable by trypan blue exclusion. In culture, cells maintained their polarity. They were arranged and grew in small and tight clusters that coalesced at confluency. When examined using transmission electron microscopy, prominent and numerous microville were identified on the apical portion of the plasma membrane. Cells were connected by well-formed desmosomes. Scanning electron microscopy revealed clusters of polyhedral cells with numerous papillary projections. Immunohistochemical studies demonstrated uniform staining of cells to keratin 35BH11 and AE1. Histochemical studies were positive for gamma-glutamyl transpeptidase and negative for glucose-6-phosphatase and albumin. Cells incorporated ({sup 3}H)uridine into intracellular proteins and ({sup 14}C)glucosamine into tissue and secreted mucous glycoproteins linearly over 2 to 24 hours. Flow cytometry studies demonstrated a consistent and reproducible number of cells (10 to 12%) at S-phase. However, the number of cells at S-phase was dramatically reduced to almost negligible as cells reached confluency. This method of culturing primary dog gallbladder epithelial cells is highly reproducible and reliable. These cells preserve their state of differentiation, polarity, histochemical and immunohistochemical profile, morphologic, and metabolic integrity with repeated passaging or after being frozen.

  20. Inhibition of monoamine oxidase-B by selegiline reduces cigarette smoke-induced oxidative stress and inflammation in airway epithelial cells.

    PubMed

    Cui, Yuting; Liu, Kenneth W K; Liang, Yingmin; Ip, Mary S M; Mak, Judith C W

    2017-02-15

    Chronic obstructive pulmonary disease (COPD) is caused by the build-up of oxidative stress-induced damages due to cigarette smoking, but how monoamine oxidase (MAO)-B signaling is involved remains unclear. This study aims to establish the involvement of MAO-B signaling pathways in cigarette smoke medium (CSM)-induced oxidative stress and inflammation in human airway epithelial cells (AECs). CSM treatment increased MAO-B activity, ROS levels and IL-8 release in AECs. Pretreatment with MAO-B selective inhibitor selegiline reversed the CSM-induced changes in MAO-B activity, ROS levels and IL-8 release in a dose-dependent manner. Selegiline also reversed CSM-induced changes of anti-oxidant enzymes superoxide dismutase (SOD) and catalase (CAT) activities, GSH/GSSG ratio, as well expression of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1). The effects of selegiline are partially driven through the nuclear factor erythroid 2 related factor 2 (Nrf2) and cytosol translocation of its negative regulator, BTB and CNC homolog 1 (Bach1). Nevertheless, selegiline fully reversed the CSM-induced effects on IKK, cytoplasmic IκB expression, and nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit. Our study demonstrated that in AECs, inhibition of MAO-B using selegiline reversed the CSM-induced oxidative stress and inflammation. These data may provide a novel strategy for therapy in COPD.

  1. 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...

  2. Bisulfite and sulfite as derivatives of sulfur dioxide alters biomechanical behaviors of airway smooth muscle cells in culture.

    PubMed

    Song, Aijing; Lin, Feng; Li, Jianming; Liao, Qingfeng; Liu, Enmei; Jiang, Xuemei; Deng, Linhong

    2014-02-01

    Sulfur dioxide (SO2) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how SO2 in vivo affects airway smooth muscle (ASM) cells of which the mechanics is known to ultimately mediate airway hyperresponsiveness (AHR) - a hallmark feature of asthma. To this end, we investigated the effects of bisulfite/sulfite (1:3 M/M in neutral fluid to simulate the in vivo derivatives of inhaled SO2 in the airways), on the viability, migration, stiffness and contractility of ASM cells cultured in vitro. The results showed that bisulfite/sulfite consistently increased viability, migration, F-actin intensity and stiffness of ASM cells in similar fashion as concentration increasing from 10(-4) to 10(-1) mmol/L. However, bisulfite/sulfite increased the ASM cell contractility induced by KCl only at the concentration between 10(-4) and 10(-3) mmol/L (p < 0.05), while having no consistent effect on that induced by histamine. At the concentration of 10(0) mmol/L, bisulfite/sulfite became acutely toxic to the ASM cells. Taken together, the data suggest that SO2 derivatives at low levels in vivo may directly increase the mass, stiffness and contractility of ASM cells, which may help understand the mechanism in which specific air pollutants contribute in vivo to the pathogenesis of asthma.

