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Sample records for airway ciliated cells

  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. 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

  3. Chibby functions to preserve normal ciliary morphology through the regulation of intraflagellar transport in airway ciliated cells.

    PubMed

    Siller, Saul S; Burke, Michael C; Li, Feng-Qian; Takemaru, Ken-Ichi

    2015-01-01

    Airway cilia provide the coordinated motive force for mucociliary transport, which prevents the accumulation of mucus, debris, pollutants, and bacteria in our respiratory tracts. As airway cilia are constantly exposed to the environment and, hence, are an integral component of the pathogenesis of several congenital and chronic pulmonary disorders, it is necessary to understand the molecular mechanisms that control ciliated cell differentiation and ciliogenesis. We have previously reported that loss of the basal body protein Chibby (Cby) results in chronic upper airway infection in mice due to a significant reduction in the number of airway cilia. In the present work, we demonstrate that Cby is required for normal ciliary structure and proper distribution of proteins involved in the bidirectional intraflagellar transport (IFT) system, which consists of 2 distinct sub-complexes, IFT-A and IFT-B, and is essential for ciliary biogenesis and maintenance. In fully differentiated ciliated cells, abnormal paddle-like cilia with dilated ciliary tips are observed in Cby-/- airways and primary cultures of mouse tracheal epithelial cells (MTECs). In addition, IFT88, an IFT-B sub-complex protein, robustly accumulates within the dilated tips of both multicilia in Cby-/- MTECs and primary cilia in Cby-/- mouse embryonic fibroblasts (MEFs). Furthermore, we show that only IFT-B components, including IFT20 and IFT57, but not IFT-A and Bardet-Biedl syndrome (BBS) proteins, amass with IFT88 in these distended tips in Cby-/- ciliated cells. Taken together, our findings suggest that Cby plays a role in the proper distribution of IFT particles to preserve normal ciliary morphology in airway ciliated cells.

  4. The differentiated airway epithelium infected by influenza viruses maintains the barrier function despite a dramatic loss of ciliated cells

    PubMed Central

    Wu, Nai-Huei; Yang, Wei; Beineke, Andreas; Dijkman, Ronald; Matrosovich, Mikhail; Baumgärtner, Wolfgang; Thiel, Volker; Valentin-Weigand, Peter; Meng, Fandan; Herrler, Georg

    2016-01-01

    Virus-host interactions in the respiratory epithelium during long term influenza virus infection are not well characterized. Therefore, we developed an air-liquid interface culture system for differentiated porcine respiratory epithelial cells to study the effect of virus-induced cellular damage. In our well-differentiated cells, α2,6-linked sialic acid is predominantly expressed on the apical surface and the basal cells mainly express α2,3-linked sialic acid. During the whole infection period, release of infectious virus was maintained at a high titre for more than seven days. The infected epithelial cells were subject to apoptosis resulting in the loss of ciliated cells together with a thinner thickness. Nevertheless, the airway epithelium maintained trans-epithelial electrical resistance and retained its barrier function. The loss of ciliated cells was compensated by the cells which contained the KRT5 basal cell marker but were not yet differentiated into ciliated cells. These specialized cells showed an increase of α2,3-linked sialic acid on the apical surface. In sum, our results help to explain the localized infection of the airway epithelium by influenza viruses. The impairment of mucociliary clearance in the epithelial cells provides an explanation why prior viral infection renders the host more susceptible to secondary co-infection by another pathogen. PMID:28004801

  5. CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner

    PubMed Central

    Jeong, Kwang-Il; Piepenhagen, Peter A.; Kishko, Michael; DiNapoli, Joshua M.; Groppo, Rachel P.; Zhang, Linong; Almond, Jeffrey; Kleanthous, Harry; Delagrave, Simon; Parrington, Mark

    2015-01-01

    Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein’s CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma. PMID:26107373

  6. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    PubMed Central

    González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

    2013-01-01

    Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

  7. 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...

  8. The role of oestrogen in the control of ciliated cells of the human endometrium.

    PubMed

    More, I A; Masterton, R G

    1976-05-01

    The incidence of ciliated cells in the human endometrium was determined. Conditions associated with an excess of oestrogenic activity were characterized by an increased incidence of ciliated cells, whilst oestrogen deficiency was associated with decreased numbers. When endometrium was cultured, addition of oestradiol-17 beta caused an increase in the ciliated cell population.

  9. [Early morphogenesis of ciliated cells in human oral cavity].

    PubMed

    Kurtova, A I; Chernikov, V P; Savel'ev, S V

    2013-01-01

    Ciliated cells were found in the epithelium of the oral cavity of human embryos and fetuses starting from the seventh week of prenatal development. At the early stages of prenatal development (until the 13th week), cells with cilia cover most of the dorsal surface of the tongue and the soft palate, whereas they are found only near the gland ducts in the circumvallate and foliate lingual papillae after 17 weeks of development. The ultrastructure of the axoneme of cilia corresponds to the structure of motile cilia and is represented by nine microtubule doublets that surround the central pair of microtubule singlets. An immunohistochemical study performed on weeks 10-12 of development identified nerve endings associated with the ciliated cells. Until the 14th week of development, the cytoplasm of ciliated cells is immunopositive for NSE. The spatial distribution of ciliated cells in the tongue epithelium until the 13th week of development is not related to the morphogenesis of lingual papillae, and their role in the human oral cavity during the first trimester of pregnancy is unclear and requires further study.

  10. Alterations of ciliate phosducin phosphorylation in Blepharisma japonicum cells.

    PubMed

    Sobierajska, Katarzyna; Fabczak, Hanna; Fabczak, Stanisław

    2005-05-13

    We have previously reported that motile photophobic response in ciliate Blepharisma japonicum correlates with dephosphorylation of a cytosolic 28 kDa phosphoprotein (PP28) exhibiting properties similar to those of phosducin. Here we demonstrate in in vivo phosphorylation assay that the light-elicited dephosphorylation of the PP28 is significantly modified by cell incubation with substances known to modulate protein phosphatase and kinase activities. Immunoblot analyses showed that incubation of ciliates with okadaic acid and calyculin A, potent inhibitors of type 1 or 2A protein phosphatases, distinctly increased phosphorylation of PP28 in dark-adapted cells and markedly weakened dephosphorylation of the ciliate phosducin following cell illumination. An enhancement of PP28 phosphorylation was also observed in dark-adapted ciliates exposed to 8-Br-cAMP and 8-Br-cGMP, slowly hydrolysable cyclic nucleotide analogs and 3-isobutyryl-1-methylxanthine (IBMX), a non-specific cyclic nucleotide phosphodiesterase (PDEs) inhibitor. Only slight changes in light-evoked dephosphorylation levels of PP28 were observed in cells treated with the cyclic nucleotide analogs and IBMX. Incubation of ciliates with H 89 or KT 5823, highly selective inhibitor of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, decreased PP28 phosphorylation levels in dark-adapted cells, whereas the extent of light-evoked dephosphorylation of the phosphoprotein was only slightly influenced. Cell treatment with higher Ca2+ concentration together with ionophore A23187 in culture medium resulted in marked increase in PP28 phosphorylation levels, while quite an opposite effect was observed in cells exposed to Ca2+ chelators, EGTA or BAPTA/AM as well as calmodulin antagonists, such as trifluoperazine (TFP), W-7 or calmidazolium. Light-dependent dephosphorylation was not considerably affected by these treatments. The experimental findings presented here suggest that an

  11. Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys

    PubMed Central

    Schaap-Nutt, Anne; Scull, Margaret A.; Schmidt, Alexander C.; Murphy, Brian R.; Pickles, Raymond J.

    2010-01-01

    Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32°C and exhibited little productive replication at 37°C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates. PMID:20139039

  12. Purinergic stimulation of rabbit ciliated airway epithelia: control by multiple calcium sources.

    PubMed Central

    Korngreen, A; Priel, Z

    1996-01-01

    1. Simultaneous measurements of average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were carried out on ciliated rabbit tracheal cells in order to determine quantitatively the role of calcium in the regulation of mucus-transporting cilia. 2. Extracellular ATP caused a rapid increase in both [Ca2+]i and CBF in the 0.1-1000 microM concentration range. The rise in [Ca2+]i levelled off to an elevated [Ca2+]i plateau while the cilia remained in a high activation state. The magnitude of the rise in [Ca2+]i and CBF as well as the value of the elevated [Ca2+]i plateau and the value of the sustained CBF were dependent on the concentration of ATP in the solution. 3. No correlation was found between the mean values of [Ca2+]i and CBF at rest but a sigmoidal relationship was found to exist between the maximal rises of these parameters following excitation with extracellular ATP. This sigmoidal correlation incorporated the experiments where [Ca2+]i rise was induced by depletion of internal calcium stores with thapsigargin or by entry of calcium induced by ionomycin. 4. Extracellular ATP caused both the release of calcium from internal stores and calcium influx from the extracellular solution. The release of calcium was identified as originating from a thapsigargin-sensitive and a thapsigargin-insensitive calcium store. It is suggested that the release of calcium from these stores induces the initial rise in CBF. 5. The sustained activation of the cilia and elevated calcium plateau were found to be the result of the extracellular ATP-induced calcium influx. This calcium influx was insensitive to the voltage-gated calcium channel inhibitors verapamil and diltiazem, but was completely eliminated by lowering the extracellular calcium concentration to 0.1 microM. 6. We propose that the initial jump in the CBF is mediated by the calcium released from a thapsigargin-insensitive calcium store adjacent to the cilia, while the later, and longer, rise in

  13. EGF-Amphiregulin Interplay in Airway Stem/Progenitor Cells Links the Pathogenesis of Smoking-Induced Lesions in the Human Airway Epithelium

    PubMed Central

    Zuo, Wu-Lin; Yang, Jing; Gomi, Kazunori; Chao, IonWa; Crystal, Ronald G.; Shaykhiev, Renat

    2017-01-01

    The airway epithelium of cigarette smokers undergoes dramatic remodeling with hyperplasia of basal cells (BC) and mucus-producing cells, squamous metaplasia, altered ciliated cell differentiation and decreased junctional barrier integrity, relevant to chronic obstructive pulmonary disease and lung cancer. In this study, we show that epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) is induced by smoking in human airway epithelium as a result of epidermal growth factor (EGF)-driven squamous differentiation of airway BC stem/progenitor cells. In turn, AREG induced a unique EGFR activation pattern in human airway BC, distinct from that evoked by EGF, leading to BC- and mucous hyperplasia, altered ciliated cell differentiation and impaired barrier integrity. Further, AREG promoted its own expression and suppressed expression of EGF, establishing an autonomous self-amplifying signaling loop in airway BC relevant for promotion of EGF-independent hyperplastic phenotypes. Thus, EGF-AREG interplay in airway BC stem/progenitor cells is one of the mechanisms that mediates the interconnected pathogenesis of all major smoking-induced lesions in the human airway epithelium. PMID:27709733

  14. Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

    SciTech Connect

    Rutten, A.A.; Beems, R.B.; Wilmer, J.W.; Feron, V.J.

    1988-09-01

    The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated (methyl-3H)-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 micron, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating (methyl-/sup 3/H)thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration.

  15. Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats.

    PubMed

    Cohen, Mitchell D; Vaughan, Joshua M; Garrett, Brittany; Prophete, Colette; Horton, Lori; Sisco, Maureen; Ghio, Andrew; Zelikoff, Judith; Lung-chi, Chen

    2015-01-01

    Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12-13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6-11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled

  16. Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats

    PubMed Central

    Cohen, Mitchell D.; Vaughan, Joshua M.; Garrett, Brittany; Prophete, Colette; Horton, Lori; Sisco, Maureen; Ghio, Andrew; Zelikoff, Judith; Lung-chi, Chen

    2015-01-01

    Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12–13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6–11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled

  17. New light sensor molecules of single-cell ciliates

    NASA Astrophysics Data System (ADS)

    Tao, Nengbing; Song, Pill-Soon

    1994-05-01

    The unicellular ciliate, Stentor coeruleus, exhibits sensitive light-avoiding behavior. The photosensor stentorin showed a (M - H)- at 591.1304, which is in accord with the formula C34H23O10. Acetylated stentorin, when FAB-desorbed as (M + H)+, shows a series of ions indicating the presence of eight hydroxyl groups. Additional confirmation is a collisionally activated decomposition (CAD) spectrum of the (M + H)+ of the octaacetate. The NMR spectrum of stentorin shows characteristic signals of isopropyl groups. Similar studies indicate that photosensor blepharismin from Blepharisma japonicum is structurally different from stentorin. Time-resolved fluorescence decays indicated that a primary event occurs within a few picoseconds. The stimulus light signal absorbed/perceived by Stentor and possibly by Blepharisma, is apparently amplified by a transient calcium influx into the cell. Preliminary studies suggest that signal transduction in both organisms utilizes G-protein(s) as an initial transducer and a cGMP-phosphodiesterase as the effector system, analogous to the visual system of higher animals.

  18. Delineating cellular interactions between ciliates and fish by co-culturing Tetrahymena thermophila with fish cells.

    PubMed

    Pinheiro, Marcel D O; Bols, Niels C

    2014-10-01

    Although several species of Tetrahymena are often described as histophagous and opportunistic pathogens of fish, little is known about ciliate/fish cell interactions, but one approach for studying these is in vitro with cell lines. In this study, T. thermophila, B1975 (wild type) and NP1 (temperature sensitive mutant for phagocytosis) were cultured on monolayers of 3 fish epithelial cell lines, CHSE-214, RTgill-W1, and ZEB2J, and the rabbit kidney epithelial cell line, RK-13. Generally the ciliates flourished, whereas the monolayers died, being completely consumed over several days. The destruction of monolayers required that the ciliates could make contact with the animal cells through swimming, which appeared to dislodge or loosen cells so that they could be phagocytosed. The ciliates internalized into food vacuoles ZEB2J from cell monolayers as well as from cell suspensions. Phagocytosis was essential for monolayer destruction as monolayers remained intact under conditions where phagocytosis was impeded, such as 37°C for NP1 and 4°C for B1975. Monolayers of fish cells supported the proliferation of ciliates. Thus T. thermophila can 'eat' animal cells or be histophagous in vitro, with the potential to be histophagous in vivo.

  19. 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

  20. Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells

    PubMed Central

    Hegan, Peter S.; Ostertag, Eric; Geurts, Aron M.; Mooseker, Mark S.

    2015-01-01

    In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost. PMID:26446290

  1. Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells.

    PubMed

    Hegan, Peter S; Ostertag, Eric; Geurts, Aron M; Mooseker, Mark S

    2015-10-01

    In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost.

  2. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

    PubMed

    Burke, Michael C; Li, Feng-Qian; Cyge, Benjamin; Arashiro, Takeshi; Brechbuhl, Heather M; Chen, Xingwang; Siller, Saul S; Weiss, Matthew A; O'Connell, Christopher B; Love, Damon; Westlake, Christopher J; Reynolds, Susan D; Kuriyama, Ryoko; Takemaru, Ken-Ichi

    2014-10-13

    Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

  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. Effects of histamine on ciliary beat frequency of ciliated cells from guinea pigs nasal mucosa.

    PubMed

    An, Fengwei; Xing, Lijun; Zhang, Zhiqiang; Chen, Lei

    2015-10-01

    We aimed to investigate the effect of histamine on ciliary beat frequency (CBF) through combining high-speed digital microscopy and patch-clamp technology. Ciliated cells were obtained from septum and turbinate of 90-120-day-old healthy male guinea pigs. Tight seal was formed by applying negative pressure on the glass electrode after the drawing and pushing progress. Then, we enrolled high-speed digital microscopy to measure CBF before and after treatment with histamine of different concentrations ranging from 10(-6) to 10(-1) mol/L in Hank's solution and D-Hank's solution as well as after administrating adenosine triphosphate. One-way ANOVA, Student's t test or Kruskal-Wallis test was used for statistical comparisons. Glass electrode fix up ciliated cell is available at tip diameter of 2-5 μm and negative pressure of 10-20 cmH2O column. The baseline CBF in Hank's solution was higher than in D-Hank's solution. Treatment with 10(-6)-l0(-3) mol/L histamine of concentrations can stimulate a rise of CBF. Nevertheless, CBF in all groups decreased to baseline CBF within 20 min. Generally, 10(-2) mol/L histamine can stimulate a rise of CBF; meanwhile, the high concentration of histamine killed 50% ciliated cell. Histamine at 10(-1) mol/L killed all ciliated cells. Ciliary beating activity decreased in Ca(2+)-free solution. Moreover, adenosine triphosphate could increase CBF effectively after the stimulation effect of histamine. We construct an effective technology integrating patch-clamp technique with CBF measurements on ciliated cells. Extracellular histamine stimulation could increase CBF effectively.

  5. Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody

    PubMed Central

    1986-01-01

    A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti- myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed. PMID:3525577

  6. 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

  7. 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

  8. Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

    PubMed Central

    Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

    2016-01-01

    ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

  9. Smoking-Associated Disordering of the Airway Basal Stem/Progenitor Cell Metabotype

    PubMed Central

    Deeb, Ruba S.; Walters, Matthew S.; Strulovici-Barel, Yael; Chen, Qiuying; Gross, Steven S.

    2016-01-01

    The airway epithelium is a complex pseudostratified multicellular layer lining the tracheobronchial tree, functioning as the primary defense against inhaled environmental contaminants. The major cell types of the airway epithelium include basal, intermediate columnar, ciliated, and secretory. Basal cells (BCs) are the proliferating stem/progenitor population that differentiate into the other specialized cell types of the airway epithelium during normal turnover and repair. Given that cigarette smoke delivers thousands of xenobiotics and high levels of reactive molecules to the lung epithelial surface, we hypothesized that cigarette smoke broadly perturbs BC metabolism. To test this hypothesis, primary airway BCs were isolated from healthy nonsmokers (n = 11) and healthy smokers (n = 7) and assessed by global metabolic profiling by liquid chromatography–mass spectrometry. The analysis identified 52 significant metabolites in BCs differentially expressed between smokers and nonsmokers (P < 0.05). These changes included metabolites associated with redox pathways, energy production, and inflammatory processes. Notably, BCs from smokers exhibited altered levels of the key enzyme cofactors/substrates nicotinamide adenine dinucleotide, flavin adenine dinucleotide, acetyl coenzyme A, and membrane phospholipid levels. Consistent with the high burden of oxidants in cigarette smoke, glutathione levels were diminished, whereas 3-nitrotyrosine levels were increased, suggesting that protection of airway epithelial cells against oxidative and nitrosative stress is significantly compromised in smoker BCs. It is likely that this altered metabotype is a reflection of, and likely contributes to, the disordered biology of airway BCs consequent to the stress cigarette smoking puts on the airway epithelium. PMID:26161876

  10. Arrest in ciliated cell expansion on the bronchial lining of adult rats caused by chronic exposure to industrial noise.

    PubMed

    Oliveira, Maria João R; Pereira, António S; Ferreira, Paula G; Guimarães, Laura; Freitas, Diamantino; Carvalho, António P O; Grande, Nuno R; Aguas, Artur P

    2005-03-01

    Workers chronically exposed to high-intensity/low-frequency noise at textile plants show increased frequency of respiratory infections. This phenomenon prompted the herein investigation on the cytology of the bronchial epithelium of Wistar rats submitted to textile noise. Workplace noise from a cotton-mill room of a textile factory was recorded and reproduced in a sound-insulated animal room. The Wistar rats were submitted to a weekly schedule of noise treatment that was similar to that of the textile workers (8h/day, 5 days/week). Scanning electron microscopy (SEM) was used to compare the fine morphology of the inner surface of the bronchi in noise-exposed and control rats. SEM quantitative cytology revealed that exposure to noise for 5-7 months caused inhibition in the natural expansion of the area occupied by ciliated cells on the bronchial epithelium as adult rats grow older. This difference between noise-exposed and age-matched control rats was statistically significant (P<0.05) and documents that the cytology of the rat bronchial epithelium is mildly altered by noise exposure. The decrease in the area of bronchial cilia may impair the mucociliar clearance of the respiratory airways and, thus, increase vulnerability to respiratory infection.

  11. Ecotoxicity assessment using ciliate cells in millifluidic droplets

    PubMed Central

    Illing, Rico; Burkart, Corinna; Pfitzner, Daniel; Jungmann, Dirk; Baraban, Larysa; Cuniberti, Gianaurelio

    2016-01-01

    Precise analysis of the aquatic cells and their responses to the toxic chemicals, i.e., water disinfective agents, is of crucial importance due to their role in the ecosystem. We demonstrate the application of the droplets based millifluidic tool for isolating and longtime monitoring of single Paramecium tetraurelia cells using a large number of water-in-oil emulsion droplets. Due to the automated monitoring of the fluorescence signal, the droplets containing cells are distinguished from the empty reservoirs. A viability indicator is used to follow the metabolic dynamic of the cells in every single droplet. Finally, we perform ecotoxicity tests in droplets, exposing the encapsulated paramecia cells to silver nitrate for determination of EC50 levels, and compare the output with the conventional microtiter plate assay. PMID:27051472

  12. Photomovements in Ciliated Protozoa

    NASA Astrophysics Data System (ADS)

    Kuhlmann, Hans-Werner

    Ciliates are unicellular, nonphotosynthetic organisms which show a number of light-induced responses. Orientation with respect to the direction of light, phototaxis, has been demonstrated in some species of ciliates. Most of these species bear conspicuous cell organelles such as subpellicular pigment granules, a colored stigma, a watchglass organelle, or a compound crystalline organelle. Several lines of evidence suggest that these kinds of organelles are prerequisites for phototactic orientation of the cells. Photoreceptor molecules presumedly mediating the photobehavior of two species have been identified. The ecological advantage of light-induced responses in ciliated protozoa is still debated. In some cases the organisms may utilize this behavior either to approach their potential prey, to escape their predators, to escape damaging light, or to meet a mating partner. Several species of ciliates display inverse phototactic behavior at different stages of their life cycle.

  13. 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

  14. 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.

  15. 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

  16. Expression of ligands for Siglec-8 and Siglec-9 in human airways and airway cells

    PubMed Central

    Jia, Yi; Yu, Huifeng; Fernandes, Steve M.; Wei, Yadong; Gonzalez-Gil, Anabel; Motari, Mary G.; Vajn, Katarina; Stevens, Whitney W.; Peters, Anju T.; Bochner, Bruce S.; Kern, Robert C.; Schleimer, Robert P.; Schnaar, Ronald L.

    2015-01-01

    Background Balanced activation and inhibition of the immune system ensures pathogen clearance while avoiding hyperinflammation. Siglecs, sialic acid binding proteins found on subsets of immune cells, often inhibit inflammation: Siglec-8 on eosinophils and Siglec-9 on neutrophils engage sialoglycan ligands on airways to diminish ongoing inflammation. The identities of human siglec ligands and their expression during inflammation are largely unknown. Objective The histological distribution, expression and molecular characteristics of siglec ligands were explored in healthy and inflamed human upper airways and in a cellular model of airway inflammation. Methods Normal and chronically inflamed upper airway tissues were stained for siglec ligands. The ligands were extracted from normal and inflamed tissues and from human Calu-3 cells for quantitative analysis by siglec blotting and isolation by siglec capture. Results Siglec-8 ligands were expressed on a subpopulation of submucosal gland cells of human inferior turbinate, whereas Siglec-9 ligands were expressed more broadly (submucosal glands, epithelium, connective tissue); both were significantly upregulated in chronic rhinosinusitis patients. Human airway (Calu-3) cells expressed Siglec-9 ligands on mucin 5B under inflammatory control via the NF-κB pathway, and mucin 5B carried sialoglycan ligands of Siglec-9 on human upper airway tissue. Conclusion Inflammation results in upregulation of immune inhibitory Siglec-8 and Siglec-9 sialoglycan ligands on human airways. Siglec-9 ligands were upregulated via the NF-κB pathway resulting in their enhanced expression on mucin 5B. Siglec sialoglycan ligand expression in inflamed cells and tissues may contribute to the control of airway inflammation. PMID:25747723

  17. 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.

  18. Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell.

    PubMed

    Kobayashi, N; Hirokawa, N

    1988-01-01

    In order to understand the cytoskeletal architecture at the terminal web of the ciliated cell, we examined chicken tracheal epithelium by quick-freeze deep-etch (QFDE) electron microscopy combined with immunocytochemistry of fodrin. At the terminal web, the cilia ended into the basal bodies and then to the rootlets. The rootlets were composed of several filaments and globular structures attached regularly to them. Decoration with myosin subfragment 1 (S1) revealed that some actin filaments ran parallel to the apical plasma membrane between the basal bodies, and other population traveled perpendicularly or obliquely, i.e., along the rootlets. Some actin filaments were connected to the surface of the basal bodies and the basal feet. Among the basal bodies and the rootlets there existed three kinds of fine crossbridges, which were not decorated with S1. In the deeper part of the terminal web, intermediate filaments were observed between the rootlets and were sometimes crosslinked with the rootlets. Immunocytochemistry combined with the QFDE method revealed that fodrin was a component of fine crossbridges associated with the basal bodies. We concluded that an extensive crosslinker system among the basal bodies and the rootlets along with networks of actin and intermediate filaments formed a structural basis for the effective beating of cilia.

  19. 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

  20. 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.

  1. 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.

  2. Connecting alveolate cell biology with trophic ecology in the marine plankton using the ciliate Favella as a model.

    PubMed

    Echevarria, Michael L; Wolfe, Gordon V; Strom, Suzanne L; Taylor, Alison R

    2014-10-01

    Planktonic alveolates (ciliates and dinoflagellates), key trophic links in marine planktonic communities, exhibit complex behaviors that are underappreciated by microbiologists and ecologists. Furthermore, the physiological mechanisms underlying these behaviors are still poorly understood except in a few freshwater model ciliates, which are significantly different in cell structure and behavior than marine planktonic species. Here, we argue for an interdisciplinary research approach to connect physiological mechanisms with population-level outcomes of behaviors. Presenting the tintinnid ciliate Favella as a model alveolate, we review its population ecology, behavior, and cellular/molecular biology in the context of sensory biology and synthesize past research and current findings to construct a conceptual model describing the sensory biology of Favella. We discuss how emerging genomic information and new technical methods for integrating research across different levels of biological organization are paving the way for rapid advance. These research approaches will yield a deeper understanding of the role that planktonic alveolates may play in biogeochemical cycles, and how they may respond to future ocean conditions.

  3. Ultrastructure of the ciliated cells of the free-swimming larva, and sessile stages, of the marine sponge Haliclona indistincta (Demospongiae: Haplosclerida).

    PubMed

    Stephens, Kelly M; Ereskovsky, Alexander; Lalor, Pierce; McCormack, Grace P

    2013-11-01

    We provide a detailed, comparative study of the ciliated cells of the marine haplosclerid sponge Haliclona indistincta, in order to make data available for future phylogenetic comparisons at the ultrastructural level. Our study focuses on the description and analysis of the larval epithelial cells, and choanocytes of the metamorphosed juvenile sponge. The ultrastructure of the two cell types is sufficiently different to prevent our ability to conclusively determine the origin of the choanocytes from the larval ciliated cells. However, ciliated, epithelial cells were observed in a migratory position within the inner cell mass of the larval stages. Some cilia were observed within the cell's cytoplasm, which is indicative of the ciliated epithelial cell undergoing transdifferentiation into a choanocyte; while traces of other ciliated epithelial cells were contained within phagosomes, suggesting they are phagocytosed. We compared our data with other species described in the literature. However, any phylogenetic inference must wait until further detailed comparisons can be made with species whose phylogenetic position has been determined by other means, such as phylogenomics, in order to more closely link genomic, and morphological information.

  4. 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...

  5. 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.

  6. Stimulus-response coupling in mammalian ciliated cells. Demonstration of two mechanisms of control for cytosolic [Ca2+

    PubMed Central

    Villalón, M; Hinds, T R; Verdugo, P

    1989-01-01

    Changes of cytosolic [Ca2+] have been proposed to couple stimulation of ciliary movement, however, quantitative measurements of fluctuations of intracellular free [Ca2+] associated with stimulation of ciliated cells have not been investigated. In primary cultures of rabbit oviductal ciliated cells, the stimulation of ciliary activity produced by micromolar concentrations of adenosine triphosphate (ATP) and prostaglandin F2 alpha (PGF2 alpha) was associated with a transient increase of intracellular [Ca2+]. Whereas the increase of cytosolic [Ca2+] and beat frequency produced by ATP were inhibited by the Ca-channel blocker LaCl3, the rise of cytosolic [Ca2+] and frequency of ciliary beat produced by PGF2 alpha was not affected by LaCl3. These results are the first direct demonstration that fluctuations of cytosolic [Ca2+] are associated with increased ciliary beat frequency in mammalian epithelial cells. The present findings suggest two different calcium-dependent mechanisms for stimulus-coupling in ciliary epithelium: ATP acting via purinergic receptor coupled to transmembrane influx of Ca2+, and PGF2 alpha acting via receptor-mediated release of intracellular sequestered Ca. PMID:2611335

  7. Photosensory transduction in unicellular eukaryotes: a comparison between related ciliates Blepharisma japonicum and Stentor coeruleus and photoreceptor cells of higher organisms.

    PubMed

    Sobierajska, Katarzyna; Fabczak, Hanna; Fabczak, Stanisław

    2006-06-01

    Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.

  8. Exploring the Transcriptome of Ciliated Cells Using In Silico Dissection of Human Tissues

    PubMed Central

    Ivliev, Alexander E.; 't Hoen, Peter A. C.; van Roon-Mom, Willeke M. C.; Peters, Dorien J. M.; Sergeeva, Marina G.

    2012-01-01

    Cilia are cell organelles that play important roles in cell motility, sensory and developmental functions and are involved in a range of human diseases, known as ciliopathies. Here, we search for novel human genes related to cilia using a strategy that exploits the previously reported tendency of cell type-specific genes to be coexpressed in the transcriptome of complex tissues. Gene coexpression networks were constructed using the noise-resistant WGCNA algorithm in 12 publicly available microarray datasets from human tissues rich in motile cilia: airways, fallopian tubes and brain. A cilia-related coexpression module was detected in 10 out of the 12 datasets. A consensus analysis of this module's gene composition recapitulated 297 known and predicted 74 novel cilia-related genes. 82% of the novel candidates were supported by tissue-specificity expression data from GEO and/or proteomic data from the Human Protein Atlas. The novel findings included a set of genes (DCDC2, DYX1C1, KIAA0319) related to a neurological disease dyslexia suggesting their potential involvement in ciliary functions. Furthermore, we searched for differences in gene composition of the ciliary module between the tissues. A multidrug-and-toxin extrusion transporter MATE2 (SLC47A2) was found as a brain-specific central gene in the ciliary module. We confirm the localization of MATE2 in cilia by immunofluorescence staining using MDCK cells as a model. While MATE2 has previously gained attention as a pharmacologically relevant transporter, its potential relation to cilia is suggested for the first time. Taken together, our large-scale analysis of gene coexpression networks identifies novel genes related to human cell cilia. PMID:22558177

  9. Cell-specific transcriptional profiling of ciliated sensory neurons reveals regulators of behavior and extracellular vesicle biogenesis

    PubMed Central

    Wang, Juan; Kaletsky, Rachel; Silva, Malan; Williams, April; Haas, Leonard; Androwski, Rebecca; Landis, Jessica; Patrick, Cory; Rashid, Alina; Santiago-Martinez, Dianaliz; Gravato-Nobre, Maria; Hodgkin, Jonathan; Hall, David H.; Murphy, Coleen T.; Barr, Maureen M.

    2015-01-01

    Summary Cilia and extracellular vesicles (EVs) are signaling organelles[1]. Cilia act as cellular sensory antennae, with defects resulting in human ciliopathies. Cilia both release and bind to EVs[1]. EVs are submicron-sized particles released by cells and function in both short and long range intercellular communication. In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation[2]. A fundamental understanding of EV biology and the relationship between the polycystins, cilia, and EVs is lacking. To define properties of a ciliated EV-releasing cell, we performed RNAseq on 27 GFP-labeled EV releasing neurons (EVNs) isolated from adult C. elegans. We identified 335 significantly overrepresented genes, of which 61 were validated by GFP reporters. The EVN transcriptional profile uncovered new pathways controlling EV biogenesis and polycystin signaling and also identified EV cargo, which included an antimicrobial peptide and ASIC channel. Tumor necrosis associated factor (TRAF) homologues trf-1 and trf-2 and the p38 mitogen-activated protein kinase (MAPK) pmk-1 acted in polycystin signaling pathways controlling male mating behaviors. pmk-1 was also required for EV biogenesis, independent of the innate immunity MAPK signaling cascade. This first high-resolution transcriptome profile of a subtype of ciliated sensory neurons isolated from adult animals reveals the functional components of an EVN. PMID:26687621

  10. 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.

  11. 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.

  12. 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...

  13. Genome-Wide Analysis of the Phosphoinositide Kinome from Two Ciliates Reveals Novel Evolutionary Links for Phosphoinositide Kinases in Eukaryotic Cells

    PubMed Central

    Leondaritis, George; Siokos, John; Skaripa, Irini; Galanopoulou, Dia

    2013-01-01

    Background The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs) that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. Methodology/Principal Findings We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. Conclusions/Significance Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs) not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory pathways of

  14. CD38 and airway hyper-responsiveness: studies on human airway smooth muscle cells and mouse models.

    PubMed

    Guedes, Alonso G P; Deshpande, Deepak A; Dileepan, Mythili; Walseth, Timothy F; Panettieri, Reynold A; Subramanian, Subbaya; Kannan, Mathur S

    2015-02-01

    Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.

  15. Aeroallergen challenge promotes dendritic cell proliferation in the airways.

    PubMed

    Veres, Tibor Z; Voedisch, Sabrina; Spies, Emma; Valtonen, Joona; Prenzler, Frauke; Braun, Armin

    2013-02-01

    Aeroallergen provocation induces the rapid accumulation of CD11c(+)MHC class II (MHC II)(+) dendritic cells (DCs) in the lungs, which is driven by an increased recruitment of blood-derived DC precursors. Recent data show, however, that well-differentiated DCs proliferate in situ in various tissues. This may also contribute to their allergen-induced expansion; therefore, we studied DC proliferation in the airways of mice in the steady state and after local aeroallergen provocation. Confocal whole-mount microscopy was used to visualize proliferating DCs in different microanatomical compartments of the lung. We demonstrate that in the steady state, CD11c(+)MHC II(+) DCs proliferate in both the epithelial and subepithelial layers of the airway mucosa as well as in the lung parenchyma. A 1-h pulse of the nucleotide 5-ethynyl-2'-deoxyuridine was sufficient to label 5% of DCs in both layers of the airway mucosa. On the level of whole-lung tissue, 3-5% of both CD11b(+) and CD11b(-) DC populations and 0.3% of CD11c(+)MHC II(low) lung macrophages incorporated 5-ethynyl-2'-deoxyuridine. Aeroallergen provocation caused a 3-fold increase in the frequency of locally proliferating DCs in the airway mucosa. This increase in mucosal DC proliferation was later followed by an elevation in the number of DCs. The recruitment of monocyte-derived inflammatory DCs contributed to the increasing number of DCs in the lung parenchyma, but not in the airway mucosa. We conclude that local proliferation significantly contributes to airway DC homeostasis in the steady state and that it is the major mechanism underlying the expansion of the mucosal epithelial/subepithelial DC network in allergic inflammation.

  16. Relationship between the flagellates and the ciliates.

    PubMed Central

    Lee, R E; Kugrens, P

    1992-01-01

    The flagellates and the ciliates have long been considered to be closely related because of their unicellular nature and the similarity in the structures of the axoneme of the flagella and cilia in both groups. Most protozoologists believe that the ciliates arose from a flagellate. The flagellates that are most similar in structure to the ciliates are the dinoflagellates and two genera of uncertain taxonomic position, Colponema and Katablepharis. Structurally, dinoflagellates have a number of similarities with ciliates. These include the similarity of the cortical alveoli in the ciliates to the thecal vesicles in the dinoflagellates, the possession of tubular cristae, the similarity of the parasomal sac of the ciliates to the pusule of the dinoflagellates, the possession of similar trichocysts and mucocysts, and some similarity in the feeding apparatus. Colponema spp. are probably related to the dinoflagellates and have many of the same similarities with the ciliates. Katablepharis spp. are very similar in structure to the swarmer (embryo) of the suctorian ciliates. Indeed, reduction in the number of cilia to two in the suctorian swarmer and elimination of the macronucleus would result in a cell that is very similar to the Katablepharis cell. The feeding apparatus of Katablepharis spp. and the rest of the ciliates consists of two concentric microtubular arrays associated with vesicles. Information available from nucleotide sequencing of rRNA places the dinoflagellates in an ancestral position to the ciliates. The rRNA of Colponema and Katablepharis spp. has not yet been investigated. The use of stop codons in mRNA is discussed in relation to phylogeny. Images PMID:1480107

  17. Mast cells in airway diseases and interstitial lung disease.

    PubMed

    Cruse, Glenn; Bradding, Peter

    2016-05-05

    Mast cells are major effector cells of inflammation and there is strong evidence that mast cells play a significant role in asthma pathophysiology. There is also a growing body of evidence that mast cells contribute to other inflammatory and fibrotic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. This review discusses the role that mast cells play in airway diseases and highlights how mast cell microlocalisation within specific lung compartments and their cellular interactions are likely to be critical for their effector function in disease.

  18. 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

  19. The DNA of ciliated protozoa.

    PubMed Central

    Prescott, D M

    1994-01-01

    Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons. Images PMID:8078435

  20. Dendritic cell-nerve clusters are sites of T cell proliferation in allergic airway inflammation.

    PubMed

    Veres, Tibor Z; Shevchenko, Marina; Krasteva, Gabriela; Spies, Emma; Prenzler, Frauke; Rochlitzer, Sabine; Tschernig, Thomas; Krug, Norbert; Kummer, Wolfgang; Braun, Armin

    2009-03-01

    Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2'-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semi-automated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.

  1. 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

  2. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia.

    PubMed

    Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer; Wong, Puisan; Huong, Tra Nguyen; Li, Chunwei; Tan, Boon Huan; Wang, De Yun; Sugrue, Richard J

    2015-10-01

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss.

  3. Cell proliferation contributes to PNEC hyperplasia after acute airway injury.

    PubMed

    Stevens, T P; McBride, J T; Peake, J L; Pinkerton, K E; Stripp, B R

    1997-03-01

    Pulmonary neuroendocrine cells (PNECs) are airway epithelial cells that are capable of secreting a variety of neuropeptides. PNECs are scattered throughout the bronchial tree either as individual cells or clusters of cells termed neuroepithelial bodies (NEBs). PNECs and their secretory peptides have been considered to play a role in fetal lung development. Although the normal physiological function of PNECs and neuropeptides in normal adult lungs and in repair from lung injury is not known, PNEC hyperplasia has been associated with chronic lung diseases, such as bronchopulmonary dysplasia, and with chronic exposures, such as hypoxia, tobacco smoke, nitrosamines, and ozone. To evaluate changes in PNEC number and distribution after acute airway injury, FVB/n mice were treated with either naphthalene or vehicle. Naphthalene is an aromatic hydrocarbon that, at the dose used in this study, selectively destroys nonciliated bronchial epithelial cells (Clara cells) through cytochrome P-450-mediated metabolic activation into cytotoxic epoxides. PNECs were identified by immunohistochemical analysis of calcitonin gene-related peptide-like immunoreactivity (CGRP-IR). Proliferating cells were marked with [(3)H]thymidine incorporation. Acute naphthalene toxicity results in PNEC hyperplasia that is detectable after 5 days of recovery. PNEC hyperplasia is characterized by increased numbers of NEBs without significant changes in the number of isolated PNECs and by increased [(3)H]thymidine labeling of CGRP-IR cells. These data show that cell proliferation contributes to PNEC hyperplasia after acute airway injury and suggest that PNECs may be capable of more rapidly increasing their number in response to injury than previously recognized.

  4. Can a fermentation gas mainly produced by rumen Isotrichidae ciliates be a potential source of biohydrogen and a fuel for a chemical fuel cell?

    PubMed

    Piela, Piotr; Michałowski, Tadeusz; Miltko, Renata; Szewczyk, Krzysztof; Sikora, Radosław; Grzesiuk, Elzbieta; Sikora, Anna

    2010-07-01

    Bacteria, fungi and protozoa inhabiting the rumen, the largest chamber of the ruminants' stomach, release large quantities of hydrogen during the fermentation of carbohydrates. The hydrogen is used by coexisting methanogens to produce methane in energy-yielding processes. This work shows, for the first time, a fundamental possibility of using a hydrogen-rich fermentation gas produced by selected rumen ciliates to feed a low-temperature hydrogen fuel cell. A biohydrogen fuel cell (BHFC) was constructed consisting of (i) a bioreactor, in which a hydrogen-rich gas was produced from glucose by rumen ciliates, mainly of the Isotrichidae family, deprived of intra- and extracellular bacteria, methanogens, and fungi, and (ii) a chemical fuel cell of the polymer-electrolyte type (PEFC). The fuel cell was used as a tester of the technical applicability of the fermentation gas produced by the rumen ciliates for power generation. The average estimated hydrogen yield was ca. 1.15 mol H2 per mol of fermented glucose. The BHFC performance was equal to the performance of the PEFC running on pure hydrogen. No fuel cell poisoning effects were detected. A maximum power density of 1.66 kW/m2 (PEFC geometric area) was obtained at room temperature. The maximum volumetric power density was 128 W/m3 but the coulombic efficiency was only ca. 3.8%. The configuration of the bioreactor limited the continuous operation time of this BHFC to ca. 14 hours.

  5. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    SciTech Connect

    Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer; Wong, Puisan; Huong, Tra Nguyen; Li, Chunwei; Tan, Boon Huan; Wang, De Yun; Sugrue, Richard J.

    2015-10-15

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function.

  6. 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

  7. Ciliates by the Slice.

    ERIC Educational Resources Information Center

    Boynton, John E.; Small, Eugene B.

    1984-01-01

    Describes new methods of collecting and examining ciliates, particularly those found in the sediments of lakes, rivers, and estuaries. Discusses extraction methods in preparation for observations in the classroom. Suggests investigations of ciliate ecology as an area of increasing research interest. (JM)

  8. Airway responses towards allergens - from the airway epithelium to T cells.

    PubMed

    Papazian, D; Hansen, S; Würtzen, P A

    2015-08-01

    The prevalence of allergic diseases such as allergic rhinitis is increasing, affecting up to 30% of the human population worldwide. Allergic sensitization arises from complex interactions between environmental exposures and genetic susceptibility, resulting in inflammatory T helper 2 (Th2) cell-derived immune responses towards environmental allergens. Emerging evidence now suggests that an epithelial dysfunction, coupled with inherent properties of environmental allergens, can be responsible for the inflammatory responses towards allergens. Several epithelial-derived cytokines, such as thymic stromal lymphopoietin (TSLP), IL-25 and IL-33, influence tissue-resident dendritic cells (DCs) as well as Th2 effector cells. Exposure to environmental allergens does not elicit Th2 inflammatory responses or any clinical symptoms in nonatopic individuals, and recent findings suggest that a nondamaged, healthy epithelium lowers the DCs' ability to induce inflammatory T-cell responses towards allergens. The purpose of this review was to summarize the current knowledge on which signals from the airway epithelium, from first contact with inhaled allergens all the way to the ensuing Th2-cell responses, influence the pathology of allergic diseases.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model

    PubMed Central

    Spaziano, Giuseppe; Piegari, Elena; Matteis, Maria; Cappetta, Donato; Esposito, Grazia; Russo, Rosa; Tartaglione, Gioia; De Palma, Raffaele; Rossi, Francesco; D’Agostino, Bruno

    2016-01-01

    Background The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs) can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce. Objectives To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model. Methods GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro. Results Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties. Conclusion Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide

  14. Mast Cells Mediate Hyperoxia-Induced Airway Hyper-reactivity in Newborn Rats

    PubMed Central

    Schultz, Eric D.; Potts, Erin N.; Mason, Stanley N.; Foster, W. Michael; Auten, Richard L.

    2011-01-01

    Premature infants are at increased risk of developing airway hyper-reactivity following oxidative stress and inflammation. Mast cells contribute to airway hyper-reactivity partly by mediator release, so we sought to determine if blocking mast cell degranulation or recruitment prevents hyperoxia-induced airway hyper-reactivity, mast cell accumulation, and airway smooth muscle changes. Rats were exposed at birth to air or 60% O2 for 14 days, inducing significantly increased airway hyper-reactivity (AHR) in the latter group, induced by nebulized methacholine challenge, measured by forced oscillometry. Daily treatment (postnatal days 1-14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal days 5-14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways, and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell. PMID:20386143

  15. 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

  16. Heterogeneity of tight junction morphology in extrapulmonary and intrapulmonary airways of the rat.

    PubMed

    Schneeberger, E E

    1980-10-01

    In the present study morphology of tight junctions was related to the various cell types lining extrapulmonary and intrapulmonary airways of the rat. Freeze fracture replicas were prepared from extrapulmonary airway epithelium derived from the cartilagenous and membranous sides of upper, middle, and lower thirds of the trachea. Intrapulmonary airway epithelium was obtained from airways less than 1 mm in diameter. Tight junction fibrils on the P fracture face were organized into three types of patterns. Type 1: parallel sparsely interconnected lumenal fibrils with large ablumenal fibril loops. Type 2: richly interconnected lumenal fibrils with large ablumenal fibril loops. Type 3: narrow network of interconnected fibrils. On the E fracture face complementary grooves were organized in a similar pattern. Ciliated cells on both sides and all levels of the trachea were associated with type 1 junctions. In intrapulmonary airways, however, the junctional pattern of ciliated cells changed to type 2. Brush cells at all levels of the airways were bounded by type 2 and occasionally by type 1 junctions. Secretory cell junctions displayed the following patterns: Mucous cells were bounded solely by type 3, serous cells by either types 2 or 3, and Clara cells predominantly by type 2. Cells tentatively identified as intermediate cells displayed all three junctional patterns. The number of parallel fibrils comprising tight junctions was higher in extrapulmonary as compared to intrapulmonary airways. No difference was seen in the various locations sampled in the trachea. Gap junctions were observed between secretory cells of extrapulmonary but not intrapulmonary airways. These observations are discussed in relation to current physiologic data.

  17. CGRP induction in cystic fibrosis airways alters the submucosal gland progenitor cell niche in mice

    PubMed Central

    Xie, Weiliang; Fisher, John T.; Lynch, Thomas J.; Luo, Meihui; Evans, Turan I.A.; Neff, Traci L.; Zhou, Weihong; Zhang, Yulong; Ou, Yi; Bunnett, Nigel W.; Russo, Andrew F.; Goodheart, Michael J.; Parekh, Kalpaj R.; Liu, Xiaoming; Engelhardt, John F.

    2011-01-01

    In cystic fibrosis (CF), a lack of functional CF transmembrane conductance regulator (CFTR) chloride channels causes defective secretion by submucosal glands (SMGs), leading to persistent bacterial infection that damages airways and necessitates tissue repair. SMGs are also important niches for slow-cycling progenitor cells (SCPCs) in the proximal airways, which may be involved in disease-related airway repair. Here, we report that calcitonin gene–related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. Since CGRP-expressing neuroendocrine cells reside in bronchiolar SCPC niches, we hypothesized that the glandular SCPC niche may be dysfunctional in CF. Consistent with this hypothesis, CFTR-deficient mice failed to maintain glandular SCPCs following airway injury. In wild-type mice, CGRP levels increased following airway injury and functioned as an injury-induced mitogen that stimulated SMG progenitor cell proliferation in vivo and altered the proliferative potential of airway progenitors in vitro. Components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation in a non-cell-autonomous manner. These findings demonstrate that CGRP-dependent pathways for CFTR activation are abnormally upregulated in CF SMGs and that this sustained mitogenic signal alters properties of the SMG progenitor cell niche in CF airways. This discovery may have important implications for injury/repair mechanisms in the CF airway. PMID:21765217

  18. 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...