  3. Probing the viscoelastic behavior of cultured airway smooth muscle cells with atomic force microscopy: stiffening induced by contractile agonist.

    PubMed

    Smith, Benjamin A; Tolloczko, Barbara; Martin, James G; Grütter, Peter

    2005-04-01

    Complex rheology of airway smooth muscle cells and its dynamic response during contractile stimulation involves many molecular processes, foremost of which are actomyosin cross-bridge cycling and actin polymerization. With an atomic force microscope, we tracked the spatial and temporal variations of the viscoelastic properties of cultured airway smooth muscle cells. Elasticity mapping identified stiff structural elements of the cytoskeletal network. Using a precisely positioned microscale probe, picoNewton forces and nanometer level indentation modulations were applied to cell surfaces at frequencies ranging from 0.5 to 100 Hz. The resulting elastic storage modulus (G') and dissipative modulus (G'') increased dramatically, with hysteresivity (eta = G''/G') showing a definitive decrease after stimulation with the contractile agonist 5-hydroxytryptamine. Frequency-dependent assays showed weak power-law structural damping behavior and universal scaling in support of the soft-glassy material description of cellular biophysics. Additionally, a high-frequency component of the loss modulus (attributed to cellular Newtonian viscosity) increased fourfold during the contractile process. The complex shear modulus showed a strong sensitivity to the degree of actin polymerization. Inhibitors of myosin light chain kinase activity had little effect on the stiffening response to contractile stimulation. Thus, our measurements appear to be particularly well suited for characterization of dynamic actin rheology during airway smooth muscle contraction.

  4. Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

    SciTech Connect

    Gutierrez-Barrera, Angelica M.; Menter, David G.; Abbruzzese, James L.; Reddy, Shrikanth A.G. . E-mail: sa08366@wotan.mdacc.tmc.edu

    2007-07-06

    Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7 organized into spheroids with what appeared to be apical and basal membranes and a luminal space. Immunostaining experiments indicated that protein kinase Akt was phosphorylated (Ser473) and CTMP, a negative Akt regulator, was expressed in both HPDE-E6E7 and cancer cells. However, a nuclear pool of CTMP was detectable in HPDE-E6E7 cells that showed a dynamic concentrated expression pattern, a feature that further distinguished HPDE-E637 cells from cancer cells. Collectively, these data suggest that 3D cultures of HPDE-E6E7 cells are useful for investigating signaling and morphological abnormalities in pancreatic cancer cells.

  5. Comparative evaluation of re-epithelialization promoted by fresh or cryopreserved cultured dermal substitute.

    PubMed

    Yamada, Naoto; Uchinuma, Eiju; Matsumoto, Yasuhiro; Kuroyanagi, Yoshimitsu

    2008-01-01

    Allogeneic cultured dermal substitute (CDS) was prepared by plating cultured fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen, followed by culturing for 1 week. The resulting fresh CDS was then cryopreserved in a freezer at -152 degrees C in accordance with conventional procedures. Fresh CDS was rinsed thoroughly with lactated Ringer's solution to remove fetal bovine serum (FBS) and was then used in the clinical study, whereas cryopreserved CDS was thawed and then rinsed with lactated Ringer's solution to remove cryoprotectant and FBS. The present study was designed to compare the efficacy of fresh and cryopreserved CDS in promoting reepithelialization at donor sites leaving behind split-thickness skin autografts. Fourteen donor sites were used for this comparative study. There were no differences in the period of complete re-epithelialization between the fresh and cryopreserved CDS. The results of this comparative study thus suggest that cryopreserved CDS is able to maintain the same level of potency to promote re-epithelialization as fresh CDS. This indicates that, although the release of growth factors is suppressed to some extent during the course of cryopreserving, thawing, and rinsing procedures, it is still sufficient to promote re-epithelialization.