  19. New Role of Adult Lung c-kit+ Cells in a Mouse Model of Airway Hyperresponsiveness

    PubMed Central

    Cappetta, Donato; Urbanek, Konrad; Esposito, Grazia; Matteis, Maria; Sgambato, Manuela; Tartaglione, Gioia; Rossi, Francesco

    2016-01-01

    Structural changes contribute to airway hyperresponsiveness and airflow obstruction in asthma. Emerging evidence points to the involvement of c-kit+ cells in lung homeostasis, although their potential role in asthma is unknown. Our aim was to isolate c-kit+ cells from normal mouse lungs and to test whether these cells can interfere with hallmarks of asthma in an animal model. Adult mouse GFP-tagged c-kit+ cells, intratracheally delivered in the ovalbumin-induced airway hyperresponsiveness, positively affected airway remodeling and improved airway function. In bronchoalveolar lavage fluid of cell-treated animals, a reduction in the number of inflammatory cells and in IL-4, IL-5, and IL-13 release, along with an increase of IL-10, was observed. In MSC-treated mice, the macrophage polarization to M2-like subset may explain, at least in part, the increment in the level of anti-inflammatory cytokine IL-10. After in vitro stimulation of c-kit+ cells with proinflammatory cytokines, the indoleamine 2,3-dioxygenase and TGFβ were upregulated. These data, together with the increased apoptosis of inflammatory cells in vivo, indicate that c-kit+ cells downregulate immune response in asthma by influencing local environment, possibly by cell-to-cell contact combined to paracrine action. In conclusion, intratracheally administered c-kit+ cells reduce inflammation, positively modulate airway remodeling, and improve function. These data document previously unrecognized properties of c-kit+ cells, able to impede pathophysiological features of experimental airway hyperresponsiveness. PMID:28090152

  20. On the evolution of the karyorelict ciliate life cycle: heterophasic ciliates and the origin of ciliate binary fission.

    PubMed

    Orias, E

    1991-01-01

    Karyorelict ciliates have near diploid somatic nuclei (macronuclei) incapable of division. If selective pressure favors nuclear division, how could such macronuclei have evolved? I propose that they initially evolved in the context of a diplophase stage that consisted entirely of a non-dividing trophont that was terminated by the induction of meiosis. The diploid macronucleus then differentiated, functioned and was destroyed in the absence of cell division. Such a life cycle would necessarily be heterophasic, i.e. with alternating haploid and diploid generations. I call these ancestors heterophasic ciliates. I further propose that the ability of this diploid trophont to undergo binary fission arose de novo. Ciliate binary fission would then be a derived characteristic, which possibly evolved indepedently in more than one heterophasic ciliate lineage. A progression of steps, leading to the reduction of the haplophase and the generation of the karyorelict life cycle, is proposed. The shared possession of nuclear dimorphism with non-dividing macronuclei, conjugation, and a putative heterophasic ancestry invites further investigation of the phylogenetic relationship between heterokaryotic foraminifera and karyorelict ciliates.

  1. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling.

    PubMed

    Salmon, M; Walsh, D A; Koto, H; Barnes, P J; Chung, K F

    1999-09-01

    Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.

  2. Adoptive transfer of induced-Treg cells effectively attenuates murine airway allergic inflammation.

    PubMed

    Xu, Wei; Lan, Qin; Chen, Maogen; Chen, Hui; Zhu, Ning; Zhou, Xiaohui; Wang, Julie; Fan, Huimin; Yan, Chun-Song; Kuang, Jiu-Long; Warburton, David; Togbe, Dieudonnée; Ryffel, Bernhard; Zheng, Song-Guo; Shi, Wei

    2012-01-01

    Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4(+)FoxP3(+)) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.

  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. Defining an olfactory receptor function in airway smooth muscle cells

    PubMed Central

    Aisenberg, William H.; Huang, Jessie; Zhu, Wanqu; Rajkumar, Premraj; Cruz, Randy; Santhanam, Lakshmi; Natarajan, Niranjana; Yong, Hwan Mee; De Santiago, Breann; Oh, Jung Jin; Yoon, A-Rum; Panettieri, Reynold A.; Homann, Oliver; Sullivan, John K.; Liggett, Stephen B.; Pluznick, Jennifer L.; An, Steven S.

    2016-01-01

    Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma. PMID:27905542

  5. Defining an olfactory receptor function in airway smooth muscle cells.

    PubMed

    Aisenberg, William H; Huang, Jessie; Zhu, Wanqu; Rajkumar, Premraj; Cruz, Randy; Santhanam, Lakshmi; Natarajan, Niranjana; Yong, Hwan Mee; De Santiago, Breann; Oh, Jung Jin; Yoon, A-Rum; Panettieri, Reynold A; Homann, Oliver; Sullivan, John K; Liggett, Stephen B; Pluznick, Jennifer L; An, Steven S

    2016-12-01

    Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.

  6. 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.

  7. Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice.

    PubMed

    Pan, Jie; Luk, Catherine; Kent, Geraldine; Cutz, Ernest; Yeger, Herman

    2006-09-01

    The amine- and peptide-producing pulmonary neuroendocrine cells (PNEC) are widely distributed within the airway mucosa of mammalian lung as solitary cells and innervated clusters, neuroepithelial bodies (NEB), which function as airway O2 sensors. These cells express Cftr and hence could play a role in the pathophysiology of cystic fibrosis (CF) lung disease. We performed confocal microscopy and morphometric analysis on lung sections from Cftr-/- (null), Cftr+/+, and Cftr+/- (control) mice at developmental stages E20, P5, P9, and P30 to determine the distribution, frequency, and innervation of PNEC/NEB, innervation and cell mass of airway smooth muscle, and neuromuscular junctions using synaptic vesicle protein 2, smooth muscle actin, and synaptophysin markers, respectively. The mean number of PNEC/NEB in Cftr-/- mice was significantly reduced compared with control mice at E20, whereas comparable or increased numbers were observed postnatally. NEB cells in Cftr null mice showed a significant reduction in intracorpuscular nerve endings compared with control mice, which is consistent with an intrinsic abnormality of the PNEC system. The airways of Cftr-/- mice showed reduced density (approximately 20-30%) of smooth muscle innervation, decreased mean airway smooth muscle mass (approximately 35%), and reduced density (approximately 20%) of nerve endings compared with control mice. We conclude that the airways of Cftr-/- mice exhibit heretofore unappreciated structural alterations affecting cellular and neural components of the PNEC system and airway smooth muscle and its innervation resulting in blunted O2 sensing and reduced airway tonus. Cftr could play a role in the development of the PNEC system, lung innervation, and airway smooth muscle.

  8. NOTCH1 is required for regeneration of Clara cells during repair of airway injury.

    PubMed

    Xing, Yiming; Li, Aimin; Borok, Zea; Li, Changgong; Minoo, Parviz

    2012-05-01

    The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the targets of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells, and repair and re-establishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell-fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/enhancer of split-5 (Hes5) and paired-box-containing gene 6 (Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild-type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression.

  9. 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...

  10. TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium.

    PubMed

    González, Claudia; Droguett, Karla; Rios, Mariana; Cohen, Noam A; Villalón, Manuel

    2016-01-01

    In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNFα) that can alter mucus viscosity; however airway ciliated cells have an autoregulatory mechanism to prevent the collapse of CBF in response to increase in mucus viscosity, mechanism that is associated with an increment in intracellular Ca(+2) level ([Ca(2+)]i). We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca(2+)]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca(2+)]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways.

  11. TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium

    PubMed Central

    Droguett, Karla; Rios, Mariana; Cohen, Noam A.

    2016-01-01

    In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNFα) that can alter mucus viscosity; however airway ciliated cells have an autoregulatory mechanism to prevent the collapse of CBF in response to increase in mucus viscosity, mechanism that is associated with an increment in intracellular Ca+2 level ([Ca2+]i). We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca2+]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca2+]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways. PMID:28025644

  12. 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

  13. α7 Nicotinic Acetylcholine Receptor Regulates Airway Epithelium Differentiation by Controlling Basal Cell Proliferation

    PubMed Central

    Maouche, Kamel; Polette, Myriam; Jolly, Thomas; Medjber, Kahina; Cloëz-Tayarani, Isabelle; Changeux, Jean-Pierre; Burlet, Henriette; Terryn, Christine; Coraux, Christelle; Zahm, Jean-Marie; Birembaut, Philippe; Tournier, Jean-Marie

    2009-01-01

    Airway epithelial basal cells are known to be critical for regenerating injured epithelium and maintaining tissue homeostasis. Recent evidence suggests that the α7 nicotinic acetylcholine receptor (nAChR), which is highly permeable to Ca2+, is involved in lung morphogenesis. Here, we have investigated the potential role of the α7 nAChR in the regulation of airway epithelial basal cell proliferation and the differentiation of the human airway epithelium. In vivo during fetal development and in vitro during the regeneration of the human airway epithelium, α7 nAChR expression coincides with epithelium differentiation. Inactivating α7 nAChR function in vitro increases cell proliferation during the initial steps of the epithelium regeneration, leading to epithelial alterations such as basal cell hyperplasia and squamous metaplasia, remodeling observed in many bronchopulmonary diseases. The regeneration of the airway epithelium after injury in α7−/− mice is delayed and characterized by a transient hyperplasia of basal cells. Moreover, 1-year-old α7−/− mice more frequently present basal cells hyperplasia. Modulating nAChR function or expression shows that only α7 nAChR, as opposed to heteropentameric αxβy nAChRs, controls the proliferation of human airway epithelial basal cells. These findings suggest that α7 nAChR is a key regulator of the plasticity of the human airway epithelium by controlling basal cell proliferation and differentiation pathway and is involved in airway remodeling during bronchopulmonary diseases. PMID:19808646

  14. Innate immune response of human pluripotent stem cell-derived airway epithelium.

    PubMed

    McIntyre, Brendan A S; Kushwah, Rahul; Mechael, Rami; Shapovalova, Zoya; Alev, Cantas; Bhatia, Mickie

    2015-07-01

    The acquisition of innate immune response is requisite to having bona fide differentiation of airway epithelium. Procedures developed to differentiate lung airway from human pluripotent stem cells (hPSCs) have demonstrated anecdotal evidence for innate immune response, but an in-depth exploration of response levels is lacking. Herein, using an established method of airway epithelial generation from hPSCs, we show that hPSC-derived epithelial cells are able to up-regulate expression of TNFα, IL8 and IL1β in response to challenge with bacterial endotoxin LPS, but lack response from genes associated with innate immune response in other cell types. Further, stimulation of cells with TNF-α resulted in auto-induction of TNFα transcript, as well as cytokine responses of IL8 and IL1β. The demonstration of innate immune induction in hPSC-derived airway epithelia gives further strength to the functionality of in vitro protocols aimed at generating differentiated airway cells that can potentially be used in a translational setting. Finally, we propose that innate immune challenge of airway epithelium from human pluripotent stem cell sources be used as a robust validation of functional in vitro differentiation.

  15. Pluripotent allospecific CD8+ effector T cells traffic to lung in murine obliterative airway disease.

    PubMed

    West, Erin E; Lavoie, Tera L; Orens, Jonathan B; Chen, Edward S; Ye, Shui Q; Finkelman, Fred D; Garcia, Joe G N; McDyer, John F

    2006-01-01

    Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+ IFN-gamma+ T cells were detected in airway allografts, with significant coexpression of TNF-alpha and granzyme B. Therefore, using IFN-gamma as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-gamma was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.

  16. 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

  17. 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.

  18. Epigenetics of Ciliates

    PubMed Central

    Chalker, Douglas L.; Meyer, Eric; Mochizuki, Kazufumi

    2013-01-01

    Research using ciliates revealed early examples of epigenetic phenomena and continues to provide novel findings. These protozoans maintain separate germline and somatic nuclei that carry transcriptionally silent and active genomes, respectively. Examining the differences in chromatin within distinct nuclei of Tetrahymena identified histone variants and established that transcriptional regulators act by modifying histones. Formation of somatic nuclei requires both transcriptional activation of silent chromatin and large-scale DNA elimination. This somatic genome remodeling is directed by homologous RNAs, acting with an RNA interference (RNAi)-related machinery. Furthermore, the content of the parental somatic genome provides a homologous template to guide this genome restructuring. The mechanisms regulating ciliate DNA rearrangements reveal the surprising power of homologous RNAs to remodel the genome and transmit information transgenerationally. PMID:24296171

  19. Equine Airway Mast Cells are Sensitive to Cell Death Induced by Lysosomotropic Agents.

    PubMed

    Wernersson, S; Riihimäki, M; Pejler, G; Waern, I

    2017-01-01

    Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine- and human-cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of in vivo-derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of equine in vivo-derived mast cells. Bronchoalveolar lavage (BAL) fluids from horses were incubated with LLME at 15-100 μm for up to 48 h. The overall cell viability was unaffected by 15 μm LLME up to 48 h, whereas a relatively modest drop in total cell counts (~30%) was seen at the highest LLME dose used. In contrast to the relatively low effect on total cell counts, LLME efficiently and dose dependently reduced the number of mast cells in BAL fluids, with an almost complete depletion (96%) of mast cells after 24 h of incubation with 100 μm LLME. A significant but less dramatic reduction (up to ~45%) of lymphocytes was also seen, whereas macrophages and neutrophils were essentially resistant. The appearance of apoptotic bodies suggested a mechanism involving apoptosis rather than necrosis. These findings suggest that equine airway mast cells are highly sensitive to lysosomotropic agents. Possibly, lysosomotropic agents could be of therapeutic value to treat disorders involving harmful accumulation of mast cells in the airways.

  20. The Endoplasmic Reticulum Resident Protein AGR3. Required for Regulation of Ciliary Beat Frequency in the Airway.

    PubMed

    Bonser, Luke R; Schroeder, Bradley W; Ostrin, Lisa A; Baumlin, Nathalie; Olson, Jean L; Salathe, Matthias; Erle, David J

    2015-10-01

    Protein disulfide isomerase (PDI) family members regulate protein folding and calcium homeostasis in the endoplasmic reticulum (ER). The PDI family member anterior gradient (AGR) 3 is expressed in the airway, but the localization, regulation, and function of AGR3 are poorly understood. Here we report that AGR3, unlike its closest homolog AGR2, is restricted to ciliated cells in the airway epithelium and is not induced by ER stress. Mice lacking AGR3 are viable and develop ciliated cells with normal-appearing cilia. However, ciliary beat frequency was lower in airways from AGR3-deficient mice compared with control mice (20% lower in the absence of stimulation and 35% lower after ATP stimulation). AGR3 deficiency had no detectable effects on ciliary beat frequency (CBF) when airways were perfused with a calcium-free solution, suggesting that AGR3 is required for calcium-mediated regulation of ciliary function. Decreased CBF was associated with impaired mucociliary clearance in AGR3-deficient airways. We conclude that AGR3 is a specialized member of the PDI family that plays an unexpected role in the regulation of CBF and mucociliary clearance in the airway.

  1. Vitamin D attenuates cytokine-induced remodeling in human fetal airway smooth muscle cells.

    PubMed

    Britt, Rodney D; Faksh, Arij; Vogel, Elizabeth R; Thompson, Michael A; Chu, Vivian; Pandya, Hitesh C; Amrani, Yassine; Martin, Richard J; Pabelick, Christina M; Prakash, Y S

    2015-06-01

    Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25-dihydroxyvitamin D3 ; 1,25(OH)2 D3 ), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro-inflammatory cytokines. Cells were pre-treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP-9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ-induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood.

  2. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

    PubMed

    Cervia, Davide; Catalani, Elisabetta; Belardinelli, Maria Cristina; Perrotta, Cristiana; Picchietti, Simona; Alimenti, Claudio; Casini, Giovanni; Fausto, Anna Maria; Vallesi, Adriana

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth.

  3. 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.

  4. Bystander suppression of allergic airway inflammation by lung resident memory CD8+ T cells

    NASA Astrophysics Data System (ADS)

    Marsland, Benjamin J.; Harris, Nicola L.; Camberis, Mali; Kopf, Manfred; Hook, Sarah M.; Le Gros, Graham

    2004-04-01

    CD8+ memory T cells have recently been recognized as playing a key role in natural immunity against unrelated viral infections, a phenomenon referred to as "heterologous antiviral immunity." We now provide data that the cellular immunological interactions that underlie such heterologous immunity can play an equally important role in regulating T helper 2 immune responses and protecting mucosal surfaces from allergen-induced inflammation. Our data show that CD8+ T cells, either retained in the lung after infection with influenza virus, or adoptively transferred via the intranasal route can suppress allergic airway inflammation. The suppression is mediated by IFN-, which acts to reduce the activation level, T helper 2 cytokine production, airways hyperresponsiveness, and migration of allergen-specific CD4+ T cells into the lung, whereas the systemic and draining lymph node responses remain unchanged. Of note, adoptive transfer of previously activated transgenic CD8+ T cells conferred protection against allergic airway inflammation, even in the absence of specific-antigen. Airway resident CD8+ T cells produced IFN- when directly exposed to conditioned media from activated dendritic cells or the proinflammatory cytokines IL-12 and IL-18. Taken together these data indicate that effector/memory CD8+ T cells present in the airways produce IFN- after inflammatory stimuli, independent of specific-antigen, and as a consequence play a key role in modifying the degree and frequency of allergic responses in the lung.

  5. Airway Hyperresponsiveness in Children With Sickle Cell Anemia

    PubMed Central

    Field, Joshua J.; Stocks, Janet; Kirkham, Fenella J.; Rosen, Carol L.; Dietzen, Dennis J.; Semon, Trisha; Kirkby, Jane; Bates, Pamela; Seicean, Sinziana; DeBaun, Michael R.; Redline, Susan

    2011-01-01

    Background: The high prevalence of airway hyperresponsiveness (AHR) among children with sickle cell anemia (SCA) remains unexplained. Methods: To determine the relationship between AHR, features of asthma, and clinical characteristics of SCA, we conducted a multicenter, prospective cohort study of children with SCA. Dose response slope (DRS) was calculated to describe methacholine responsiveness, because 30% of participants did not achieve a 20% decrease in FEV1 after inhalation of the highest methacholine concentration, 25 mg/mL. Multiple linear regression analysis was done to identify independent predictors of DRS. Results: Methacholine challenge was performed in 99 children with SCA aged 5.6 to 19.9 years (median, 12.8 years). Fifty-four (55%) children had a provocative concentration of methacholine producing a 20% decrease in FEV1 < 4 mg/mL. In a multivariate analysis, independent associations were found between increased methacholine responsiveness and age (P < .001), IgE (P = .009), and lactate dehydrogenase (LDH) levels (P = .005). There was no association between methacholine responsiveness and a parent report of a doctor diagnosis of asthma (P = .986). Other characteristics of asthma were not associated with methacholine responsiveness, including positive skin tests to aeroallergens, exhaled nitric oxide, peripheral blood eosinophil count, and pulmonary function measures indicating airflow obstruction. Conclusions: In children with SCA, AHR to methacholine is prevalent. Younger age, serum IgE concentration, and LDH level, a marker of hemolysis, are associated with AHR. With the exception of serum IgE, no signs or symptoms of an allergic diathesis are associated with AHR. Although the relationship between methacholine responsiveness and LDH suggests that factors related to SCA may contribute to AHR, these results will need to be validated in future studies. PMID:20724735

  6. Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung

    PubMed Central

    Thornton, Emily E.; Looney, Mark R.; Bose, Oishee; Sen, Debasish; Sheppard, Dean; Locksley, Richard; Huang, Xiaozhu

    2012-01-01

    Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b+ dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung. PMID:22585735

  7. 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.

  8. A critical role for dendritic cells in the evolution of IL-1β-mediated murine airway disease

    PubMed Central

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Goodsell, Amanda; Ma, Royce; Moermans, Catherine; McKnelly, Kate J.; Baron, Jody L.; Krummel, Matthew F.; Nishimura, Stephen L.

    2015-01-01

    Chronic airway inflammation and fibrosis, known as airway remodeling, are defining features of chronic obstructive pulmonary disease (COPD) and are refractory to current treatments. How and if chronic inflammation contributes to airway fibrosis remains controversial. Here, we use a model of COPD airway disease utilizing adenoviral (Ad) delivery of IL-1β to determine that adaptive T-cell immunity is required for airway remodeling since mice deficient in α/β T-cells (tcra −/−) are protected. Dendritic cells (DCs) accumulate around COPD airways and are critical to prime adaptive immunity, but have not been shown to directly influence airway remodeling. We show that DC depletion or deficiency in the crucial DC chemokine receptor, ccr6, both protect from Ad-IL-1β-induced airway adaptive T-cell immune responses, and fibrosis in mice. These results provide evidence that chronic airway inflammation, mediated by accumulation of α/β T-cells and driven by DCs, is critical to airway fibrosis. PMID:25786688

  9. Insights into Group 2 Innate Lymphoid Cells in Human Airway Disease.

    PubMed

    Karta, Maya R; Broide, David H; Doherty, Taylor A

    2016-01-01

    Recent discoveries have led to the identification of a novel group of immune cells, the innate lymphoid cells (ILCs). The members of this group are divided into three subpopulations: ILC1s, ILC2s, and ILC3s. ILC2s produce Th2 cytokines, IL-4, IL-5, and IL-13, upon activation by epithelial cell-derived cytokines, lipid mediators (cysteinyl leukotrienes and prostaglandin D2), and TNF family member TL1A and promote structural and immune cell responses in the airways after antigen exposure. In addition, ILC2 function is also influenced by inducible T cell costimulator (ICOS)/ICOS-ligand (ICOS-L) interactions via direct contact between immune cells. The most common airway antigens are allergens and viruses which are highly linked to the induction of airway diseases with underlying type 2 inflammation including asthma and allergic rhinitis. Based on recent findings linking ILC2s and airway Th2 responses, there is intensive investigation into the role of ILC2s in human disease with the hope of a better understanding of the pathophysiology and the discovery of novel potential therapeutic targets. This review summarizes the recent advances made in elucidating ILC2 involvement in human Th2 airway disease.

  10. 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.

  11. Mast cell mediators in citric acid-induced airway constriction of guinea pigs

    SciTech Connect

    Lin, C.-H.; Lai, Y.-L. . E-mail: tiger@ha.mc.ntu.edu.tw

    2005-08-15

    We demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. In this study, we further investigated the underlying mediator(s) for this type of airway constriction. At first, to examine effects caused by blocking agents, 67 young Hartley guinea pigs were divided into 7 groups: saline + CA; methysergide (serotonin receptor antagonist) + CA; MK-886 (leukotriene synthesis inhibitor) + CA; mepyramine (histamine H{sub 1} receptor antagonist) + CA; indomethacin (cyclooxygenase inhibitor) + CA; cromolyn sodium (mast cell stabilizer) + CA; and compound 48/80 (mast cell degranulating agent) + CA. Then, we tested whether leukotriene C{sub 4} (LTC{sub 4}) or histamine enhances CA-induced airway constriction in compound 48/80-pretreated guinea pigs. We measured dynamic respiratory compliance (Crs) and forced expiratory volume in 0.1 s (FEV{sub 0.1}) during either baseline or recovery period. In addition, we detected histamine level, an index of pulmonary mast cell degranulation, in bronchoalveolar lavage (BAL) samples. Citric acid aerosol inhalation caused decreases in Crs and FEV{sub 0.1}, indicating airway constriction in the control group. This airway constriction was significantly attenuated by MK-886, mepyramine, cromolyn sodium, and compound 48/80, but not by either methysergide or indomethacin. Both LTC{sub 4} and histamine infusion significantly increased the magnitude of CA-induced airway constriction in compound 48/80-pretreated guinea pigs. Citric acid inhalation caused significant increase in histamine level in the BAL sample, which was significantly suppressed by compound 48/80. These results suggest that leukotrienes and histamine originating from mast cells play an important role in CA inhalation-induced noncholinergic airway constriction.

  12. Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

    SciTech Connect

    Chen, Ming; Lv, Zhiqiang; Huang, Linjie; Zhang, Wei; Lin, Xiaoling; Shi, Jianting; Zhang, Wei; Liang, Ruiyun; Jiang, Shanping

    2015-02-15

    Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolide significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.

  13. Effects of second hand smoke on airway secretion and mucociliary clearance

    PubMed Central

    Liu, Yanyan; Di, Y. Peter

    2012-01-01

    The airway acts as the first defense against inhaled pathogens and particulate matter from the environment. One major way for the airway to clear inhaled foreign objects is through mucociliary clearance (MCC), an important component of the respiratory innate immune defense against lung disease. MCC is characterized by the upward movement of mucus by ciliary motion that requires a balance between the volume and composition of the mucus, adequate periciliary liquid (PCL) volume, and normal ciliary beat frequency (CBF). Airway surface fluid (ASL) is a thin layer liquid that consists of the highly viscous mucus upper “gel” layer, and the watery lubricating lower “sol” layer. Mucus production, secretion and clearance are considered to play a critical role in maintenance of airway health because it maintains hydration in the airway and traps particulates, bacteria, and viruses. Different types of epithelial cells, including secretory cells, and ciliated cells, contribute to the MCC function. Cigarette smoke (CS) contains chemicals and particulates that significantly affect airway secretion. Active and passive CS-induced chronic obstructive pulmonary disease (COPD) is frequently associated with hyperplasia of goblet cells and submucosal glands (SMGs), thus increasing the secretory capacity of the airways that impairs MCC. PMID:22973232

  14. 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.

  15. 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

  16. Post-transcriptional regulation of interleukin-10 in peripheral B cells of airway allergy patients

    PubMed Central

    Luo, Xiang-Qian; Yang, Shao-Bo; Qiu, Shu-Qi; Xie, Rui-Di; Yang, Li-Tao; Ke, Yu-Xing; Zhao, Hong-Xia; Geng, Xiao-Rui; Yang, Gui; Liu, Zhi-Qiang; Liu, Jiang-Qi; Wang, Shuai; Liu, Da-Bo; Liu, Jun

    2016-01-01

    The dysfunction of peripheral immune tolerance plays an important role in the pathogenesis of allergic diseases. Recent reports indicate that micro RNA (miR)-98 is associated with the process of aberrant immune responses. This study aims to test a hypothesis that miR-98 is associated with the pathogenesis of airway allergy via interfering with the development of regulatory B cells (Breg). In this study, patients with airway allergy were recruited into this study. The frequency of Bregs was assessed by flow cytometry. The levels of miR-98 in peripheral B cells were determined by RT-qPCR. A cell-culture model of B cells was developed to test the role of miR-98 in the repressing of interleukin (IL)-10 in B cells. The results showed that the levels of IL-10 in peripheral B cells were significantly lower in patients with airway allergy as compared with healthy subjects. High levels of miR-98 (one of the miR-98 members) were detected in peripheral B cells of patients with airway allergy, which was mimicked by stimulating B cells with IL-4. Histone acetyltransferase p300 was involved in the IL-4-induced miR-98 expression. miR-98 mediated the IL-4-inhibited IL-10 expression in B cells. In conclusion, miR-98 affects the expression of IL-10 in B cells and may be a novel therapeutic target for the treatment of allergic diseases. PMID:28078048

  17. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration.

    PubMed

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping; Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (KATP) channels have been identified in ASMCs. Mount evidence has suggested that KATP channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K(+) channels triggers K(+) efflux, which leading to membrane hyperpolarization, preventing Ca(2+)entry through closing voltage-operated Ca(2+) channels. Intracellular Ca(2+) is the most important regulator of muscle contraction, cell proliferation and migration. K(+) efflux decreases Ca(2+) influx, which consequently influences ASMCs proliferation and migration. As a KATP channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca(2+)/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective KATP channel antagonist. These findings provide a strong evidence to support that Ipt antagonize the proliferating and migrating effects of PDGF-BB on

  18. 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.

  19. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system

    SciTech Connect

    Cervia, Davide; Catalani, Elisabetta; Belardinelli, Maria Cristina; Perrotta, Cristiana; Picchietti, Simona; Alimenti, Claudio; Casini, Giovanni; Fausto, Anna Maria; Vallesi, Adriana

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

  20. Interactive effect of cigarette smoke extract and world trade center dust particles on airway cell cytotoxicity.

    PubMed

    Xu, Alice; Prophete, Colette; Chen, Lung-chi; Emala, Charles W; Cohen, Mitchell D

    2011-01-01

    Rescue workers and residents exposed to the environment surrounding the collapse of the World Trade Center (WTC) on September 11, 2001, have suffered a disproportionate incidence of chronic lung disease attributed to the inhalation of airborne dust. To date, the pathophysiology of this lung disease is poorly understood. The aim of this study was to examine whether airborne dust contaminants recovered from the surrounding area 24-48 h after the collapse of the WTC demonstrate direct cytotoxicity to two airway cell types that were most directly exposed to inhaled dust, airway epithelial and smooth muscle cells. It was also of interest to determine whether the presence of these dusts could modulate the effects of cigarette smoke on these cell types in that some of the individuals who responded to the collapse site were also smokers. Human cultured airway epithelial (BEAS-2B) cells were exposed to 10% cigarette smoke extract (CSE), WTC dust particles (10-53 μm; 0.01-0.5 μg/μl), or a combination of the two for 2-24 h. Cell viability was measured by determining mitochondrial integrity (MTT assays) and apoptosis (poly-ADP-ribose polymerase [PARP] immunoblotting). Conditioned cell culture media recovered from the CSE- and/or WTC dust-exposed BEAS-2B cells were then applied to cultured human airway smooth muscle cells that were subsequently assayed for mitochondrial integrity and their ability to synthesize cyclic AMP (a regulator of airway smooth muscle constriction). BEAS-2B cells underwent necrotic cell death following exposure to WTC dust or CSE for 2-24 h without evidence of apoptosis. Smooth muscle cells demonstrated cellular toxicity and enhanced cyclic AMP synthesis following exposure to conditioned media from WTC- or CSE-exposed epithelial cells. These acute toxicity assays of WTC dust and CSE offer insights into lung cell toxicity that may contribute to the pathophysiology of chronic lung disease in workers and residents exposed to WTC dust. These studies

  1. TGF-β-dependent dendritic cell chemokinesis in murine models of airway disease

    PubMed Central

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Ma, Royce; Murray, Lynne A.; Tsui, Ping; Lou, Jianlong; Marks, James D.; Baron, Jody L.; Krummel, Matthew F.; Nishimura, Stephen L.

    2015-01-01

    Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of antigen surveillance and antigen presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1β) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 μm from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin αvβ8, a critical activator of TGF-β αvβ8-mediated TGF-β activation is known to enhance IL-1β-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which αvβ8, ccl20 and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli. PMID:26109638

  2. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia and airway mucus formation in vitro and in vivo

    PubMed Central

    Gundavarapu, Sravanthi; Wilder, Julie A.; Mishra, Neerad C.; Rir-sima-ah, Jules; Langley, Raymond J.; Singh, Shashi P.; Saeed, Ali Imran; Jaramillo, Richard J.; Gott, Katherine M.; Peña-Philippides, Juan Carlos; Harrod, Kevin S.; McIntosh, J. Michael; Buch, Shilpa; Sopori, Mohan L.

    2012-01-01

    Background Airway mucus hypersecretion is a key pathophysiological feature in number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. Objectives Characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. Methods IL-13 and gamma-aminobutyric acid receptors (GABAARs) are implicated in airway mucus. We examined the role of IL-13 and GABAARs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells, and secondhand cigarette smoke and/or ovalbumin-induced mucus formation in vivo. Results Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABAAR antagonist picrotoxin (PIC). Airway epithelial cells express α7/α9/α10 nicotinic acetylcholine receptors (nAChRs) and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells and/or in mouse airways. Conclusions Nicotine-induced airway mucus formation is independent of IL-13 and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various pro-mucoid agents, including IL-13. In the absence of nicotine, acetylcholine may be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABAARα2 in the MUC5AC pathway. Acetylcholine and α-7-nAChRs may serve as therapeutic targets to control airway mucus. PMID:22578901

  3. 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.

  4. Single-Cell Analysis of Mast Cell Degranulation Induced by Airway Smooth Muscle-Secreted Chemokines

    PubMed Central

    Manning, Benjamin M.; Meyer, Audrey F.; Gruba, Sarah M.; Haynes, Christy L.

    2015-01-01

    Background Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma. Methods Carbon fiber microelectrode amperometry was used to study the effects of ASM–secreted chemokines on mouse peritoneal MC degranulation. Results MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin released per granule was correlated with increased spike half-width and rise-time values. Conclusions MCs are directly activated with ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery. General Significance The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation. PMID:25986989

  5. Detection of a novel stem cell probably involved in normal turnover of the lung airway epithelium.

    PubMed

    Ortega-Martínez, Marta; Rodríguez-Flores, Laura E; de-la-Garza-González, Carlos; Ancer-Rodríguez, Jesús; Jaramillo-Rangel, Gilberto

    2015-11-01

    Regeneration of the lung airway epithelium after injury has been extensively studied. In contrast, analysis of its turnover in healthy adulthood has received little attention. In the classical view, this epithelium is maintained in the steady-state by the infrequent proliferation of basal or Clara cells. The intermediate filament protein nestin was initially identified as a marker for neural stem cells, but its expression has also been detected in other stem cells. Lungs from CD1 mice at the age of 2, 6, 12, 18 or 24 months were fixed in neutral-buffered formalin and paraffin-embedded. Nestin expression was examined by an immunohistochemical peroxidase-based method. Nestin-positive cells were detected in perivascular areas and in connective tissue that were in close proximity of the airway epithelium. Also, nestin-positive cells were found among the cells lining the airway epithelium. These findings suggest that nestin-positive stem cells circulate in the bloodstream, transmigrate through blood vessels and localize in the lung airway epithelium to participate in its turnover. We previously reported the existence of similar cells able to differentiate into lung chondrocytes. Thus, the stem cell reported here might be a bone marrow-derived mesenchymal stem cell (BMDMSC) able to generate several types of lung tissues. In conclusion, our findings indicate that there exist a BMDMSC in healthy adulthood that participates in the turnover of the lung airway epithelium. These findings may improve our knowledge about the lung stem cell biology and also provide novel approaches to therapy for devastating pulmonary diseases.

  6. 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.

  7. Human airway epithelia express catalytically active NEU3 sialidase.

    PubMed

    Lillehoj, Erik P; Hyun, Sang Won; Feng, Chiguang; Zhang, Lei; Liu, Anguo; Guang, Wei; Nguyen, Chinh; Sun, Wenji; Luzina, Irina G; Webb, Tonya J; Atamas, Sergei P; Passaniti, Antonino; Twaddell, William S; Puché, Adam C; Wang, Lai-Xi; Cross, Alan S; Goldblum, Simeon E

    2014-05-01

    Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

  8. 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...

  9. 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

  10. What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia.

    PubMed

    Walentek, Peter; Quigley, Ian K

    2017-01-01

    Over the past years, the Xenopus embryo has emerged as an incredibly useful model organism for studying the formation and function of cilia and ciliated epithelia in vivo. This has led to a variety of findings elucidating the molecular mechanisms of ciliated cell specification, basal body biogenesis, cilia assembly, and ciliary motility. These findings also revealed the deep functional conservation of signaling, transcriptional, post-transcriptional, and protein networks employed in the formation and function of vertebrate ciliated cells. Therefore, Xenopus research can contribute crucial insights not only into developmental and cell biology, but also into the molecular mechanisms underlying cilia related diseases (ciliopathies) as well as diseases affecting the ciliated epithelium of the respiratory tract in humans (e.g., chronic lung diseases). Additionally, systems biology approaches including transcriptomics, genomics, and proteomics have been rapidly adapted for use in Xenopus, and broaden the applications for current and future translational biomedical research. This review aims to present the advantages of using Xenopus for cilia research, highlight some of the evolutionarily conserved key concepts and mechanisms of ciliated cell biology that were elucidated using the Xenopus model, and describe the potential for Xenopus research to address unresolved questions regarding the molecular mechanisms of ciliopathies and airway diseases.

  11. 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

  12. Matrix metalloproteinase expression and activity in human airway smooth muscle cells

    PubMed Central

    Elshaw, Shona R; Henderson, Neil; Knox, Alan J; Watson, Susan A; Buttle, David J; Johnson, Simon R

    2004-01-01

    Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells.MMPs and TIMPs were examined using quantitative real-time RT–PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity.The most abundant MMPs were pro-MMP-2, pro- MMP-3, active MMP-3 and MT1-MMP. TIMP-1 and TIMP-2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM-conditioned medium to inhibit recombinant MMP-2 in a QF assay. Thrombin increased MMP activity by activation of pro-MMP-2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4.In conclusion, ASM cells express pro-MMP-2, pro and active MMP-3, MMP-9 and MT1-MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP-2 can be activated by thrombin which may contribute to airway remodelling. PMID:15265805

  13. 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

  14. 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.

  15. TLR4 signalling in pulmonary stromal cells is critical for inflammation and immunity in the airways.

    PubMed

    Perros, Frederic; Lambrecht, Bart N; Hammad, Hamida

    2011-09-24

    Inflammation of the airways, which is often associated with life-threatening infection by Gram-negative bacteria or presence of endotoxin in the bioaerosol, is still a major cause of severe airway diseases. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to protein rich fluid leak in the alveolar space. Mammalian Toll-like receptors (TLRs) are important signalling receptors in innate host defense. Among these receptors, TLR4 plays a critical role in the response to endotoxin. Lungs are a complex compartmentalized organ with separate barriers, namely the alveolar-capillary barrier, the microvascular endothelium, and the alveolar epithelium. An emerging theme in the field of lung immunology is that structural cells (SCs) of the airways such as epithelial cells (ECs), endothelial cells, fibroblasts and other stromal cells produce activating cytokines that determine the quantity and quality of the lung immune response. This review focuses on the role of TLR4 in the innate and adaptive immune functions of the pulmonary SCs.

  16. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

    SciTech Connect

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation and migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that Ipt

  17. Airway cooling and mucosal injury during cold weather exercise.

    PubMed

    Davis, M S; Lockard, A J; Marlin, D J; Freed, A N

    2002-09-01

    In human subjects that exercise strenuously in cold weather, there is evidence that hyperventilation with cold air leads to peripheral airway cooling, desiccation and mucosal injury. Our hypothesis was that hyperventilation with cold air can result in penetration of unconditioned air (air that is not completely warmed and humidified) into the peripheral airways of exercising horses, resulting in peripheral airway mucosal injury. To test this hypothesis, a thermister-tipped catheter was inserted through the midcervical trachea and advanced into a sublobar bronchus in three horses that cantered on a treadmill at 6.6 m/s while breathing cold (5 degrees C) air. The mean (+/- s.e.) intra-airway temperature during cantering was 33.3 +/- 0.4 degrees C, a value comparable to the bronchial lumen temperatures measured in man during maximal exercise while breathing subfreezing dry air. In a second experiment, 6 fit Thoroughbred racehorses with satisfactory performance were used to determine whether strenuous exercise in cold conditions can produce airway injury. Horses were assigned to Exercise (E) or Control (C) groups in a random crossover design. Samples of bronchoalveolar lavage fluid (BALF) in the E treatment were recovered within 30 min of galloping exercise in 4 degrees C, 100% relative humidity (E), while in C BALF samples were obtained when the horses had not performed any exercise for at least 48 h prior. Ciliated epithelial cells in BALF were higher in E than in the C treatment. Similar results have been found in human athletes and laboratory animal models of cold weather exercise. These results support the hypothesis that, similar to man, horses that exercise in cold weather experience peripheral airway mucosal injury due to the penetration of unconditioned air. Furthermore, these results suggest that airway cooling and desiccation may be a factor in airway inflammation commonly found in equine athletes.

  18. 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.

  19. 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

  20. 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...

  1. 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.

  2. 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, ...

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  6. 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

  7. 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.

  8. 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

  9. α-Galactosylceramide-induced airway eosinophilia is mediated through the activation of NKT cells.

    PubMed

    Chuang, Ya-Hui; Wang, Tzu-Chun; Jen, Hsiao-Yu; Yu, Alice L; Chiang, Bor-Luen

    2011-04-15

    Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.

  10. Pycnogenol Ameliorates Asthmatic Airway Inflammation and Inhibits the Function of Goblet Cells.

    PubMed

    Liu, Zhaoe; Han, Bo; Chen, Xing; Wu, Qiaoling; Wang, Lijun; Li, Gang

    2016-11-01

    Pycnogenol(®) (PYC) is utilized in the treatment of various diseases ranging from chronic inflammation to circulatory diseases, but its efficacy and functional mechanism in pediatric asthma continue to remain obscure. Therefore, the purpose of this study was to investigate the effectiveness and molecular mechanism of PYC on regulation of asthmatic airway inflammation. We found that PYC with tail intravenous injection of 50 mg/kg or intragastric administration of 100 mg/kg all reduced ovalbumin (OVA)-induced airway injury. Pharmacokinetics of PYC was evaluated by high-performance liquid chromatography assay, indicating that PYC was quickly absorbed into the blood after intragastric administration, and PYC metabolism was later improved gradually with increase of time after PYC administration. PYC has a higher bioavailability of 71.96%, and it was more easily absorbed by the body. PYC inhibited the number of total inflammatory cells and levels of interleukin (IL)-4, IL-5, IL-9, and IL-13 in bronchoalveolar lavage fluid of OVA-induced mice. PYC inhibited IL-13 secretion from the Th2 cells, thereby causing a reduction in expression of the signaling molecules in JAK/STAT6 pathway in airway epithelial cells. STAT6 silence suppressed IL-13-increased acetylcholine level. STAT6 overexpression promoted expression of goblet cell metaplasia-associated molecules (FOXA3, SPDEF, and Muc5ac). PYC suppressed OVA-induced expression of FOXA3, SPDEF, and Muc5ac in lung. Our findings indicate that PYC has a higher bioavailability and it prevents emergence of OVA-induced airway injury and airway inflammation in mice by inhibiting IL-13/JAK/STAT6 pathway and blocking release of acetylcholine to reduce goblet cell metaplasia.

  11. Airway basal cells of healthy smokers express an embryonic stem cell signature relevant to lung cancer.

    PubMed

    Shaykhiev, Renat; Wang, Rui; Zwick, Rachel K; Hackett, Neil R; Leung, Roland; Moore, Malcolm A S; Sima, Camelia S; Chao, Ion Wa; Downey, Robert J; Strulovici-Barel, Yael; Salit, Jacqueline; Crystal, Ronald G

    2013-09-01

    Activation of the human embryonic stem cell (hESC) signature genes has been observed in various epithelial cancers. In this study, we found that the hESC signature is selectively induced in the airway basal stem/progenitor cell population of healthy smokers (BC-S), with a pattern similar to that activated in all major types of human lung cancer. We further identified a subset of 6 BC-S hESC genes, whose coherent overexpression in lung adenocarcinoma (AdCa) was associated with reduced lung function, poorer differentiation grade, more advanced tumor stage, remarkably shorter survival, and higher frequency of TP53 mutations. BC-S shared with hESC and a considerable subset of lung carcinomas a common TP53 inactivation molecular pattern which strongly correlated with the BC-S hESC gene expression. These data provide transcriptome-based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might represent a common early molecular event in the development of aggressive lung carcinomas in humans.

  12. 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

  13. 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.

  14. Omega-3 polyunsaturated fatty acids accelerate airway repair by activating FFA4 in club cells.

    PubMed

    Lee, Kyoung-Pil; Park, Soo-Jin; Kang, Saeromi; Koh, Jung-Min; Sato, Koichi; Chung, Hae Young; Okajima, Fumikazu; Im, Dong-Soon

    2017-03-17

    A GPCR named FFA4 (also known as GPR120) was found to act as a GPCR for omega-3 polyunsaturated fatty acids. Its expression has been reported in lung epithelial club cells. The authors investigated whether supplementation of the omega-3 fatty acids benefits lung health. Omacor® (7.75 mg kg-1), clinically prescribed preparation of omega-3 fatty acids and FFA4-knockout mice were utilized in a naphthalene-induced mouse model of acute airway injury (one injection of 30 mg kg-1, i.p.). Naphthalene injection induced complete destruction of bronchiolar epithelial cells within a day. Appearance of bronchiolar epithelial cells was observed after 21 days in control mice. It was found, however, that supplementation of omacor accelerated the recovery. The appearance of bronchiolar epithelial cells was observed between 7 and 14 days after naphthalene injury in omacor-treated mice. In isolated club cells, omega-3 fatty acids were found to stimulate cell proliferation and migration but to inhibit cell differentiation. Using pharmacological tools and FFA4-knockout mice, FFA4 was found to be responsible for omega-3 fatty acids-induced proliferation in vitro in club cells. Furthermore, accelerated recovery from naphthalene-induced airway injury in omacor-treated mice was not observed in FFA4-knockout mice in vivo. Present findings indicate that omega-3 fatty acids-induced proliferation of bronchiole epithelial cells through FFA4 is responsible for omacor-induced accelerated recovery from airway injury. Therefore, intermittent administration of omacor needs to be tested for acute airway injury, because omega-3 fatty acids stimulate proliferation but inhibits differentiation of club cells.

  15. Near Equilibrium Calculus of Stem Cells in Application to the Airway Epithelium Lineage

    PubMed Central

    Sun, Zheng; Plikus, Maksim V.; Komarova, Natalia L.

    2016-01-01

    Homeostatic maintenance of tissues is orchestrated by well tuned networks of cellular signaling. Such networks regulate, in a stochastic manner, fates of all cells within the respective lineages. Processes such as symmetric and asymmetric divisions, differentiation, de-differentiation, and death have to be controlled in a dynamic fashion, such that the cell population is maintained at a stable equilibrium, has a sufficiently low level of stochastic variation, and is capable of responding efficiently to external damage. Cellular lineages in real tissues may consist of a number of different cell types, connected by hierarchical relationships, albeit not necessarily linear, and engaged in a number of different processes. Here we develop a general mathematical methodology for near equilibrium studies of arbitrarily complex hierarchical cell populations, under regulation by a control network. This methodology allows us to (1) determine stability properties of the network, (2) calculate the stochastic variance, and (3) predict how different control mechanisms affect stability and robustness of the system. We demonstrate the versatility of this tool by using the example of the airway epithelium lineage. Recent research shows that airway epithelium stem cells divide mostly asymmetrically, while the so-called secretory cells divide predominantly symmetrically. It further provides quantitative data on the recovery dynamics of the airway epithelium, which can include secretory cell de-differentiation. Using our new methodology, we demonstrate that while a number of regulatory networks can be compatible with the observed recovery behavior, the observed division patterns of cells are the most optimal from the viewpoint of homeostatic lineage stability and minimizing the variation of the cell population size. This not only explains the observed yet poorly understood features of airway tissue architecture, but also helps to deduce the information on the still largely hypothetical

  16. 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

  17. Pseudomonas aeruginosa Airway Infection Recruits and Modulates Neutrophilic Myeloid-Derived Suppressor Cells

    PubMed Central

    Öz, Hasan H.; Zhou, Benyuan; Voss, Pina; Carevic, Melanie; Schroth, Carolin; Frey, Nina; Rieber, Nikolaus; Hector, Andreas; Hartl, Dominik

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes infections mainly in patients with cystic fibrosis (CF) lung disease. Despite innate and adaptive immune responses upon infection, P. aeruginosa is capable of efficiently escaping host defenses, but the underlying immune mechanisms remain poorly understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that are functionally characterized by their potential to suppress T- and natural killer (NK)-cell responses. Here we demonstrate, using an airway in vivo infection model, that P. aeruginosa recruits and activates neutrophilic MDSCs, which functionally suppress T-cell responses. We further show that the CF gene defect (CF transmembrane conductance regulator, CFTR) modulates the functionality, but not the recruitment or generation of neutrophilic MDSCs. Collectively, we define a mechanism by which P. aeruginosa airway infection undermines host immunity by modulating neutrophilic MDSCs in vivo. PMID:27965936

  18. 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.

  19. Lung airway-surveilling CXCR3(hi) memory CD8(+) T cells are critical for protection against influenza A virus.

    PubMed

    Slütter, Bram; Pewe, Lecia L; Kaech, Susan M; Harty, John T

    2013-11-14

    Inducing memory CD8(+) T cells specific for conserved antigens from influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8(+) T cells in the airways played an important role in early control of IAV. Expression of the chemokine receptor CXCR3 was critical for memory CD8(+) T cells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8(+) T cells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8(+) T cells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12 enhanced CXCR3 expression, sustained airway localization of memory CD8(+) T cells, and resulted in superior protection against IAV.