  6. MCP-1 expression by rat type II alveolar epithelial cells in primary culture.

    PubMed

    Paine, R; Rolfe, M W; Standiford, T J; Burdick, M D; Rollins, B J; Strieter, R M

    1993-05-15

    Recruitment and activation of mononuclear phagocytes are potentially critical regulatory events for control of pulmonary inflammation. Located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells are ideally situated to regulate the recruitment and activation of mononuclear phagocytes through the production of cytokines in response to inflammatory stimulation from the alveolar space. To test this hypothesis, we investigated the production of monocyte chemotactic polypeptide-1 (MCP-1), a protein that is chemotactic for and that activates monocytes, by rat type II alveolar epithelial cells in primary culture. Immunocytochemical staining using anti-murine JE, an antibody recognizing rat MCP-1, demonstrated cell-associated MCP-1 Ag throughout the monolayer. The intensity of staining was increased in response to IL-1 beta. When type II epithelial cells formed a tight monolayer on a filter support, there was polar secretion of MCP-1 Ag into the apical compartment by both control and IL-1-stimulated cells as measured by specific MCP-1 ELISA. Northern blot analysis revealed that IL-1 and TNF-alpha stimulated MCP-1 mRNA expression in a dose-dependent manner, whereas dexamethasone blocked MCP-1 expression by cells stimulated with IL-1. In contrast to previous results using transformed epithelial cell lines, MCP-1 mRNA was induced in these primary cultures directly by stimulation with LPS. These data suggest that alveolar epithelial cells may have an important and previously unrecognized role in the initiation and maintenance of inflammatory processes in the lung by recruiting and activating circulating monocytes through the production of MCP-1.

  7. Thick airway surface liquid volume and weak mucin expression in pendrin-deficient human airway epithelia

    PubMed Central

    Lee, Hyun Jae; Yoo, Jee Eun; Namkung, Wan; Cho, Hyung-Ju; Kim, Kyubo; Kang, Joo Wan; Yoon, Joo-Heon; Choi, Jae Young

    2015-01-01

    Pendrin is an anion exchanger whose mutations are known to cause hearing loss. However, recent data support the linkage between pendrin expression and airway diseases, such as asthma. To evaluate the role of pendrin in the regulation of the airway surface liquid (ASL) volume and mucin expression, we investigated the function and expression of pendrin and ion channels and anion exchangers. Human nasal epithelial cells were cultured from 16 deaf patients carrying pendrin mutations (DFNB4) and 17 controls. The cells were treated with IL-13 to induce mucus hypersecretion. Airway surface liquid thickness was measured and real-time polymerase chain reaction was performed targeting various transporters and MUC5AC. Anion exchanger activity was measured using a pH-sensitive fluorescent probe. Periodic acid-Schiff staining was performed on the cultured cells and inferior turbinate tissues. The ASL layer of the nasal epithelia from DFNB4 subjects was thicker than the controls, and the difference became more prominent following IL-13 stimulation. There was no difference in anion exchange activity after IL-13 treatment in the cells from DFNB4 patients, while it increased in the controls. Goblet cell metaplasia induced by IL-13 treatment seen in the controls was not observed in the DFNB4 cells. Furthermore, the periodic acid-Schiff staining-positive area was lesser in the inferior turbinate tissues from DFNB4 patients that those from controls. Pendrin plays a critical role in ASL volume regulation and mucin expression as pendrin-deficient airway epithelial cells are refractory to stimulation with IL-13. Specific blockers targeting pendrin in the airways may therefore have therapeutic potential in the treatment of allergic airway diseases. PMID:26243215

  8. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures

    PubMed Central

    Doyle, Timothy E.; Goodrich, Jeffrey B.; Ambrose, Brady J.; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H.

    2010-01-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  9. Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

    PubMed

    Kobayashi, Takeshi; Shiraishi, Atsushi; Hara, Yuko; Kadota, Yuko; Yang, Lujun; Inoue, Tomoyuki; Shirakata, Yuji; Ohashi, Yuichi

    2015-06-01

    Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-β1, TGF-β2, and TGF-β3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-β family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell

  10. Isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture.

    PubMed

    Xu, Kun; Buchsbaum, Rachel J

    2012-04-30

    While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors(1-2), much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension(3-4). Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture(3). While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis. The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized(4). Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells(5). Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals(6). Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors(7). Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion(8-15). We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three

  11. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  12. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    PubMed

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

  13. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses

    PubMed Central

    Ozbun, Michelle A.; Patterson, Nicole A.

    2014-01-01

    Papillomaviruses have a strict tropism for epithelial cells and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro wherein virion morphogenesis occurs under cooperative viral and cellular cues requires the cultivation of epithelium. Presented in the first section of this unit is a protocol for growing differentiating epithelial tissues, whose structure and function mimics many important morphological and biochemical aspects of normal skin. The technique, pioneered by Asslineau and Pruniéras (Asselineau and Prunieras 1984) and modified by Kopan et al. (Kopan et al. 1987), involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname “raft” cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, as well as keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single step virus growth

  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.