  20. CD4(+) Th2 cells are directly regulated by IL-10 during allergic airway inflammation.

    PubMed

    Coomes, S M; Kannan, Y; Pelly, V S; Entwistle, L J; Guidi, R; Perez-Lloret, J; Nikolov, N; Müller, W; Wilson, M S

    2017-01-01

    Interleukin-10 (IL-10) is an important regulatory cytokine required to control allergy and asthma. IL-10-mediated regulation of T cell-mediated responses was previously thought to occur indirectly via antigen-presenting cells. However, IL-10 can act directly on regulatory T cells and T helper type 17 (Th17) cells. In the context of allergy, it is therefore unclear whether IL-10 can directly regulate T helper type 2 (Th2) cells and whether this is an important regulatory axis during allergic responses. We sought to determine whether IL-10 signaling in CD4(+) Th2 cells was an important mechanism of immune regulation during airway allergy. We demonstrate that IL-10 directly limits Th2 cell differentiation and survival in vitro and in vivo. Ablation of IL-10 signaling in Th2 cells led to enhanced Th2 cell survival and exacerbated pulmonary inflammation in a murine model of house dust mite allergy. Mechanistically, IL-10R signaling regulated the expression of several genes in Th2 cells, including granzyme B. Indeed, IL-10 increased granzyme B expression in Th2 cells and led to increased Th2 cell death, identifying an IL-10-regulated granzyme B axis in Th2 cells controlling Th2 cell survival. This study provides clear evidence that IL-10 exerts direct effects on Th2 cells, regulating the survival of Th2 cells and severity of Th2-mediated allergic airway inflammation.

  1. 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

  2. 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.

  3. "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

  4. 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

  5. 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.

  6. 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.

  7. 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.

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

    PubMed Central

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

    2016-01-01

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

  9. Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance

    PubMed Central

    Soroosh, Pejman; Doherty, Taylor A.; Duan, Wei; Mehta, Amit Kumar; Choi, Heonsik; Adams, Yan Fei; Mikulski, Zbigniew; Khorram, Naseem; Rosenthal, Peter; Broide, David H.

    2013-01-01

    Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MØs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3+ iTreg cells is unclear. Here, we show that lung-resident tissue MØs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3+ Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MØs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MØs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MØs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma. PMID:23547101

  10. EVIDENCE FOR A CRYPTOMONAD SYMBIONT IN THE CILIATE, CYCLOTRICHIUM MEUNIERI(1).

    PubMed

    Barber, R T; White, A W; Siegelman, H W

    1969-03-01

    Extracts of the marine ciliate Cyclotrichium meunieri contained chlorophylls a and c, carotenoids, and a phycoerythrin with a single absorbance maximum at 542 nm. This assemblage of pigments suggests that the numerous photosynthetic symbionts present in each ciliate cell belong to the Cryptophyceae.

  11. 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.

  12. 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.

  13. The Role of Bitter and Sweet Taste Receptors in Upper Airway Immunity

    PubMed Central

    Workman, Alan D.; Palmer, James N.; Adappa, Nithin D.

    2016-01-01

    Over the past several years, taste receptors have emerged as key players in the regulation of innate immune defenses in the mammalian respiratory tract. Several cell types in the airway, including ciliated epithelial cells, solitary chemosensory cells, and bronchial smooth muscle cells, all display chemoresponsive properties that utilize taste receptors. A variety of bitter products secreted by microbes are detected with resultant downstream inflammation, increased mucous clearance, antimicrobial peptide secretion, and direct bacterial killing. Genetic variation of bitter taste receptors also appears to play a role in the susceptibility to infection in respiratory disease states, including that of chronic rhinosinusitis. Ongoing taste receptor research may yield new therapeutics that harness innate immune defenses in the respiratory tract and may offer alternatives to antibiotic treatment. The present review discusses taste receptor-protective responses and analyzes the role these receptors play in mediating airway immune function. PMID:26492878

  14. The Role of Bitter and Sweet Taste Receptors in Upper Airway Immunity.

    PubMed

    Workman, Alan D; Palmer, James N; Adappa, Nithin D; Cohen, Noam A

    2015-12-01

    Over the past several years, taste receptors have emerged as key players in the regulation of innate immune defenses in the mammalian respiratory tract. Several cell types in the airway, including ciliated epithelial cells, solitary chemosensory cells, and bronchial smooth muscle cells, all display chemoresponsive properties that utilize taste receptors. A variety of bitter products secreted by microbes are detected with resultant downstream inflammation, increased mucous clearance, antimicrobial peptide secretion, and direct bacterial killing. Genetic variation of bitter taste receptors also appears to play a role in the susceptibility to infection in respiratory disease states, including that of chronic rhinosinusitis. Ongoing taste receptor research may yield new therapeutics that harness innate immune defenses in the respiratory tract and may offer alternatives to antibiotic treatment. The present review discusses taste receptor-protective responses and analyzes the role these receptors play in mediating airway immune function.

  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 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

  17. 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.

  18. The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

    PubMed Central

    Hayman, Yvette A.; Williamson, James D.; Hart, Simon P.; Morice, Alyn H.

    2017-01-01

    Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR) and aspiration events. The observation of lipid-laden macrophages (LLMs) within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD) pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways. PMID:28344981

  19. Effects of cigarette smoke and asbestos on airway, vascular and mesothelial cell proliferation.

    PubMed Central

    Sekhon, H.; Wright, J.; Churg, A.

    1995-01-01

    In order to determine whether exposure to both cigarette smoke and asbestos leads to enhanced cell proliferation, and whether pleura cell proliferation reflects the presence of fibres at or near the pleura, rats were exposed to air (control), daily cigarette smoke, a single intratracheal instillation of amosite asbestos, or a combination of smoke and asbestos. Dividing cells were labelled with bromodeoxyuridine (BrdU) and animals were sacrificed at 1, 2, 7 or 14 days. Both cigarette smoke and asbestos produced increases in the labelling index of small airway wall, epithelial cells and pulmonary artery cells. In the small airways there was a brief marked positive synergistic interaction between these two agents, but synergism was not seen in the vessels. Cigarette smoke did not increase the labelling of mesothelial or submesothelial cells, whereas asbestos caused a persisting increase in mesothelial cell labelling. There was no correlation between the number of BrdU labelled mesothelial or submesothelial cells and the number of fibres touching the pleura, or located within 180 microns of the pleura. We conclude that the combination of cigarette smoke and asbestos exposure produces a complex set of interactions and has the potential to markedly increase cell proliferation in the parenchyma, an effect that may be important in both fibrogenesis and carcinogenesis. In contrast to the diminishing effects over time of a single dose of asbestos on cell proliferation in the small airways and vessels, the same dose of asbestos leads to sustained mesothelial cell proliferation. However, the latter process does not correlate with local accumulation of asbestos fibres. Images Figure 1 PMID:8652361

  20. 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.

  1. 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

  2. Parasitic Nematode-Induced CD4+Foxp3+T Cells Can Ameliorate Allergic Airway Inflammation

    PubMed Central

    Kang, Shin Ae; Park, Mi-Kyung; Cho, Min Kyoung; Park, Sang Kyun; Jang, Min Seong; Yang, Bo-Gie; Jang, Myoung Ho; Kim, Dong-Hee; Yu, Hak Sun

    2014-01-01

    Background The recruitment of CD4+CD25+Foxp3+T (Treg) cells is one of the most important mechanisms by which parasites down-regulate the immune system. Methodology/Principal Findings We compared the effects of Treg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced Treg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4+Foxp3+ cells from T. spiralis-infected [Inf(+)Foxp3+] or uninfected [Inf(-)Foxp3+] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3+ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3+ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3+ cells migrated to inflammation sites in the lung and expressed higher levels of Treg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3+ cells. Conclusion/Significance T. spiralis infection promotes the proliferation and functional activation of Treg cells. Parasite-induced Treg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced Treg cells. The adoptive transfer of Inf(+)Foxp3+ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment

  3. Unjamming and cell shape in the asthmatic airway epithelium

    NASA Astrophysics Data System (ADS)

    Park, Jin-Ah; Kim, Jae Hun; Bi, Dapeng; Mitchel, Jennifer A.; Qazvini, Nader Taheri; Tantisira, Kelan; Park, Chan Young; McGill, Maureen; Kim, Sae-Hoon; Gweon, Bomi; Notbohm, Jacob; Steward, Robert, Jr.; Burger, Stephanie; Randell, Scott H.; Kho, Alvin T.; Tambe, Dhananjay T.; Hardin, Corey; Shore, Stephanie A.; Israel, Elliot; Weitz, David A.; Tschumperlin, Daniel J.; Henske, Elizabeth P.; Weiss, Scott T.; Manning, M. Lisa; Butler, James P.; Drazen, Jeffrey M.; Fredberg, Jeffrey J.

    2015-10-01

    From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems--both inert and living--have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell-cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.

  4. Ozone-Induced Type 2 Immunity in Nasal Airways. Development and Lymphoid Cell Dependence in Mice.

    PubMed

    Ong, Chee Bing; Kumagai, Kazuyoshi; Brooks, Phillip T; Brandenberger, Christina; Lewandowski, Ryan P; Jackson-Humbles, Daven N; Nault, Rance; Zacharewski, Timothy R; Wagner, James G; Harkema, Jack R

    2016-03-01

    Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone.

  5. Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

    PubMed Central

    Devente, Savannah R.; Kirk, Michelle R.; Seedorf, Henning; Dehority, Burk A.

    2015-01-01

    The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups. PMID:25616800

  6. Baicalin inhibits PDGF-induced proliferation and migration of airway smooth muscle cells.

    PubMed

    Yang, Guang; Li, Jian-Qiang; Bo, Jian-Ping; Wang, Bei; Tian, Xin-Rui; Liu, Tan-Zhen; Liu, Zhuo-La

    2015-01-01

    Airway smooth muscle (ASM) cell proliferation and migration play important roles in airway remodeling in asthma. In vitro platelet-derived growth factor (PDGF) induced ASM cell proliferation and migration. Baicalin is one of flavonoid extracts from Scutellaria baicalensis, which has an anti-asthma effect. However, little is known about its role in PDGF-induced proliferation and migration in rat ASM (RASM) cells. In this study, we aimed to investigate the effects of baicalin on PDGF-induced RASM cell proliferation and migration. We also identified the signaling pathway by which baicalin influences RASM cell proliferation and migration. In the current study, we demonstrated that baicalin suppressed PDGF-induced RASM cell proliferation, arrested PDGF-induced cell-cycle progression. It also suppressed PDGF-induced RASM cell migration. Furthermore, baicalin suppressed PDGF-induced expression of phosphorylated p38, ERK1/2 and JNK in RASM cells. In summary, our study is the first to show that baicalin pretreatment can significantly inhibit PDGF-induced RASM cell proliferation and migration by suppressing the MAPK signaling pathway, and baicalin may be a useful chemotherapeutic agent for asthma.

  7. 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.

  8. Role of non-coding RNAs in maintaining primary airway smooth muscle cells

    PubMed Central

    2014-01-01

    Background The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases. Objective We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS). Methods mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR. Results A small number of miRNAs (including miR-150, −371-5p, −718, −940, −1181, −1207-5p, −1915, and −3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA ‘sponges’ for 4 miRNAs (miR-150, −371-5p, −940, −1207-5p). Conclusion This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). PMID:24886442

  9. 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.

  10. 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

  11. Sialo-oligosaccharide receptors for Mycoplasma pneumoniae and related oligosaccharides of poly-N-acetyllactosamine series are polarized at the cilia and apical-microvillar domains of the ciliated cells in human bronchial epithelium.

    PubMed

    Loveless, R W; Feizi, T

    1989-04-01

    The occurrence and distribution of the sialo-oligosaccharide receptors (sialosyl-I and sialosyl-i) for Mycoplasma pneumoniae as well as other related oligosaccharide structures of poly-N-acetyllactosamine type, and their short-chain analogs based on galactose linked beta 1-4 or beta 1-3 to N-acetylglucosamine (Gal beta 1-4GlcNAc or Gal beta 1-3GlcNAc, respectively) were investigated in the human bronchial epithelium by histochemistry by using sequence-specific monoclonal antibodies and lectins. Among the mature epithelial cells, only ciliated cells were found to express the long-chain antigens, whereas mucus-secreting cells contained the short-chain antigens associated with mucus globules. The long-chain oligosaccharides were found to be highly polarized at the luminal aspects of the ciliated cells where the branched structures (I and sialosyl-I antigens) were detected both at the apical-microvillar border and on the cilia, but the linear structures (i, sialosyl-i, and VIM-2 antigens) were detected exclusively at the apical-microvillar border. These observations provide the first in situ visualization of the receptor structures for M. pneumoniae at the primary site of infection. The lack of sialo-oligosaccharide receptors in secretory cells and the mucus they produce provides a biochemical basis for evasion by this microorganism of the secreted mucus barrier.

  12. 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.

  13. Cytoskeletal discontinuities in the cell body cortex initiate basal body assembly and oral development in the ciliate Stentor.

    PubMed

    de Terra, N

    1985-06-01

    My previous work has shown that disconnecting the oral apparatus of Stentor into two parts induces mass assembly of basal bodies on the ventral cell surface and thus initiates oral development. This operation severs the extensive microtubule tracts joining the oral membranelles at their bases. To determine whether basal body assembly and oral development are also induced by permanently disconnecting the longitudinal microtubule fibre tracts (mt fibre tracts) of the cell body cortex, I interposed a ring of inverted (heteropolar) cortex between the anterior and posterior halves of interphase stentors. When successful, this operation made it impossible for these fibre tracts to rejoin at the heteropolar boundaries and always induced basal body assembly and oral development in the graft complex. By contrast, tripartite homopolar graft complexes rarely initiated oral development; when they did, it was apparently in response to the presence of disproportionately small oral structures, which is the normal stimulus for oral development in Stentor. The mt fibre tracts of tripartite homopolar grafts also eventually became continuous. These results support the hypothesis that permanent, extensive discontinuities anywhere within the cortical cytoskeleton can trigger basal body assembly and oral development. Since the onset of these processes is known to initiate cell division in Stentor, the results also suggest that development of discontinuities within the cortical cytoskeleton during interphase growth may be the endogenous stimulus initiating cell division in Stentor.

  14. Apigenin inhibits TGF-β1-induced proliferation and migration of airway smooth muscle cells.

    PubMed

    Li, Li-Hua; Lu, Bin; Wu, Hong-Ke; Zhang, Hao; Yao, Fei-Fei

    2015-01-01

    It is well known that the proliferation and migration of ASM cells (ASMCs) plays an important role in the pathogenesis of airway remodeling in asthma. Previous studies reported that apigenin can inhibit airway remodeling in a mouse asthma model. However, its effects on the proliferation and migration of ASMCs in asthma remain unknown. Therefore, the aim of our present study was to investigate the effects of apigenin on ASMC proliferation and migration, and explore the possible molecular mechanism. We found that apigenin inhibited transforming growth factor-β1 (TGF-β1)-induced ASMC proliferation. The cell cycle was blocked at G1/S-interphase by apigenin. It also suppressed TGF-β1-induced ASMCs migration. Furthermore, apigenin inhibited TGF-β1-induced Smad 2 and Smad 3 phosphorylation in ASMCs. Taken together, these results suggested that apigenin inhibited the proliferation and migration of TGF-β1-stimulated ASMCs by inhibiting Smad signaling pathway. These data might provide useful information for treating asthma and show that apigenin has potential for attenuating airway remodeling.

  15. 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

  16. Effects of cigarette smoke extract on human airway smooth muscle cells in COPD.

    PubMed

    Chen, Ling; Ge, Qi; Tjin, Gavin; Alkhouri, Hatem; Deng, Linghong; Brandsma, Corry-Anke; Adcock, Ian; Timens, Wim; Postma, Dirkje; Burgess, Janette K; Black, Judith L; Oliver, Brian G G

    2014-09-01

    We hypothesised that the response to cigarette smoke in airway smooth muscle (ASM) cells from smokers with chronic obstructive pulmonary disease (COPD) would be intrinsically different from smokers without COPD, producing greater pro-inflammatory mediators and factors relating to airway remodelling. ASM cells were obtained from smokers with or without COPD, and then stimulated with cigarette smoke extract (CSE) or transforming growth factor-β1. The production of chemokines and matrix metalloproteinases (MMPs) were measured by ELISA, and the deposition of collagens by extracellular matrix ELISA. The effects of CSE on cell attachment and wound healing were measured by toluidine blue attachment and cell tracker green wound healing assays. CSE increased the release of CXCL8 and CXCL1 from human ASM cells, and cells from smokers with COPD produced more CSE-induced CXCL1. The production of MMP-1, -3 and -10, and the deposition of collagen VIII alpha 1 (COL8A1) were increased by CSE, especially in the COPD group which had higher production of MMP-1 and deposition of COL8A1. CSE decreased ASM cell attachment and wound healing in the COPD group only. ASM cells from smokers with COPD were more sensitive to CSE stimulation, which may explain, in part, why some smokers develop COPD.

  17. The Ciliate Colpoda: "Instant" Protozoan

    ERIC Educational Resources Information Center

    Smith, Anne Muller; Giese, Arthur C.

    1973-01-01

    Describes the characteristics of Colpoda, a ciliated protozoan which is able to survive in a dry, encysted state for long periods of time. Outlines the procedures for culturing the organism and producing cyst preparations, and recommends its use in the high school biology laboratory. (JR)

  18. 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

  19. Mature cystic fibrosis airway neutrophils suppress T cell function: evidence for a role of arginase 1 but not programmed death-ligand 1.

    PubMed

    Ingersoll, Sarah A; Laval, Julie; Forrest, Osric A; Preininger, Marcela; Brown, Milton R; Arafat, Dalia; Gibson, Greg; Tangpricha, Vin; Tirouvanziam, Rabindra

    2015-06-01

    Bacteria colonize cystic fibrosis (CF) airways, and although T cells with appropriate Ag specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T cell function therein. Programmed death-ligand 1 (PD-L1), which exerts T cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1(hi) and PD-L1(lo) subsets, whereas healthy control (HC) airway PMNs were uniformly PD-L1(hi). Blood PMNs incubated in CF airway fluid lost PD-L1 over time; in coculture, Ab blockade of PD-L1 failed to inhibit the suppression of T cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T cell suppression in CF, likely hampering resolution of infection and inflammation.

  20. MATURE CYSTIC FIBROSIS AIRWAY NEUTROPHILS SUPPRESS T-CELL FUNCTION: EVIDENCE FOR A ROLE OF ARGINASE 1, BUT NOT PROGRAMMED DEATH-LIGAND 1

    PubMed Central

    Ingersoll, Sarah A.; Laval, Julie; Forrest, Osric A.; Preininger, Marcela; Brown, Milton R.; Arafat, Dalia; Gibson, Greg; Tangpricha, Vin; Tirouvanziam, Rabindra

    2015-01-01

    Bacteria colonize cystic fibrosis (CF) airways, and while T cells with appropriate antigen specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T-cell function therein. Programmed Death-Ligand 1 (PD-L1), which exerts T-cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1hi and PD-L1lo subsets, while healthy control (HC) airway PMNs were uniformly PD-L1hi. Blood PMNs incubated in CF airway fluid lost PD-L1 over time, and in coculture, antibody blockade of PD-L1 failed to inhibit the suppression of T-cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T-cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T-cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T-cell suppression in CF, likely hampering resolution of infection and inflammation. PMID:25926674

  1. 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.

  2. NK cells contribute to persistent airway inflammation and AHR during the later stage of RSV infection in mice.

    PubMed

    Long, Xiaoru; Xie, Jun; Zhao, Keting; Li, Wei; Tang, Wei; Chen, Sisi; Zang, Na; Ren, Luo; Deng, Yu; Xie, Xiaohong; Wang, Lijia; Fu, Zhou; Liu, Enmei

    2016-10-01

    RSV can lead to persistent airway inflammation and AHR and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. There are high numbers of NK cells in the lung, which not only play important roles in the acute stage of RSV infection, but also are pivotal in regulating the pathogenesis of asthma. Therefore, in this study, we assumed that NK cells might contribute to persistent airway disease during the later stage of RSV infection. Mice were killed at serial time points after RSV infection to collect samples. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. Cytokines were detected by ELISA, and NK cells were determined by flow cytometry. Rabbit anti-mouse asialo-GM-1 antibodies and resveratrol were used to deplete or suppress NK cells. Inflammatory cells in BALF, lung tissue damage and AHR were persistent for 60 days post-RSV infection. Type 2 cytokines and NK cells were significantly increased during the later stage of infection. When NK cells were decreased by the antibodies or resveratrol, type 2 cytokines, the persistent airway inflammation and AHR were all markedly reduced. NK cells can contribute to the RSV-associated persistent airway inflammation and AHR at least partially by promoting type 2 cytokines. Therefore, therapeutic targeting of NK cells may provide a novel approach to alleviating the recurrent wheezing subsequent to RSV infection.

  3. 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.

  4. 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

  5. Functional invariant NKT cells in pig lungs regulate the airway hyperreactivity: a potential animal model.

    PubMed

    Renukaradhya, Gourapura J; Manickam, Cordelia; Khatri, Mahesh; Rauf, Abdul; Li, Xiangming; Tsuji, Moriya; Rajashekara, Gireesh; Dwivedi, Varun

    2011-04-01

    Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4(+) cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.

  6. Do cell junction protein mutations cause an airway phenotype in mice or humans?

    PubMed

    Chang, Eugene H; Pezzulo, Alejandro A; Zabner, Joseph

    2011-08-01

    Cell junction proteins connect epithelial cells to each other and to the basement membrane. Genetic mutations of these proteins can cause alterations in some epithelia leading to varied phenotypes such as deafness, renal disease, skin disorders, and cancer. This review examines if genetic mutations in these proteins affect the function of lung airway epithelia. We review cell junction proteins with examples of disease mutation phenotypes in humans and in mouse knockout models. We also review which of these genes are expressed in airway epithelium by microarray expression profiling and immunocytochemistry. Last, we present a comprehensive literature review to find the lung phenotype when cell junction and adhesion genes are mutated or subject to targeted deletion. We found that in murine models, targeted deletion of cell junction and adhesion genes rarely result in a lung phenotype. Moreover, mutations in these genes in humans have no obvious lung phenotype. Our research suggests that simply because a cell junction or adhesion protein is expressed in an organ does not imply that it will exhibit a drastic phenotype when mutated. One explanation is that because a functioning lung is critical to survival, redundancy in the system is expected. Therefore mutations in a single gene might be compensated by a related function of a similar gene product. Further studies in human and animal models will help us understand the overlap in the function of cell junction gene products. Finally, it is possible that the human lung phenotype is subtle and has not yet been described.

  7. Intestinal ciliate composition found in the feces of racing horses from Izmir, Turkey.

    PubMed

    Gürelli, Gözde; Göçmen, Bayram

    2012-08-01

    Species composition and distribution of intestinal ciliates were investigated in the feces from 15 racing horses living near Izmir, Turkey. Thirty-seven species belonging to 21 genera were identified. Although no new species were observed, this is the first report on intestinal ciliates in racing horses living in Turkey. The mean number of ciliates was 26.4 ± 13.9 × 10(4) cells ml(-1) of feces and the mean number of ciliate species per host was 18.8 ± 7.1. No ciliates were observed in one horse. Bundleia and Polymorphella were found to be the two dominant genera, occurring in high proportions. In contrast, Didesmis and Prorodonopsis were only observed at a low frequency. Bundleia nana, Blepharoconus hemiciliatus, Paraisotrichopsis composita, Prorodonopsis coli and Spirodinium equi were newly recorded from Turkey.

  8. Abundance, variability, and potential grazing impact of planktonic ciliates in the open subaratic Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Strom, Suzanne L.; Postel, James R.; Booth, Beatrice C.

    The abundance and variability of planktonic ciliates in the open subarctic Pacific were determined during four month-long cruises in 1987 and 1988. The ciliate community, numerically dominated by relatively small aloricate choreotrichs, was comparable in abundance to communities in a range of oceanic and neritic environments, including waters with much higher average chlorophyll concentrations. Integrated (0-80m) ciliate biomass was typically 100-200mgC m -2, although 3- to 4-fold higher levels were observed on two occasions in spring. Ciliate community biomass, in general, was dominated by large (>20 μm width) individuals, although in August 1988 the biomass of smaller cells was as great or greater. The estimated grazing impact of the ciliate community averaged 20% of the primary production. On one instance in May 1988, however, a large biomass of ciliates led to an estimated grazing impact equivalent to 55% of phytoplankton production. While ciliates may be major phytoplankton grazers during sporadic ciliate “blooms”, dino- and other heterotrophic flagellates, which make up the bulk of microheterotroph biomass, must normally be of equal or greater importance as herbivores in this ocean region.

  9. 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.

  10. Using Optical Tweezers to Study Cell Mechanics during Airway Reopening

    NASA Astrophysics Data System (ADS)

    Yalcin, Huseyin; Wang, Jing; Ghadiali, Samir; Ou-Yang, H. Daniel

    2006-03-01

    Patients suffering from the acute respiratory distress syndrome (ARDS) must be mechanically ventilated in order to survive. However, these ventilation protocols may generate injurious hydrodynamic stresses especially during low tidal volume (VT) ventilation when the flow of micron-sized air bubbles displace the surrounding liquid. In-vitro studies in our lab revealed that microbubble flows can severally damage lung epithelial cells (EC). The degree of injury was elevated for sub-confluent monolayers in small channel heights. Under these conditions, the micromechanics of individual EC may influence the degree of cellular injury. To investigate the role of cell mechanics, we used an oscillating Optical Tweezers (OT) technique to measure the intrinsic mechanical properties of EC before and after the flow of microbubbles. Knowledge of how the EC's micromechanical properties influence cell viability may lead to the development of novel treatment therapies that enhance the EC's ability to withstand injurious hydrodynamic stresses during ventilation treatment.

  11. Chronic exposure of rats to cotton-mill-room noise changes the cell composition of the tracheal epithelium.

    PubMed

    Oliveira, Maria João R; Pereira, António S; Guimarães, Laura; Freitas, Diamantino; Carvalho, António P O; Grande, Nuno R; Aguas, Artur P

    2002-12-01

    The work environment of cotton mill rooms of modern textile plants is characterized by noise pollution. We have taped and reproduced this noisy environment to study its effects on experimentally exposed rats. Because we have previously documented that chronic noise causes alterations in the respiratory epithelium, we have focused our investigation on the morphology of the tracheal lining. Wistar rats were exposed to the textile-type noise from 1 up to 7 months, with an average 40 hours weekly exposure of the animals. The rats were sacrificed monthly and the tracheas were studied by scanning electron microscopy (SEM) to quantify the areas of the airway lining that were covered by ciliated, serous or other cells of the epithelium. We found that noise exposure of the rats caused a significant loss of tracheal ciliated cells; an increased density of serous cells on the epithelium balanced this change. This modification of the rat trachea was already established after 1 month of noise treatment of the animals; it did not change significantly throughout the 7-month course of the herein investigation. Loss of ciliated cells was more intense in areas of the tracheal epithelium located between the regions of cartilage rings. We conclude that the ciliated cell is an elective target for damage caused on the respiratory epithelium by the workplace noise occurring in cotton mill rooms. This modification of the respiratory epithelium is likely to impair clearance of the airways since this function depends on the activity of ciliated cells.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Chlamydia pneumoniae infection induced allergic airway sensitization is controlled by regulatory T-cells and plasmacytoid dendritic cells.

    PubMed

    Crother, Timothy R; Schröder, Nicolas W J; Karlin, Justin; Chen, Shuang; Shimada, Kenichi; Slepenkin, Anatoly; Alsabeh, Randa; Peterson, Ellena; Arditi, Moshe

    2011-01-01

    Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.

  18. Multipotent versus differentiated cell fate selection in the developing Drosophila airways

    PubMed Central

    Matsuda, Ryo; Hosono, Chie; Samakovlis, Christos; Saigo, Kaoru

    2015-01-01

    Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway. DOI: http://dx.doi.org/10.7554/eLife.09646.001 PMID:26633813

  19. A Cryptic Marine Ciliate Feeds on Progametes of Noctiluca scintillans.

    PubMed

    Zhang, Shuwen; Chan, Kit Yu Karen; Shen, Zhuo; Cheung, Shunyan; Landry, Michael R; Liu, Hongbin

    2017-02-01

    The dinoflagellate Noctiluca scintillans (Noctiluca) has the ability to reproduce sexually, which may help to increase or restore its population size during periods of blooms or environmental stress. Here, we documented for the first time a marine ciliate Strombidium sp. that feeds on Noctiluca's progametes undergoing stages 5 to 9 of nuclear division. This ciliate frequently swam on or around gametogenic and some vegetative Noctiluca cells. The ciliates associated with gametogenic cells had significantly lower swimming speed and changed direction more frequently than those associated with vegetative cells, which overall increased their time spent around the food patches (progametes). This trophic interaction constitutes an upside-down predator-prey link, in which ciliates within the typical size range of Noctiluca prey, become the predators. Based on the phylogenetic tree (maximum-likelihood), there are 14 environmental clones similar to Strombidium sp. found in other coastal waters, where Noctiluca presence or blooms have been reported. This novel predator-prey relationship could therefore be common in other Noctiluca habitats. Additional studies are needed to assess the magnitude of its impacts on Noctiluca population dynamics and plankton bloom succession.

  20. Complement-Mediated Death of Ciliate Tetrahymena pyriformis Caused by Human Blood Serum.

    PubMed

    Ivanov, P A; Faktor, M I; Karpova, N S; Cheremnykh, E G; Brusov, O S

    2016-04-01

    Toxicity of human blood serum for ciliate Tetrahymena pyriformis is determined by the complement system. When ciliate are dying after being exposed to blood serum, cell membrane permeability for low-molecular-weight compounds significantly increases, probably due to pore formation. Serine protease inhibitors or exposure to physical factors inducing complement inactivation (e.g., heating up to 56°C) completely prevented ciliate death under the effect of human serum. Activation of serum complement upon interaction with Tetrahymena cells occurred by the classical or lectin pathway, while the contribution of the alternative activation pathway was negligible.

  1. Characterization and expression of the gene encoding En-MAPK1, an intestinal cell kinase (ICK)-like kinase activated by the autocrine pheromone-signaling loop in the Polar Ciliate, Euplotes nobilii.

    PubMed

    Candelori, Annalisa; Luporini, Pierangelo; Alimenti, Claudio; Vallesi, Adriana

    2013-04-03

    In the protozoan ciliate Euplotes, a transduction pathway resulting in a mitogenic cell growth response is activated by autocrine receptor binding of cell type-specific, water-borne signaling protein pheromones. In Euplotes raikovi, a marine species of temperate waters, this transduction pathway was previously shown to involve the phosphorylation of a nuclear protein kinase structurally similar to the intestinal-cell and male germ cell-associated kinases described in mammals. In E. nobilii, which is phylogenetically closely related to E. raikovi but inhabits Antarctic and Arctic waters, we have now characterized a gene encoding a structurally homologous kinase. The expression of this gene requires +1 translational frameshifting and a process of intron splicing for the production of the active protein, designated En-MAPK1, which contains amino acid substitutions of potential significance for cold-adaptation.

  2. A comparative in-silico analysis of autophagy proteins in ciliates

    PubMed Central

    Küçükoğlu, Nurçin; Arslanyolu, Muhittin

    2017-01-01

    Autophagy serves as a turnover mechanism for the recycling of redundant and/or damaged macromolecules present in eukaryotic cells to re-use them under starvation conditions via a double-membrane structure known as autophagosome. A set of eukaryotic genes called autophagy-related genes (ATGs) orchestrate this highly elaborative process. The existence of these genes and the role they play in different eukaryotes are well-characterized. However, little is known of their role in some eukaryotes such as ciliates. Here, we report the computational analyses of ATG genes in five ciliate genomes to understand their diversity. Our results show that Oxytricha trifallax is the sole ciliate which has a conserved Atg12 conjugation system (Atg5-Atg12-Atg16). Interestingly, Oxytricha Atg16 protein includes WD repeats in addition to its N-terminal Atg16 domain as is the case in multicellular organisms. Additionally, phylogenetic analyses revealed that E2-like conjugating protein Atg10 is only present in Tetrahymena thermophila. We fail to find critical autophagy components Atg5, Atg7 and Atg8 in the parasitic ciliate Ichthyophthirius multifiliis. Contrary to previous reports, we also find that ciliate genomes do not encode typical Atg1 since all the candidate sequences lack an Atg1-specific C-terminal domain which is essential for Atg1 complex formation. Consistent with the absence of Atg1, ciliates also lack other members of the Atg1 complex. However, the presence of Atg6 in all ciliates examined here may rise the possibility that autophagosome formation could be operated through Atg6 in ciliates, since Atg6 has been shown as an alternative autophagy inducer. In conclusion, our results highlight that Atg proteins are partially conserved in ciliates. This may provide a better understanding for the autophagic destruction of the parental macronucleus, a developmental process also known as programmed nuclear death in ciliates. PMID:28123910

  3. Prostaglandin E2 induces expression of MAPK phosphatase 1 (MKP-1) in airway smooth muscle cells.

    PubMed

    Rumzhum, Nowshin N; Ammit, Alaina J

    2016-07-05

    Prostaglandin E2 (PGE2) is a prostanoid with diverse actions in health and disease. In chronic respiratory diseases driven by inflammation, PGE2 has both positive and negative effects. An enhanced understanding of the receptor-mediated cellular signalling pathways induced by PGE2 may help us separate the beneficial properties from unwanted actions of this important prostaglandin. PGE2 is known to exert anti-inflammatory and bronchoprotective actions in human airways. To date however, whether PGE2 increases production of the anti-inflammatory protein MAPK phosphatase 1 (MKP-1) was unknown. We address this herein and use primary cultures of human airway smooth muscle (ASM) cells to show that PGE2 increases MKP-1 mRNA and protein upregulation in a concentration-dependent manner. We explore the signalling pathways responsible and show that PGE2-induces CREB phosphorylation, not p38 MAPK activation, in ASM cells. Moreover, we utilize selective antagonists of EP2 (PF-04418948) and EP4 receptors (GW 627368X) to begin to identify EP-mediated functional outcomes in ASM cells in vitro. Taken together with earlier studies, our data suggest that PGE2 increases production of the anti-inflammatory protein MKP-1 via cAMP/CREB-mediated cellular signalling in ASM cells and demonstrates that EP2 may, in part, be involved.

  4. 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

  5. Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells.

    PubMed

    Berntsen, P; Park, C Y; Rothen-Rutishauser, B; Tsuda, A; Sager, T M; Molina, R M; Donaghey, T C; Alencar, A M; Kasahara, D I; Ericsson, T; Millet, E J; Swenson, J; Tschumperlin, D J; Butler, J P; Brain, J D; Fredberg, J J; Gehr, P; Zhou, E H

    2010-06-06

    The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

  6. B cells play key roles in th2-type airway immune responses in mice exposed to natural airborne allergens.

    PubMed

    Drake, Li Yin; Iijima, Koji; Hara, Kenichiro; Kobayashi, Takao; Kephart, Gail M; Kita, Hirohito

    2015-01-01

    Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH-/- mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH-/- mice. The allergen-induced expansion of CD4+ T cells was impaired in the lungs and draining lymph nodes of JH-/- mice. Furthermore, lymphocytes from JH-/- mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

    PubMed

    Böttcher-Friebertshäuser, Eva; Klenk, Hans-Dieter; Garten, Wolfgang

    2013-11-01

    Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment.

  12. 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

  13. 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

  14. Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation

    SciTech Connect

    Svensson Holm, Ann-Charlotte B.; Bengtsson, Torbjoern; Grenegard, Magnus; Lindstroem, Eva G.

    2012-03-10

    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Spatial and temporal traction response in human airway smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Tolic-Norrelykke, Iva Marija; Butler, James P.; Chen, Jianxin; Wang, Ning

    2002-01-01

    Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

  20. The effect of humic acid on uptake/adsorption of copper by a marine bacterium and two marine ciliates.

    PubMed

    Lores, E M; Snyder, R A; Pennock, J R

    1999-01-01

    The effect of humic acid (HA) on Cu uptake by a bacterium and two bacterivorus ciliates was investigated. The presence of HA resulted in a statistically significant (p<0.001) decrease in Cu associated with bacteria that were exposed to 67 microg Cu L(-1). Complexation of Cu appears to lower the availability of Cu with respect to bacterial cell surface binding and uptake. For ciliates, 10 mg HA L(-1) significantly reduced uptake of Cu by Uronema, but did not reduce uptake of Cu by Pleuronema. Uronema exposed to 67 microg Cu L(-1) accumulated 54% less Cu when 10 mg HA L(-1) was present (0.50 pg ciliate(-1) vs 0.23 pg ciliate(-1)). Uronema feeding on V. natriegens, took up less than half as much Cu as unfed Uronema when exposed to Cu without HA (0.41 pg Cu fed ciliate(-1) vs 0.86 pg Cu unfed ciliate(-1), but only 40% less when exposed to Cu and HA (0.31 pg Cu fed ciliate(-1) vs 0.51 pg Cu unfed ciliate(-1)). The lower % reduction attributable to fed ciliates in the presence of HA suggests that some of the Cu associated with HA is available through trophic processes.

  1. Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

    PubMed Central

    Kalbe, Benjamin; Knobloch, Jürgen; Schulz, Viola M.; Wecker, Christine; Schlimm, Marian; Scholz, Paul; Jansen, Fabian; Stoelben, Erich; Philippou, Stathis; Hecker, Erich; Lübbert, Hermann; Koch, Andrea; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca2+ increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases. PMID:27540365

  2. 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

  3. Human influenza is more effective than avian influenza at antiviral suppression in airway cells.

    PubMed

    Hsu, Alan Chen-Yu; Barr, Ian; Hansbro, Philip M; Wark, Peter A

    2011-06-01

    Airway epithelial cells are the initial site of infection with influenza viruses. The innate immune responses of airway epithelial cells to infection are important in limiting virus replication and spread. However, relatively little is known about the importance of this innate antiviral response to infection. Avian influenza viruses are a potential source of future pandemics; therefore, it is critical to examine the effectiveness of the host antiviral system to different influenza viruses. We used a human influenza (H3N2) and a low-pathogenic avian influenza (H11N9) to assess and compare the antiviral responses of Calu-3 cells. After infection, H3N2 replicated more effectively than the H11N9 in Calu-3 cells. This was not due to differential expression of sialic acid residues on Calu-3 cells, but was attributed to the interference of host antiviral responses by H3N2. H3N2 induced a delayed antiviral signaling and impaired type I and type III IFN induction compared with the H11N9. The gene encoding for nonstructural (NS) 1 protein was transfected into the bronchial epithelial cells (BECs), and the H3N2 NS1 induced a greater inhibition of antiviral responses compared with the H11N9 NS1. Although the low-pathogenic avian influenza virus was capable of infecting BECs, the human influenza virus replicated more effectively than avian influenza virus in BECs, and this was due to a differential ability of the two NS1 proteins to inhibit antiviral responses. This suggests that the subversion of human antiviral responses may be an important requirement for influenza viruses to adapt to the human host and cause disease.

  4. 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...

  5. 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

  6. 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

  7. 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.

  8. Toll-like Receptors, Triggering Receptor Expressed on Myeloid Cells Family Members and Receptor for Advanced Glycation End-products in Allergic Airway Inflammation

    PubMed Central

    Hall, Sannette C.; Agrawal, Devendra K.

    2016-01-01

    Asthma is a chronic disorder of the airways characterized by cellular infiltration, airway hyper-responsive and airway inflammation. Innate immune cells are the first line of defense against endogenous and exogenous signals in the airways and as such possess a diverse array of pattern recognition receptors. Toll-like receptors are crucial sentinels which when activated, can either promote or ameliorate the inflammatory response in predisposed individuals. The recently discovered triggering receptor expressed on myeloid cells family members are emerging mediators of inflammation. These receptors are believed to modulate inflammatory responses by collaborating with classic PRRs. Endogenous signals like HMGB-1, signaling through the receptor for advanced glycation end products, also promotes inflammation, however, its contribution to inflammation in the airways is not well known. Here, we discuss the role of each receptor in airway inflammation and highlight potential synergistic mechanisms, which contribute to disease pathogenesis in allergic asthma. PMID:26678062

  9. p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration.

    PubMed

    Zuo, Wei; Zhang, Ting; Wu, Daniel Zheng'An; Guan, Shou Ping; Liew, Audrey-Ann; Yamamoto, Yusuke; Wang, Xia; Lim, Siew Joo; Vincent, Matthew; Lessard, Mark; Crum, Christopher P; Xian, Wa; McKeon, Frank

    2015-01-29

    Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells expressing Trp63 (p63) and keratin 5, called DASC(p63/Krt5), to this process. Here we show that pre-existing, intrinsically committed DASC(p63/Krt5) undergo a proliferative expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASC(p63/Krt5) in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASC(p63/Krt5)-derived pedigrees differentiate to type I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic lung diseases.

  10. 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.

  11. 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

  12. Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells.

    PubMed

    Takahara, Norihiro; Ito, Satoru; Furuya, Kishio; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-12-01

    Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca(2+)-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

  13. 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

  14. Adoptive transfer of dendritic cells isolated from helminth-infected mice enhanced T regulatory cell responses in airway allergic inflammation.

    PubMed

    Liu, J-Y; Li, L-Y; Yang, X-Z; Li, J; Zhong, G; Wang, J; Li, L-J; Ji, B; Wu, Z-Q; Liu, H; Yang, X; Liu, P-M

    2011-10-01

    Our and others' previous studies have shown that Schistosoma japonicum (SJ) infection can inhibit allergic reactions. Moreover, we found that adoptive transfer of dendritic cells (DCs) from inhibited mice showed a similar inhibitory effect on allergy, suggesting a critical role of DCs in SJ-infected mediated inhibition of allergy. In this study, we further examined the mechanism by which DCs contribute to inhibition of allergy. Our results showed that DCs from SJ-infected mice (SJDCs) produced significantly higher levels of IL-10 compared to those from naive control mice (NDCs). Adoptive transfer of SJDCs, unlike NDCs, significantly increased CD4+CD25+Foxp3+ T cells and CD4+CD25+IL-10+ T cells regulatory T-cell responses in vivo. This was correlated with significantly reduced production of IL-4 and IL-5 by CD4+ T cells, eotaxin in lung tissues and reduced airway allergic inflammation in the SJDC recipients following allergen sensitization and challenge. These data suggest that helminth infection may induce tolerogenic DCs that can inhibit the development of airway allergic inflammation through enhancing T regulatory cell responses.

  15. Mucociliary clearance defects in a murine in vitro model of pneumococcal airway infection.

    PubMed

    Fliegauf, Manfred; Sonnen, Andreas F-P; Kremer, Bernhard; Henneke, Philipp

    2013-01-01

    Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where--in pneumococcal infection--persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.

  16. Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells

    PubMed Central

    Crother, Timothy R.; Schröder, Nicolas W. J.; Karlin, Justin; Chen, Shuang; Shimada, Kenichi; Slepenkin, Anatoly; Alsabeh, Randa; Peterson, Ellena; Arditi, Moshe

    2011-01-01

    Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs. PMID:21695198

  17. Type 2 innate lymphoid cells: friends or foes-role in airway allergic inflammation and asthma.

    PubMed

    Pishdadian, Abbas; Varasteh, Abdol-Reza; Sankian, Mojtaba

    2012-01-01

    Innate-like lymphocytes (ILLs) and innate lymphoid cells (ILCs) are two newly characterized families of lymphocytes with limited and no rearranged antigen receptors, respectively. These soldiers provide a first line of defense against foreign insults by triggering a prompt innate immune response and bridging the gap of innate and adaptive immunity. Type 2 innate lymphoid cells (ILCs2) are newly identified members of the ILC family that play a key role in type 2 immune responses by prompt production of type 2 cytokines (especially IL-5 and IL-13) in response to antigen-induced IL-25/33 and by recruiting type 2 "immune franchise." Regarding the two different roles of type 2 cytokines, helminth expulsion and type 2-related diseases, here we review the latest advances in ILC2 biology and examine the pivotal role of resident ILCs2 in allergen-specific airway inflammation and asthma.

  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. Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling.

    PubMed

    Paul, Manash K; Bisht, Bharti; Darmawan, Daphne O; Chiou, Richard; Ha, Vi L; Wallace, William D; Chon, Andrew T; Hegab, Ahmed E; Grogan, Tristan; Elashoff, David A; Alva-Ornelas, Jackelyn A; Gomperts, Brigitte N

    2014-08-07

    Airways are exposed to myriad environmental and damaging agents such as reactive oxygen species (ROS), which also have physiological roles as signaling molecules that regulate stem cell function. However, the functional significance of both steady and dynamically changing ROS levels in different stem cell populations, as well as downstream mechanisms that integrate ROS sensing into decisions regarding stem cell homeostasis, are unclear. Here, we show in mouse and human airway basal stem cells (ABSCs) that intracellular flux from low to moderate ROS levels is required for stem cell self-renewal and proliferation. Changing ROS levels activate Nrf2, which activates the Notch pathway to stimulate ABSC self-renewal and an antioxidant program that scavenges intracellular ROS, returning overall ROS levels to a low state to maintain homeostatic balance. This redox-mediated regulation of lung stem cell function has significant implications for stem cell biology, repair of lung injuries, and diseases such as cancer.

  20. Effect of murine recombinant interleukin-5 on the cell population in guinea-pig airways.

    PubMed Central

    Iwama, T.; Nagai, H.; Suda, H.; Tsuruoka, N.; Koda, A.

    1992-01-01

    1. An intratracheal injection of murine recombinant interleukin 5 (mrIL-5, 2-15 microgram/0.25 ml/animal) induced a dose-dependent increase in the number of macrophages, eosinophils, neutrophils and epithelial cells in the bronchoalveolar lavage fluid (BALF) of guinea-pigs 24 h after administration. Bovine serum albumin (15 micrograms/0.25 ml/animal), used as a reference material, did not cause any change of this type. 2. The intratracheal administration of mrIL-5 at a dose of 15 microgram showed a tendency to increase the number of these pulmonary inflammatory cells and epithelial cells in the BALF at 12 h with a significant increase observed at 24 h. 3. Prednisolone (20 mg kg-1, i.p.) inhibited the mrIL-5-induced increase in macrophages, eosinophils, neutrophils and epithelial cells. Ketotifen (2 mg kg-1, i.p.) reduced the mrIL-5-induced increase in the eosinophil, neutrophil and epithelial cell populations. The simultaneous injection of 2% disodium cromoglycate (DSCG) into the trachea prevented the mrIL-5-induced increase in the number of airway epithelial cells, without affecting changes in the other inflammatory leukocytes. 4. These results suggest that mrIL-5 is a potent inducer of lung inflammation, in terms of increased inflammatory leukocytes and epithelial cells in guinea-pig BALF. Prednisolone, DSCG and ketotifen are effective against mrIL-5-induced pulmonary inflammation, especially the desquamation of bronchial epithelial cells. PMID:1596681

  1. 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

  2. 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

  3. 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

  4. The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells.

    PubMed

    Yang, Chao-Huei; Tsao, Chiung-Fang; Ko, Wang-Sheng; Chiou, Ya-Ling

    2016-01-09

    In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%-99% after 48 h (p < 0.05) and induced G₁/G₀ cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials.

  5. The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells

    PubMed Central

    Yang, Chao-Huei; Tsao, Chiung-Fang; Ko, Wang-Sheng; Chiou, Ya-Ling

    2016-01-01

    In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05) and induced G1/G0 cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials. PMID:26761017

  6. 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

  7. 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.