  15. Enhanced growth medium and method for culturing human mammary epithelial cells

    DOEpatents

    Stampfer, Martha R.; Smith, Helene S.; Hackett, Adeline J.

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  16. The biomedical aspects of oral mucosal epithelial cell culture in mammals.

    PubMed

    Bryja, A; Dyszkiewicz-Konwińska, M; Budna, J; Kranc, W; Chachuła, A; Borys, S; Ciesiółka, S; Sokalski, J; Prylinski, M; Bukowska, D; Antosik, P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

    2017-01-01

    In recent years, there has been a growing interest in epithelial cell tissue culture, particularly oral mucosa and its application utilizing in vitro cell culture in medicine. This involves tests using animal models to better understand oral mucosa function, and the differences in its construction in various animal models. The use of buccal pouch mucosal cell culture provides insight into the processes of trans mucosal transport and regeneration of the oral epithelium. The processes associated with epithelium regeneration is the base for stem cell research and/or oral cancer investigation. These artificially cultured tissue equivalents are used in transplant surgery for the treatment of a variety of tissue dysfunctions, i.e. eye, esophagus, or urethra. In this review, the most recent results from studies carried out on in animal models, which may be applied in areas such as regenerative medicine and reconstructive surgery, were explored.

  17. PRIMARY CULTURE OF CHOROIDAL EPITHELIAL CELLS: CHARACTERIZATION OF AN IN VITRO MODEL OF BLOOD-CSF BARRIER

    PubMed Central

    ZHENG, WEI; ZHAO, QIUQU; GRAZIANO, JOSEPH H.

    2016-01-01

    Summary A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2–5 × 104 cells from pooled plexuses of three to four rats, and a viability of 77–85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2–3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 ± 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood–CSF barrier. PMID:9542634

  18. Inhibition of airway surface fluid absorption by cholinergic stimulation

    PubMed Central

    Joo, Nam Soo; Krouse, Mauri E.; Choi, Jae Young; Cho, Hyung-Ju; Wine, Jeffrey J.

    2016-01-01

    In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20–70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways. PMID:26846701

  19. Exposure to Mexicali PM10 Induces IL-8 Expression Through an Alternative NFKB Mechanism In Human Airway Epithelial Cells

    EPA Science Inventory

    Studies have shown associations between exposure to ambient air particulate matter (PM) and increased rates of cardio-pulmonary morbidity and mortality. The aim of this study was to examine the signaling events involved in the expression of inflammatory genes in cultured human ai...

  20. Effects of steroids on the secretion of immunoregulatory factors by thymic epithelial cell cultures.

    PubMed Central

    Stimson, W H; Crilly, P J

    1981-01-01

    Rat thymic epithelial cells were cultured for 39 days in the presence of various concentrations of oestradiol, testosterone, progesterone and corticosterone and the supernatants assessed for effects on the stimulation of cells from the thymus, bone marrow, lymph nodes and spleen, with several agents. All the steroids, except progesterone, were found to significantly regulate the secretion of immunoregulatory factors by the epithelial cells at physiological levels but the effects were dose dependent. Fractionation of active supernatants indicated that the capacity to enhance or depress cellular proliferation was mainly associated with substances having molecular weights greater than 30,000 or less than 1000, respectively. This study supports the idea that certain steroids can influence the immune response indirectly through the thymus. PMID:7298074

  1. The TAK1→IKKβ→TPL2→MKK1/MKK2 Signaling Cascade Regulates IL-33 Expression in Cystic Fibrosis Airway Epithelial Cells Following Infection by Pseudomonas aeruginosa

    PubMed Central

    Farias, Raquel; Rousseau, Simon

    2016-01-01

    In cystic fibrosis (CF), chronic respiratory infections result in an exaggerated and uncontrolled inflammatory response that ultimately lead to a decrease in pulmonary function. We have previously described the presence of the alarmin IL-33 in lung explants from CF patients. The signals regulating IL-33 expression in the airway epithelium following a gram-negative bacterial infection are currently unknown. Our objective was to characterize the pathways in CF airway epithelial cells (AECs) leading to an increase in IL-33 expression. We found that, in CF AECs expressing a deletion of a phenylalanine at position 508 of the gene coding for Cystic Fibrosis Transmembrane Conductance Regulator (CFTRdelF508), exposure to live Pseudomonas aeruginosa upregulates IL-33 via the TLR2 and TLR5 signaling pathways. This up-regulation can be partially or fully reverted by pre-incubating CFTRdelF508 AECs with a CFTR corrector (VX-809) and/or a CFTR potentiator (VX-770). Similarly, incubation with the CFTR corrector and/or the CFTR potentiator also decreased IL-8 expression in response to infection. Moreover, using different protein kinase inhibitors that target elements downstream of TLR signaling, we show that the TAK1→IKKβ→TPL2→MKK1/MKK2 pathway regulates IL-33 expression following an infection with P. aeruginosa. Our findings represent the first characterization of the signals regulating IL-33 expression in CF airway epithelial cells in response to a bacterial infection. PMID:26793709