  8. Hydrogen sulfide inhalation ameliorates allergen induced airway hypereactivity by modulating mast cell activation.

    PubMed

    Roviezzo, Fiorentina; Bertolino, Antonio; Sorrentino, Rosalinda; Terlizzi, Michela; Matteis, Maria; Calderone, Vincenzo; Mattera, Valentina; Martelli, Alma; Spaziano, Giuseppe; Pinto, Aldo; D'Agostino, Bruno; Cirino, Giuseppe

    2015-10-01

    Compelling evidence suggests that hydrogen sulfide represents an important gaseous transmitter in the mammalian respiratory system. In the present study, we have evaluated the role of mast cells in hydrogen sulfide-induced effects on airways in a mouse model of asthma. Mice were sensitized to ovalbumin and received aerosol of a hydrogen sulfide donor (NaHS; 100 ppm) starting at day 7 after ovalbumin challenge. Exposure to hydrogen sulfide abrogated ovalbumin-induced bronchial hypereactivity as well as the increase in lung resistance. Concomitantly, hydrogen sulfide prevented mast cell activity as well as FGF-2 and IL-13 upregulation. Conversely, pulmonary inflammation and the increase in plasmatic IgE levels were not affected by hydrogen sulfide. A lack of hydrogen sulfide effects in mast cell deficient mice occurred. Primary fibroblasts harvested from ovalbumin-sensitized mice showed an increased proliferation rate that was inhibited by hydrogen sulfide aerosol. Furthermore, ovalbumin-induced transdifferentiation of pulmonary fibroblasts into myofibroblasts was reversed. Finally, hydrogen sulfide did abrogate in vitro the degranulation of the mast cell-like RBL-2H3 cell line. Similarly to the in vivo experiments the inhibitory effect was present only when the cells were activated by antigen exposure. In conclusion, inhaled hydrogen sulfide improves lung function and inhibits bronchial hyper-reactivity by modulating mast cells and in turn fibroblast activation.

  9. 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.

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

    SciTech Connect

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

    2008-04-25

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

  11. Airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain.

    PubMed

    Fairbank, Nigel J; Connolly, Sarah C; Mackinnon, James D; Wehry, Kathrin; Deng, Linhong; Maksym, Geoffrey N

    2008-09-01

    Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and "relaxability" (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness.

  12. FOXJ1 prevents cilia growth inhibition by cigarette smoke in human airway epithelium in vitro.

    PubMed

    Brekman, Angelika; Walters, Matthew S; Tilley, Ann E; Crystal, Ronald G

    2014-11-01

    Airway epithelium ciliated cells play a central role in clearing the lung of inhaled pathogens and xenobiotics, and cilia length and coordinated beating are important for airway clearance. Based on in vivo studies showing that the airway epithelium of healthy smokers has shorter cilia than that of healthy nonsmokers, we investigated the mechanisms involved in cigarette smoke-mediated inhibition of ciliogenesis by assessing normal human airway basal cell differentiation in air-liquid interface (ALI) cultures in the presence of nontoxic concentrations of cigarette smoke extract (CSE). Measurements of cilia length from Day 28 ALI cultures demonstrated that CSE exposure was associated with shorter cilia (P < 0.05), reproducing the effect of cigarette smoking on cilia length observed in vivo. This phenotype correlated with a broad CSE-mediated suppression of genes involved in cilia-related transcriptional regulation, intraflagellar transport, cilia motility, structural integrity, and basal body development but not of control genes or epithelial barrier integrity. The CSE-mediated inhibition of cilia growth could be prevented by lentivirus-mediated overexpression of FOXJ1, the major cilia-related transcription factor, which led to partial reversal of expression of cilia-related genes suppressed by CSE. Together, the data suggest that components of cigarette smoke are responsible for a broad suppression of genes involved in cilia growth, but, by stimulating ciliogenesis with the transcription factor FOXJ1, it may be possible to maintain close to normal cilia length despite the stress of cigarette smoking.

  13. EFFECT OF INHALED ENDOTOXIN ON AIRWAY AND CIRCULATING INFLAMMATORY CELL PHAGOCYTOSIS AND CD11B EXPRESSION IN ATOPIC ASTHMATIC SUBJECTS

    EPA Science Inventory

    Effect of inhaled endotoxin on airway and circulating inflammatory cell phagocytosis and CD11b expression in atopic asthmatic subjects

    Neil E. Alexis, PhD, Marlowe W. Eldridge, MD, David B. Peden, MD, MS

    Chapel Hill and Research Triangle Park, NC

    Backgrou...

  14. 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...

  15. 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...

  16. 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...

  17. Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

    PubMed

    Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

    2005-09-01

    Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

  18. 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

  19. Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells.

    PubMed

    Foxman, Ellen F; Storer, James A; Fitzgerald, Megan E; Wasik, Bethany R; Hou, Lin; Zhao, Hongyu; Turner, Paul E; Pyle, Anna Marie; Iwasaki, Akiko

    2015-01-20

    Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33-35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I-like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

  20. Ultrastructure of extrusomes in hypotrichous ciliate Pseudourostyla nova

    NASA Astrophysics Data System (ADS)

    Zhou, Yao; Wang, Zhengjun; Zhang, Jun; Gu, Fukang

    2011-01-01

    The ultrastructure of extrusomes of the hypotrichous ciliate Pseudourostyla nova was observed in scanning and transmission electron microscopy and enzyme-cytochemistry. The results show that the distribution, morphological characteristics, morphogenesis process, and extrusive process of the extrusomes in P. nova are different from the trichocysts in Paramecium, suggesting that the extrusomes of P. nova can respond to environmental stimuli, play an important role in the defense of this species, and cannot be regarded as "trichocysts". The results also suggest that the extrusomes might be originated from the Golgi apparatus and mature in the cytoplasm; after the extrusion of mature extrusomes, the residual substance might be reabsorbed and reused by the ciliate cell via food vacuoles, and take part in material recycling of the cell.

  1. Pulmonary neuroendocrine cells function as airway sensors to control lung immune response

    PubMed Central

    Branchfield, Kelsey; Nantie, Leah; Verheyden, Jamie M.; Sui, Pengfei; Wienhold, Mark D.; Sun, Xin

    2016-01-01

    The lung is constantly exposed to environmental atmospheric cues. How it senses and responds to these cues is poorly defined. Here, we show that Roundabout receptor (Robo) genes are expressed in pulmonary neuroendocrine cells (PNECs), a rare, innervated epithelial population. Robo inactivation in mouse lung results in an inability of PNECs to cluster into sensory organoids and triggers increased neuropeptide production upon exposure to air. Excess neuropeptides lead to an increase in immune infiltrates, which in turn remodel the matrix and irreversibly simplify the alveoli. We demonstrate in vivo that PNECs act as precise airway sensors that elicit immune responses via neuropeptides. These findings suggest that the PNEC and neuropeptide abnormalities documented in a wide array of pulmonary diseases may profoundly affect symptoms and progression. PMID:26743624

  2. A role for CCL28-CCR3 in T-cell homing to the human upper airway mucosa.

    PubMed

    Danilova, E; Skrindo, I; Gran, E; Hales, B J; Smith, W A; Jahnsen, J; Johansen, F E; Jahnsen, F L; Baekkevold, E S

    2015-01-01

    Lymphocyte recruitment to peripheral tissues is fundamental for immune surveillance and homeostasis, but the chemokines and chemokine receptors responsible for tissue-specific homing of T cells to the upper airway mucosa have not been determined. To address this, we analyzed the chemokines expressed in the normal human nasal mucosa and found that CCL28 is preferentially expressed at a high level on the lumenal face of vascular endothelial cells in the mucosa. Analysis of the cognate chemokine receptors revealed that close to 50% of the CD4(+) T cells in the human nasal mucosa expressed the CCL28 receptor CCR3, whereas CCR3 was hardly detectable on T cells in the small intestine and skin. In the circulation, CCR3(+) T cells comprised a small subset that did not express homing receptors to the intestine or skin. Moreover, depletion of CCR3(+)CD4(+) T cells abrogated the proliferative response of human blood CD4(+) T cells against the opportunistic nasopharyngeal pathogen Haemophilus influenzae, indicating that the CCR3(+)CD4(+) T-cell subset in the circulation contains antigen specificities relevant for the upper airways. Together, these findings indicate that CCL28-CCR3 interactions are involved in the homeostatic trafficking of CD4(+) T cells to the upper airways.

  3. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

    PubMed

    Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

    2015-07-15

    Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. Transcriptional Classification and Functional Characterization of Human Airway Macrophage and Dendritic Cell Subsets.

    PubMed

    Patel, Vineet I; Booth, J Leland; Duggan, Elizabeth S; Cate, Steven; White, Vicky L; Hutchings, David; Kovats, Susan; Burian, Dennis M; Dozmorov, Mikhail; Metcalf, Jordan P

    2017-02-01

    The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Owing to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole-lung bronchoalveolar lavage. Six subsets of phagocytic APC (HLA-DR(+)) were consistently observed. Aside from alveolar macrophages, subsets of Langerin(+), BDCA1(-)CD14(+), BDCA1(+)CD14(+), BDCA1(+)CD14(-), and BDCA1(-)CD14(-) cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared with E. coli Alveolar macrophages and CD14(+) cells were overall more efficient at particle internalization compared with the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole-genome transcriptional profiling revealed a clade of "true dendritic cells" consisting of Langerin(+), BDCA1(+)CD14(+), and BDCA1(+)CD14(-) cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell-associated genes, including CD1, FLT3, CX3CR1, and CCR6 Each clade, and each member of both clades, was discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.

  11. Comparison of ciliature microtubule organelles in three hypotrichous ciliate species

    NASA Astrophysics Data System (ADS)

    Li, Yisong; Shi, Lei; Gu, Fukang

    2010-05-01

    We examined the structure and spatial organization of ciliature base-associated microtubules (BAM) in three hypotrichous ciliates ( Stylonychia mytilus, Pseudourostyla cristata, Euplotes woodruffi) in fluorescence microscopy. The results revealed that BAM, including the anterior (ALM), posterior longitudinal microtubule (PLM) and the transverse microtubule (TM) bands, are composed of tubulin. The respective microtubular bands have cytoplasmic polarization patterns that are significantly asymmetric. The BAM of the midventral files in P. cristata appear cord-shaped compared with the ALM bands of transverse cirri in both S. mytilus and E. woodruffi, which extend to the left anterior side of the cell before converging. The TM bands of the left marginal cirri (MC) in S. mytilus extend to the right side of the cell, while those of the right MC bands extend to the left. Our observations suggest that BAM traits are common in hypotrichous ciliates even though different species possess different microtubule arrangements related to the conserved cirral morphogenetic patterns in the respective species. The differing development of BAM in the three ciliate suggests that the microtubules may be conserved in different hypotrichs. We have also demonstrated that the BAM, which appear polar and asymmetric, are localized in specific cytoskeletal positions and extend in different orientations within the cortex to connect with other ciliature-associated structures and, thus, strengthen the cortex. These BAM features indicate that they are directly associated with cell motion.

  12. Allergic airway disease in mice alters T and B cell responses during an acute respiratory poxvirus infection.

    PubMed

    Walline, Crystal C; Sehra, Sarita; Fisher, Amanda J; Guindon, Lynette M; Kratzke, Ian M; Montgomery, Jessica B; Lipking, Kelsey P; Glosson, Nicole L; Benson, Heather L; Sandusky, George E; Wilkes, David S; Brutkiewicz, Randy R; Kaplan, Mark H; Blum, Janice S

    2013-01-01

    Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8(+) T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8(+) T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4(+) T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. Myeloid cell HIF-1α regulates asthma airway resistance and eosinophil function

    PubMed Central

    Akong-Moore, Kathryn; Feldstein, Stephanie; Johansson, Per; Nyugen, Anh; McEachern, Elisa K.; Nicatia, Shari; Cowburn, Andrew S.; Olson, Joshua; Cho, Jae Youn; Isaacs, Hart; Johnson, Randall S.; Broide, David H.; Nizet, Victor

    2013-01-01

    Hypoxia-inducible factor (HIF)-1α is a master regulator of inflammatory activities of myeloid cells, including neutrophils and macrophages. These studies examine the role of myeloid cell HIF-1α in regulating asthma induction and pathogenesis, and for the first time, evaluate the roles of HIF-1α and HIF-2α in the chemotactic properties of eosinophils, the myeloid cells most associated with asthma. Wild-type (WT) and myeloid cell-specific HIF-1α knockout (KO) C57BL/6 mice were studied in an ovalbumin (OVA) model of asthma. Administration of the pharmacological HIF-1α antagonist YC-1 was used to corroborate findings from the genetic model. WT, HIF-1α, and HIF-2α KO eosinophils underwent in vitro chemotaxis assays. We found that deletion of HIF-1α in myeloid cells and systemic treatment with YC-1 during asthma induction decreased airway hyperresponsiveness (AHR). Deletion of HIF-1α in myeloid cells in OVA-induced asthma also reduced eosinophil infiltration, goblet cell hyperplasia, and levels 34 of cytokines IL-4, IL-5, and IL-13 in the lung. HIF-1α inhibition with YC-1 during asthma induction decreased eosinophilia in bronchoalveolar lavage, lung parenchyma, and blood, as well as decreased total lung inflammation, IL-5, and serum OVA-specific IgE levels. Deletion of HIF-1α in eosinophils decreased their chemotaxis, while deletion of the isoform HIF-2α led to increased chemotaxis. This work demonstrates that HIF-1α in myeloid cells plays a role in asthma pathogenesis, particularly in AHR development. Additionally, treatment with HIF-1α inhibitors during asthma induction decreases AHR and eosinophilia. Finally, we show that HIF- 1α and HIF-2α regulate eosinophil migration in opposing ways. PMID:23250618

  20. 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.

  1. Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus.

    PubMed

    Zhang, Liqun; Collins, Peter L; Lamb, Robert A; Pickles, Raymond J

    2011-12-05

    Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.

  2. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression.

    PubMed

    Matusovsky, Oleg S; Nakada, Emily M; Kachmar, Linda; Fixman, Elizabeth D; Lauzon, Anne-Marie

    2014-07-15

    Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T-helper 2 (Th2) inflammatory response. CD4(+) T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4(+) T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen-stimulated CD4(+) T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co-incubation with antigen-stimulated CD4(+) T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4(+) T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co-incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper-responsiveness and asthma.

  8. Ciliated protozoa of two antarctic lakes: analysis by quantitative protargol staining and examination of artificial substrates

    NASA Technical Reports Server (NTRS)

    Kepner, R. L. Jr; Wharton, R. A. Jr; Coats, D. W.; Wharton RA, J. r. (Principal Investigator)

    1999-01-01

    Planktonic and artificial substrate-associated ciliates have been identified in two perennially ice-covered antarctic lakes of the McMurdo Dry Valleys. Abundances estimated by quantitative protargol staining ranged from < 5 to 31690 cells l-1, levels that are comparable to those previously obtained using other methods. Nineteen ciliate taxa were identified from these lakes, with the most frequently encountered genera being Plagiocampa, Askenasia, Monodinium, Sphaerophrya and Vorticella. The taxonomic findings compare favorably with those of previous investigators; however four previously unreported genera were observed in both Lakes Fryxell and Hoare. The variability in the depth distributions of ciliates in Lake Fryxell is explained in terms of lake physicochemical properties and ciliate prey distributions, while factors related to temporal succession in the Lake Hoare assemblage remain unexplained. Local marine or temperate zone freshwater habitats are a more likely source than the surrounding dry valleys soils for present ciliate colonists in these lakes. Although the taxonomic uncertainties require further examination, our results suggest that ciliate populations in these antarctic lakes undergo significant fluctuations and are more diverse than was previously recognized.

  9. Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

    NASA Astrophysics Data System (ADS)

    Youta Dombu, Christophe; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier

    2010-09-01

    A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 °C compared with 37 °C, or by NaN3 treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

  10. 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

  11. 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.

  12. 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.

  13. Endotoxin Augments Myeloid Dendritic Cell Influx into the Airways in Patients with Allergic Asthma

    PubMed Central

    Schaumann, Frank; Müller, Meike; Braun, Armin; Luettig, Birgit; Peden, David B.; Hohlfeld, Jens M.; Krug, Norbert

    2008-01-01

    Rationale: Epidemiologic studies have shown that exacerbation of asthma is modulated by environmental endotoxin. High levels of endotoxin are associated with asthma symptoms and the current use of asthma medication. However, the underlying mechanisms by which endotoxin modulates asthma are not completely understood. Objectives: The aim of the study was to test whether endotoxin enhances the response of individuals with allergic asthma to allergen, and to determine if this interaction is associated with increased numbers of antigen-presenting cells in the airways. Methods: Seventeen subjects with mild allergic asthma underwent segmental challenge with allergen, endotoxin, and the combination of both in three different lung segments via bronchoscopy. The cellular influx including monocytes, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs), as well as the level of cytokines, were assessed in bronchoalveolar lavage fluid obtained 24 hours after segmental challenge. Monocytes, mDCs, and pDCs were isolated and their capacity to induce T cell proliferation was determined. Measurements and Main Results: Endotoxin enhanced the cellular response to allergen. The combination of allergen and endotoxin resulted in increased numbers of total cells, lymphocytes, neutrophils, eosinophils, monocytes, and mDCs, as well as increased levels of lipopolysaccharide-binding protein, IL-1α, IL-6, and tumor necrosis factor–α in the bronchoalveolar lavage fluid compared with allergen alone. Isolated mDCs but not pDCs induced a strong T cell proliferation in vitro. Conclusions: Endotoxin augments the allergic inflammation in the lungs of individuals with asthma, and induces an enhanced influx of monocytes and functionally active antigen-presenting mDCs into the respiratory tract. PMID:18388357

  14. 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

  15. 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.

  16. Chrysin inhibits human airway smooth muscle cells proliferation through the extracellular signal-regulated kinase 1/2 signaling pathway.

    PubMed

    Yao, Jing; Zhang, Yun-Shi; Feng, Gan-Zhu; Du, Qiang

    2015-11-01

    Asthma is a chronic airway inflammatory disease characterized by an increased mass of airway smooth muscle (ASM). Chrysin (5,7-dihydroxyflavone), a natural flavonoid, has been shown to exert multiple biological activities, including anti-inflammatory, anti-proliferative and anti-oxidant effects, as well as the potency to ameliorate asthma in animal models. The objective of the present study was to identify the underlying mechanism of the therapeutic effects of chrysin. The impact of chrysin on basal and platelet-derived growth factor (PDGF)-induced proliferation and apoptosis of human airway smooth muscle cells (HASMCs) was investigated. Furthermore, the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway was evaluated in HASMCs. The results revealed that chrysin significantly inhibited basal as well as PDGF-induced HASMC proliferation, most likely through the suppression of ERK1/2 phosphorylation. However, chrysin did not significantly reduce PDGF-induced apoptosis of HASMCs. The present study indicated that chrysin may be a promising medication for controlling airway remodeling and clinical manifestations of asthma.

  17. Ciliate communities consistently associated with coral diseases

    NASA Astrophysics Data System (ADS)

    Sweet, M. J.; Séré, M. G.

    2016-07-01

    Incidences of coral disease are increasing. Most studies which focus on diseases in these organisms routinely assess variations in bacterial associates. However, other microorganism groups such as viruses, fungi and protozoa are only recently starting to receive attention. This study aimed at assessing the diversity of ciliates associated with coral diseases over a wide geographical range. Here we show that a wide variety of ciliates are associated with all nine coral diseases assessed. Many of these ciliates such as Trochilia petrani and Glauconema trihymene feed on the bacteria which are likely colonizing the bare skeleton exposed by the advancing disease lesion or the necrotic tissue itself. Others such as Pseudokeronopsis and Licnophora macfarlandi are common predators of other protozoans and will be attracted by the increase in other ciliate species to the lesion interface. However, a few ciliate species (namely Varistrombidium kielum, Philaster lucinda, Philaster guamense, a Euplotes sp., a Trachelotractus sp. and a Condylostoma sp.) appear to harbor symbiotic algae, potentially from the coral themselves, a result which may indicate that they play some role in the disease pathology at the very least. Although, from this study alone we are not able to discern what roles any of these ciliates play in disease causation, the consistent presence of such communities with disease lesion interfaces warrants further investigation.

  18. 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

  19. Tbet Deficiency Causes T Helper Cell Dependent Airways Eosinophilia and Mucus Hypersecretion in Response to Rhinovirus Infection

    PubMed Central

    Glanville, Nicholas; Schröder, Armin; Walton, Ross P.; Johnston, Sebastian L.

    2016-01-01

    Current understanding of adaptive immune, particularly T cell, responses to human rhinoviruses (RV) is limited. Memory T cells are thought to be of a primarily T helper 1 type, but both T helper 1 and T helper 2 memory cells have been described, and heightened T helper 2/ lessened T helper 1 responses have been associated with increased RV-induced asthma exacerbation severity. We examined the contribution of T helper 1 cells to RV-induced airways inflammation using mice deficient in the transcription factor T-Box Expressed In T Cells (Tbet), a critical controller of T helper 1 cell differentiation. Using flow cytometry we showed that Tbet deficient mice lacked the T helper 1 response of wild type mice and instead developed mixed T helper 2/T helper 17 responses to RV infection, evidenced by increased numbers of GATA binding protein 3 (GATA-3) and RAR-related orphan receptor gamma t (RORγt), and interleukin-13 and interleukin-17A expressing CD4+ T cells in the lung. Forkhead box P3 (FOXP3) and interleukin-10 expressing T cell numbers were unaffected. Tbet deficient mice also displayed deficiencies in lung Natural Killer, Natural Killer T cell and γδT cell responses, and serum neutralising antibody responses. Tbet deficient mice exhibited pronounced airways eosinophilia and mucus production in response to RV infection that, by utilising a CD4+ cell depleting antibody, were found to be T helper cell dependent. RV induction of T helper 2 and T helper 17 responses may therefore have an important role in directly driving features of allergic airways disease such as eosinophilia and mucus hypersecretion during asthma exacerbations. PMID:27683080

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

    PubMed Central

    Salmon, Michael; Walsh, David A; Huang, Tung-Jung; Barnes, Peter J; Leonard, Thomas B; Hay, Douglas W P; Chung, K Fan

    1999-01-01

    Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin-exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline-exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5-lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen-induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure. PMID:10455261

  5. 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.

  6. 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.

  7. Type 2 innate lymphoid cells-new members of the "type 2 franchise" that mediate allergic airway inflammation.

    PubMed

    Mjösberg, Jenny; Spits, Hergen

    2012-05-01

    Type 2 innate lymphoid cells (ILC2s) are members of an ILC family, which contains NK cells and Rorγt(+) ILCs, the latter including lymphoid tissue inducer (LTi) cells and ILCs producing IL-17 and IL-22. ILC2s are dedicated to the production of IL-5 and IL-13 and, as such, ILC2s provide an early and important source of type 2 cytokines critical for helminth expulsion in the gut. Several studies have also demonstrated a role for ILC2s in airway inflammation. In this issue of the European Journal of Immunology, Klein Wolterink et al. [Eur. J. Immunol. 2012. 42: 1106-1116] show that ILC2s are instrumental in several models of experimental asthma where they significantly contribute to production of IL-5 and IL-13, key cytokines in airway inflammation. This study sheds light over the relative contribution of ILC2s versus T helper type 2 cells (Th2) in type 2 mediated allergen-specific inflammation in the airways as discussed in this commentary.

  8. Bioassay of environmental nickel dusts in a particle feeding ciliate

    SciTech Connect

    Smith-Sonneborn, J.; Leibovitz, B.; Donathan, R.; Fisher, G.L.

    1986-01-01

    The ciliated protozoan Paramecium was used to quantitate cytotoxic and genotoxic effects of nickel particles. The biological response of these eukaryotic cells to pure nickel powder and iron-nickel powder was assayed and compared to the effect of the inorganic carcinogen nickel subsulfide. Cytotoxicity was determined by the percent survival of treated cells. Genotoxicity was indicated by significant increases in the fraction of nonviable offspring (presumed index of lethal mutations) found after self-fertilization (autogamy) in parents from the nickel-treated versus neutral control groups. The cells were exposed to the dusts and the biological effects determined. Only the nickel subsulfide consistently showed a significant increase in offspring lethality.

  9. Kv7 potassium channels in airway smooth muscle cells: signal transduction intermediates and pharmacological targets for bronchodilator therapy

    PubMed Central

    Brueggemann, Lioubov I.; Kakad, Priyanka P.; Love, Robert B.; Solway, Julian; Dowell, Maria L.; Cribbs, Leanne L.

    2012-01-01

    Expression and function of Kv7 (KCNQ) voltage-activated potassium channels in guinea pig and human airway smooth muscle cells (ASMCs) were investigated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), patch-clamp electrophysiology, and precision-cut lung slices. qRT-PCR revealed expression of multiple KCNQ genes in both guinea pig and human ASMCs. Currents with electrophysiological and pharmacological characteristics of Kv7 currents were measured in freshly isolated guinea pig and human ASMCs. In guinea pig ASMCs, Kv7 currents were significantly suppressed by application of the bronchoconstrictor agonists methacholine (100 nM) or histamine (30 μM), but current amplitudes were restored by addition of a Kv7 channel activator, flupirtine (10 μM). Kv7 currents in guinea pig ASMCs were also significantly enhanced by another Kv7.2–7.5 channel activator, retigabine, and by celecoxib and 2,5-dimethyl celecoxib. In precision-cut human lung slices, constriction of airways by histamine was significantly reduced in the presence of flupirtine. Kv7 currents in both guinea pig and human ASMCs were inhibited by the Kv7 channel blocker XE991. In human lung slices, XE991 induced robust airway constriction, which was completely reversed by addition of the calcium channel blocker verapamil. These findings suggest that Kv7 channels in ASMCs play an essential role in the regulation of airway diameter and may be targeted pharmacologically to relieve airway hyperconstriction induced by elevated concentrations of bronchoconstrictor agonists. PMID:21964407

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Beta-agonists and secretory cell number and intracellular glycoproteins in airway epithelium. The effect of isoproterenol and salbutamol.

    PubMed Central

    Jones, R.; Reid, L.

    1979-01-01

    This study describes the effect of systemic administration of the beta-adrenergic agonists isoproterenol and salbutamol on the secretory cell populations in seven regions of rat airway epithelium (three extrapulmonary and four intrapulmonary) and on the size of salivary glands and heart. Isoproterenol (a nonselective beta-adrenergic agonist) significantly increases secretory cell number in all airway regions except the midtrachea; salbutamol (a selective beta 2 agonist) increases secretory cell number only in proximal and peripheral regions. The absolute number of secretory cells is greatest in the most peripheral region after isoproterenol administration and in the most proximal region after salbutamol, although both drugs produce the greatest relative increase at the periphery. In proximal and, particularly, peripheral regions, the increase by isoproterenol (less than 3- and 14-fold, respectively) is greater than by salbutamol (less than 2- and less than 3-fold, respectively). In all airway regions, both drugs modify intracellular glycoprotein in the secretory cell population; within a given region, modification is much the same. In the most proximal region, the population of cells synthesizing only granules of neutral glycoprotein significantly increases while in other regions increase is in cells synthesizing only granules of acid. A significant shift in glycoprotein synthesis occurs whether or not the secretory cell population is increased, which suggests that existing as well as newly appearing cells modify their product. Isoproterenol significantly increases the size of the parotid and submaxillary glands; salbutamol increases the size of the parotid only. Isoproterenol significantly increases the weight of both ventricles of the heart; salbutamol has no such effect. PMID:36762

  17. Differential responses of human dendritic cells to metabolites from the oral/airway microbiome.

    PubMed

    Whiteson, K; Agrawal, S; Agrawal, A

    2017-02-14

    Small molecule metabolites that are produced or altered by host-associated microbial communities are emerging as significant immune response modifiers. However, there is a key gap in our knowledge of how oral microbial metabolites affect the immune response. Here, we examined the effects of metabolites from five bacterial strains found commonly in the oral/airway microbial communities of humans. The five strains, each isolated from cystic fibrosis patient sputum, were Pseudomonas aeruginosa FLR01 non-mucoid (P1) and FLR02 mucoid (P2) forms, Streptococcus pneumoniae (Sp), S. salivarius (Ss) and Rothia mucilaginosa (Rm). The effect of bacterial metabolites on dendritic cell (DC) activation, T cell priming and cytokine secretion was determined by exposing DCs to bacterial supernatants and individual metabolites of interest. Supernatants from P1 and P2 induced high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-12 and IL-6 from DCs and primed T cells to secrete interferon (IFN)-γ, IL-22 compared to supernatants from Sp, Ss and Rm. Investigations into the composition of supernatants using gas chromatography-mass spectroscopy (GC-MS) revealed signature metabolites for each of the strains. Supernatants from P1 and P2 contained high levels of putrescine and glucose, while Sp and Ss contained high levels of 2,3-butanediol. The individual metabolites replicated the results of whole supernatants, although the magnitudes of their effects were reduced significantly. Altogether, our data demonstrate for the first time that the signature metabolites produced by different bacteria have different effects on DC functions. The identification of signature metabolites and their effects on the host immune system can provide mechanistic insights into diseases and may also be developed as biomarkers.

  18. 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.

  19. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

    SciTech Connect

    Plopper, C.G. . E-mail: cgplopper@ucdavis.edu; Mango, G.W.; Hatch, G.E.; Wong, V.J.; Toskala, E.; Reynolds, S.D.; Tarkington, B.K.; Stripp, B.R.

    2006-05-15

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O{sub 3}). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

  20. Release of biologically active TGF-beta from airway smooth muscle cells induces autocrine synthesis of collagen.

    PubMed

    Coutts, A; Chen, G; Stephens, N; Hirst, S; Douglas, D; Eichholtz, T; Khalil, N

    2001-05-01

    In severe or chronic asthma, there is an increase in airway smooth muscle cell (ASMC) mass as well as an increase in connective tissue proteins in the smooth muscle layer of airways. Transforming growth factor-beta (TGF-beta) exists in three isoforms in mammals and is a potent regulator of connective tissue protein synthesis. Using immunohistochemistry, we had previously demonstrated that ASMCs contain large quantities of TGF-beta1-3. In this study, we demonstrate that bovine ASMC-derived TGF-beta associates with the TGF-beta latency binding protein-1 (LTBP-1) expressed by the same cells. The TGF-beta associated with LTBP-1 localizes TGF-beta extracellularly. Furthermore, plasmin, a serine protease, regulates the secretion of a biologically active form of TGF-beta by ASMCs as well as the release of extracellular TGF-beta. The biologically active TGF-beta released by plasmin induces ASMCs to synthesize collagen I in an autocrine manner. The autocrine induction of collagen expression by ASMCs may contribute to the irreversible fibrosis and remodeling seen in the airways of some asthmatics.

  1. Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, and Histopathology in a Mouse Model of Occupational Asthma

    PubMed Central

    Martínez-González, Itziar; Moreno, Rafael; Morell, Ferran; Muñoz, Xavier

    2014-01-01

    Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1×106 cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA. PMID:24798370

  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. 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.

  4. 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.

  5. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium.

    PubMed

    Rawlins, Emma L; Okubo, Tadashi; Xue, Yan; Brass, David M; Auten, Richard L; Hasegawa, Hiroshi; Wang, Fan; Hogan, Brigid L M

    2009-06-05

    To directly test the contribution of Scgb1a1(+) Clara cells to postnatal growth, homeostasis, and repair of lung epithelium, we generated a Scgb1a1-CreER "knockin" mouse for lineage-tracing these cells. Under all conditions tested, the majority of Clara cells in the bronchioles both self-renews and generates ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung, it has been proposed that bronchioalveolar stem cells (BASCs) which coexpress Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis, or repair. Rather, our results support a model in which the trachea, bronchioles, and alveoli are maintained by distinct populations of epithelial progenitor cells.

  6. 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.

  7. THE CONTRACTILE PROCESS IN THE CILIATE, STENTOR COERULEUS

    PubMed Central

    Huang, B.; Pitelka, D. R.

    1973-01-01

    The structural basis for the function of microtubules and filaments in cell body contractility in the ciliate Stentor coeruleus was investigated. Cells in the extended state were obtained for ultrastructural analysis by treatment before fixation with a solution containing 10 mM EGTA, 50–80 mM Tris, 3 mM MgSO4, 7.5 mM NH4Cl, 10 mM phosphate buffer (pH 7.1). The response of Stentor to changes in the divalent cation concentrations in this solution suggests that Ca+2 and Mg+2 are physiologically important in the regulation of ciliate contractility. The generation of motive force for changes in cell length in Stentor resides in two distinct longitudinal cortical fiber systems, the km fibers and myonemes. Cyclic changes in cell length are associated with (a) the relative sliding of parallel, overlapping microtubule ribbons in the km fibers, and (b) a distinct alteration in the structure of the contractile filaments constituting the myonemes. The microtubule and filament systems are distinguished functionally as antagonistic contractile elements. The development of motive force for cell extension is accomplished by active microtubule-to-microtubule sliding generated by specific intertubule bridges. Evidence is presented which suggests that active shortening of contractile filaments, reflected in a reversible structural transformation of dense 4-nm filaments to tubular 10–12-nm filaments, provides the basis for rapid cell contraction. PMID:4633444

  8. Microstome--macrostome transformation in the polymorphic ciliate Tetrahymena vorax leads to mechanosensitivity associated with prey-capture behaviour.

    PubMed

    Grønlien, Heidi K; Hagen, Bjarne; Sand, Olav

    2011-07-01

    Ciliates feed by phagocytosis. Some ciliate species, such as Tetrahymena vorax, are polymorphic, a strategy that provides more flexible food utilization. Cells of the microstomal morph of T. vorax feed on bacteria, organic particles and organic solutes in a non-selective manner, whereas macrostome cells are predators that consume specific prey ciliates. In the present study, we investigated a possible correlation between phagocytosis and mechanosensitivity in macrostome T. vorax. Microstome cells seem to be insensitive to mechanical stimulation whereas macrostome cells depolarise in response to mechanical stimulation of the anterior part of the cell. The amplitude of the receptor potential induced by either a prey ciliate or a 5 μm push by a glass needle was sufficient to elicit a regenerative Ca²⁺ spike. The difference in mechanosensitivity of the two forms correlates with the swimming behaviour when hitting an obstacle; microstome cells swim alongside the obstacle whereas macrostome cells swim backwards, turn and resume forward swimming. Macrostome cells prevented from backward swimming and the subsequent turn failed to capture prey cells in their pouch. Macrostome cells consumed heterospecific prey ciliates preferentially over conspecific microstome cells. This selectivity is not due to electrical membrane responses elicited by physical contact. Both microstome and macrostome cells accumulated in an area containing putative substances released from heterospecific prey ciliates, but the substances did not elicit any electrophysiological membrane responses. We conclude that the mechanosensitivity of macrostome cells is associated with the prey-capture behaviour, whereas the selective phagocytosis is probably due to chemo-attraction to heterospecific prey ciliates.

  9. Cyclooxygenase-2 and microRNA-155 expression are elevated in asthmatic airway smooth muscle cells.

    PubMed

    Comer, Brian S; Camoretti-Mercado, Blanca; Kogut, Paul C; Halayko, Andrew J; Solway, Julian; Gerthoffer, William T

    2015-04-01

    Cyclooxygenase-2 (COX-2) expression and PGE2 secretion from human airway smooth muscle cells (hASMCs) may contribute to β2-adrenoceptor hyporesponsiveness, a clinical feature observed in some patients with asthma. hASMCs from patients with asthma exhibit elevated expression of cytokine-responsive genes, and in some instances this is attributable to an altered histone code and/or microRNA expression. We hypothesized that COX-2 expression and PGE2 secretion might be elevated in asthmatic hASMCs in response to proinflammatory signals in part due to altered histone acetylation and/or microRNA expression. hASMCs obtained from nonasthmatic and asthmatic human subjects were treated with cytomix (IL-1β, TNF-α, and IFN-γ). A greater elevation of COX-2 mRNA, COX-2 protein, and PGE2 secretion was observed in the asthmatic cells. We investigated histone H3/H4-acetylation, transcription factor binding, mRNA stability, p38 mitogen-activated protein kinase signaling, and microRNA (miR)-155 expression as potential mechanisms responsible for the differential elevation of COX-2 expression. We found that histone H3/H4-acetylation and transcription factor binding to the COX-2 promoter were similar in both groups, and histone H3/H4-acetylation did not increase after cytomix treatment. Cytomix treatment elevated NF-κB and RNA polymerase II binding to similar levels in both groups. COX-2 mRNA stability was increased in asthmatic cells. MiR-155 expression was higher in cytomix-treated asthmatic cells, and we show it enhances COX-2 expression and PGE2 secretion in asthmatic and nonasthmatic hASMCs. Thus, miR-155 expression positively correlates with COX-2 expression in the asthmatic hASMCs and may contribute to the elevated expression observed in these cells. These findings may explain, at least in part, β2-adrenoceptor hyporesponsiveness in patients with asthma.

  10. Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility

    SciTech Connect

    Xu, Guang-Ni; Yang, Kai; Xu, Zu-Peng; Zhu, Liang; Hou, Li-Na; Qi, Hong; Chen, Hong-Zhuan Cui, Yong-Yao

    2012-07-01

    Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclin D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation. -- Highlights: ► CSE induces tracheal cell proliferation, hyper-contractility and α-SMA expression. ► Anisodamine reverses CSE-induced tracheal hyper-contractility and cell proliferation. ► ERK, PI3K, and NF-κB pathways and cyclin D1 contribute to the reversal effect.

  11. Dectin-2 promotes house dust mite-induced T helper type 2 and type 17 cell differentiation and allergic airway inflammation in mice.

    PubMed

    Norimoto, Ayako; Hirose, Koichi; Iwata, Arifumi; Tamachi, Tomohiro; Yokota, Masaya; Takahashi, Kentaro; Saijo, Shinobu; Iwakura, Yoichiro; Nakajima, Hiroshi

    2014-08-01

    The fact that sensitization against fungi is closely related to the severity of asthma suggests that immune systems recognizing fungi are involved in the pathogenesis of severe asthma. Recently, Dectin-2 (gene symbol, Clec4n), a C-type lectin receptor, has been shown to function as not only a major pattern-recognition receptor for fungi, but also a receptor for some components of house dust mite (HDM) extract, a major allergen for asthma. However, the roles of Dectin-2 in the induction of HDM-induced allergic airway inflammation remain largely unknown. Our objective was to determine the roles of Dectin-2 in HDM-induced allergic airway inflammation. We examined the roles of Dectin-2 in the induction of HDM-induced T helper (Th) 2 and Th17 cell differentiation and subsequent allergic airway inflammation by using Clec4n-deficient (Clec4n(-/-)) mice. We also investigated Dectin-2-expressing cells in the lung and their roles in HDM-induced allergic airway inflammation. Clec4n(-/-) mice showed significantly attenuated HDM-induced allergic airway inflammation and decreased Th2 and Th17 cell differentiation. Dectin-2 mRNA, together with Dectin-3 and Fc receptor-γ mRNAs, was expressed in CD11b(+) dendritic cells (DCs), but not in CD4(+) T cells or epithelial cells in the lung. CD11b(+) DCs isolated from Clec4n(-/-) mice expressed lower amounts of proinflammatory cytokines and costimulatory molecules, which could lead to Th2 and Th17 cell differentiation than those from wild-type mice. HDM-pulsed Clec4n(-/-) DCs were less efficient for the induction of allergic airway inflammation than HDM-pulsed wild-type DCs. In conclusion, Dectin-2 expressed on CD11b(+) DCs promotes HDM-induced Th2 and Th17 cell differentiation and allergic airway inflammation.

  12. Rumen ciliate protozoa of domestic sheep (Ovis aries) and goat (Capra aegagrus hircus) in Kyrgyzstan.

    PubMed

    Gürelli, Gözde; Canbulat, Savaş; Aldayarov, Nurbek; Dehority, Burk A

    2016-03-01

    Species composition and concentration of rumen ciliate protozoa were investigated in the rumen contents of 14 domestic sheep and 1 goat living in Bishkek, Kyrgyzstan. This is the first report on rumen ciliates from ruminants living in Kyrgyzstan. In sheep 12 genera, 28 species and 12 morphotypes were detected, whereas in goat 8 genera, 12 species and 4 morphotypes were detected. The density of ciliates in sheep was (28.1 ± 20.0) × 10(4) cells mL(-1) and in goat was 37.0 × 10(4) cells mL(-1). Dasytricha ruminantium, Isotricha prostoma, Entodinium simulans and Ophryoscolex caudatus were major species (100%) in sheep, and for the first time, Diplodinium rangiferi was detected in a domestic goat.

  13. Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice.

    PubMed

    Zhang, Bei; An, Jun; Shimada, Takashi; Liu, Shuang; Maeyama, Kazutaka

    2012-08-01

    Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.

  14. Streptomycin treatment alters the intestinal microbiome, pulmonary T cell profile and airway hyperresponsiveness in a cystic fibrosis mouse model

    PubMed Central

    Bazett, Mark; Bergeron, Marie-Eve; Haston, Christina K.

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator deficient mouse models develop phenotypes of relevance to clinical cystic fibrosis (CF) including airway hyperresponsiveness, small intestinal bacterial overgrowth and an altered intestinal microbiome. As dysbiosis of the intestinal microbiota has been recognized as an important contributor to many systemic diseases, herein we investigated whether altering the intestinal microbiome of BALB/c Cftrtm1UNC mice and wild-type littermates, through treatment with the antibiotic streptomycin, affects the CF lung, intestinal and bone disease. We demonstrate that streptomycin treatment reduced the intestinal bacterial overgrowth in Cftrtm1UNC mice and altered the intestinal microbiome similarly in Cftrtm1UNC and wild-type mice, principally by affecting Lactobacillus levels. Airway hyperresponsiveness of Cftrtm1UNC mice was ameliorated with streptomycin, and correlated with Lactobacillus abundance in the intestine. Additionally, streptomycin treated Cftrtm1UNC and wild-type mice displayed an increased percentage of pulmonary and mesenteric lymph node Th17, CD8 + IL-17+ and CD8 + IFNγ+ lymphocytes, while the CF-specific increase in respiratory IL-17 producing γδ T cells was decreased in streptomycin treated Cftrtm1UNC mice. Bone disease and intestinal phenotypes were not affected by streptomycin treatment. The airway hyperresponsiveness and lymphocyte profile of BALB/c Cftrtm1UNC mice were affected by streptomycin treatment, revealing a potential intestinal microbiome influence on lung response in BALB/c Cftrtm1UNC mice. PMID:26754178

  15. Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

    PubMed

    Savoia, Carlo P; Liu, Qing-Hua; Zheng, Yun-Min; Yadav, Vishal; Zhang, Zhen; Wu, Ling-Gang; Wang, Yong-Xiao

    2014-11-15

    Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

  16. Are freshwater mixotrophic ciliates less sensitive to solar ultraviolet radiation than heterotrophic ones?

    PubMed

    Sonntag, Bettina; Summerer, Monika; Sommaruga, Ruben

    2011-01-01

    We tested whether mixotrophic ciliates are more resistant to solar ultraviolet radiation (UVR) than heterotrophic ones because symbiotic algae can provide self-shading by cell matter absorption and eventually by direct UV screening from mycosporine-like amino acids (MAAs). Sensitivity of a natural assemblage to solar radiation was tested in experiments in the original lake and in a more UV transparent alpine lake after transplantation of the ciliates. In both lakes, the assemblage was exposed either to full sunlight, to photosynthetically active radiation only, or kept in the dark. In each lake, exposure was for 5 h at the surface and at the depth corresponding to the 10% attenuation depth at 320 nm. Overall, when the assemblage was exposed to surface UVR, only one out of four dominant mixotrophic ciliates, Vorticella chlorellata, was more resistant than heterotrophic species. The higher UV resistance in V. chlorellata was related to the presence of MAAs and the high percentage of ciliate volume occupied by algal symbionts. Our results indicate that effects of UVR were species-specific and depended on efficient screening of these wavelengths, but also on the depth preference of the ciliates and thus, on their previous exposure history to UVR.

  17. Are Freshwater Mixotrophic Ciliates Less Sensitive to Solar Ultraviolet Radiation than Heterotrophic Ones?1

    PubMed Central

    SONNTAG, BETTINA; SUMMERER, MONIKA; SOMMARUGA, RUBEN

    2011-01-01

    We tested whether mixotrophic ciliates are more resistant to solar ultraviolet radiation (UVR) than heterotrophic ones because symbiotic algae can provide self-shading by cell matter absorption and eventually by direct UV screening from mycosporine-like amino acids (MAAs). Sensitivity of a natural assemblage to solar radiation was tested in experiments in the original lake and in a more UV transparent alpine lake after transplantation of the ciliates. In both lakes, the assemblage was exposed either to full sunlight, to photosynthetically active radiation only, or kept in the dark. In each lake, exposure was for 5 h at the surface and at the depth corresponding to the 10% attenuation depth at 320 nm. Overall, when the assemblage was exposed to surface UVR, only one out of four dominant mixotrophic ciliates, Vorticella chlorellata, was more resistant than heterotrophic species. The higher UV resistance in V. chlorellata was related to the presence of MAAs and the high percentage of ciliate volume occupied by algal symbionts. Our results indicate that effects of UVR were species-specific and depended on efficient screening of these wavelengths, but also on the depth preference of the ciliates and thus, on their previous exposure history to UVR. PMID:21414057

  18. 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.

  19. T-cell-intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy.

    PubMed

    Perez-Lloret, Jimena; Okoye, Isobel S; Guidi, Riccardo; Kannan, Yashaswini; Coomes, Stephanie M; Czieso, Stephanie; Mengus, Gabrielle; Davidson, Irwin; Wilson, Mark S

    2016-02-02

    There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ(+)CD4(+) T-helper 2 (TH2) cells orchestrate the type-2-driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081-E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4(gfp+)αβ(+)CD4(+) TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell-intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24(-/-) T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1-regulated signaling. Following this prediction, we found that Trim24(-/-) T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β-mediated activation in vitro and in vivo, and fail to respond to IL-1β-exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell-intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity.

  20. 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

  1. Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus).

    PubMed

    Grieves, Jessica L; Yin, Zhiwei; Durbin, Russell K; Durbin, Joan E

    2015-08-01

    Infection with respiratory syncytial virus (RSV) generally presents as a mild, upper airway disease in human patients but may cause severe lower airway disease in the very young and very old. Progress toward understanding the mechanisms of RSV pathogenesis has been hampered by a lack of relevant rodent models. Mice, the species most commonly used in RSV research, are resistant to upper respiratory infection and do not recapitulate the pattern of virus spread in the human host. To address the need for better rodent models of RSV infection, we have characterized the acute and chronic pathology of RSV infection of a relatively permissive host, cotton rats (Sigmodon hispidus). We demonstrate that virus delivered to the upper airway results in widespread RSV replication in the ciliated respiratory epithelial cells of the nasal cavity and, to a lesser extent, of the lung. Although acute inflammation is relatively mild and rapidly eliminated after viral clearance, chronic, eosinophilic lung pathology persists. These data support the use of cotton rats as a robust rodent model of human RSV disease, including the association between RSV pneumonia and subsequent development of allergic asthma.

  2. Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus)

    PubMed Central

    Grieves, Jessica L; Yin, Zhiwei; Durbin, Russell K; Durbin, Joan E

    2015-01-01

    Infection with respiratory syncytial virus (RSV) generally presents as a mild, upper airway disease in human patients but may cause severe lower airway disease in the very young and very old. Progress toward understanding the mechanisms of RSV pathogenesis has been hampered by a lack of relevant rodent models. Mice, the species most commonly used in RSV research, are resistant to upper respiratory infection and do not recapitulate the pattern of virus spread in the human host. To address the need for better rodent models of RSV infection, we have characterized the acute and chronic pathology of RSV infection of a relatively permissive host, cotton rats (Sigmodon hispidus). We demonstrate that virus delivered to the upper airway results in widespread RSV replication in the ciliated respiratory epithelial cells of the nasal cavity and, to a lesser extent, of the lung. Although acute inflammation is relatively mild and rapidly eliminated after viral clearance, chronic, eosinophilic lung pathology persists. These data support the use of cotton rats as a robust rodent model of human RSV disease, including the association between RSV pneumonia and subsequent development of allergic asthma. PMID:26310461

  3. 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.

  4. Revisiting fifty years of research on pheromone signaling in ciliates.

    PubMed

    Luporini, Pierangelo; Pedrini, Bill; Alimenti, Claudio; Vallesi, Adriana

    2016-08-01

    Among protists, pheromones have been identified in a great variety of algal species for their activity in driving gamete-gamete interactions for fertilization. Analogously in ciliates, pheromones have been identified for their activity in inducing the sexual phenomenon of conjugation. Although this identification was pioneered by Kimball more than fifty years ago, an effective isolation and chemical characterization of ciliate pheromones has remained confined to species of