  2. The level of p38α mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.

    PubMed

    Beaudoin, Trevor; LaFayette, Shantelle; Roussel, Lucie; Bérubé, Julie; Desrosiers, Martin; Nguyen, Dao; Rousseau, Simon

    2013-05-15

    Biofilm microcolonies of Pseudomonas aeruginosa chronically infect the airways of patients with cystic fibrosis and fuel ongoing destructive inflammation, yet the impact of the switch from planktonic to biofilm growth on host responses is poorly understood. We report that in airway epithelial cells a threshold of p38α mitogen-activated protein kinase (MAPK) activation was required to trigger neutrophil recruitment, which is influenced by extrinsic and intrinsic factors. Planktonic P. aeruginosa diffusible material (PsaDM) induced stronger p38α MAPK activation as compared to biofilm PsaDM. Biofilm PsaDM activated p38α MAPK in a Toll-like receptor-independent fashion via the lasI/lasR quorum-sensing system, but this activation was insufficient to recruit neutrophils. However, in airway epithelial cells from patients with cystic fibrosis with hypersensitivity to injurious stimuli, biofilm PsaDM activated p38α MAPK strongly enough to recruit neutrophils, which can contribute to lung injury.

  3. CDK9-dependent transcriptional elongation in the innate interferon-stimulated gene response to respiratory syncytial virus infection in airway epithelial cells.

    PubMed

    Tian, Bing; Zhao, Yingxin; Kalita, Mridul; Edeh, Chukwudi B; Paessler, Slobodan; Casola, Antonella; Teng, Michael N; Garofalo, Roberto P; Brasier, Allan R

    2013-06-01

    Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3(-/-) MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser(2) carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.

  4. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    PubMed Central

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  5. Intestinal epithelial culture under an air-liquid interface: a tool for studying human and mouse esophagi.

    PubMed

    Yokobori, T; Suzuki, S; Miyazaki, T; Sohda, M; Sakai, M; Tanaka, N; Ozawa, D; Hara, K; Honjo, H; Altan, B; Fukuchi, M; Ishii, H; Iwatsuki, M; Sugimachi, K; Sudo, T; Iwaya, T; Nishida, N; Mimori, K; Kuwano, H; Mori, M

    2016-10-01

    This study investigated whether an intestinal epithelial culture method can be applied to mouse and human esophageal cultures. The esophagi harvested from 1-day-old mice and adult humans were maintained in collagen gels. A commercially available culture medium for human embryonic stem cells was used for the human esophageal culture. We discovered that the intestinal epithelial culture method can be successfully applied to both mouse and human esophageal cultures. The long-term cultured esophageal organoids were rod-like luminal structures lined with myofibroblasts. We discovered that regeneration of the esophageal mucosal surface can be almost completely achieved in vitro, and the advantage of this method is that organoid cultures may be generated using host-derived fibroblasts as a niche. This method is a promising tool for mouse and human research in intestinal biology, carcinogenesis, and regenerative medicine.

  6. 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

    2015-07-03

    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.

  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.

  8. 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

  9. 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.

  10. 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+}.

  11. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    NASA Technical Reports Server (NTRS)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  12. Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.

    PubMed

    Cravero, Diego; Diego, Cravero; Martignani, Eugenio; Eugenio, Martignani; Miretti, Silvia; Silvia, Miretti; Macchi, Elisabetta; Elisabetta, Macchi; Accornero, Paolo; Paolo, Accornero; Baratta, Mario; Mario, Baratta

    2014-10-01

    The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P < 0.5) only after day 20. In both conditions, after prolonged culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible.

  13. Gene Delivery to the Airway

    PubMed Central

    Keiser, Nicholas W.; Engelhardt, John F.

    2013-01-01

    This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols. PMID:23853081

  14. Effects and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial</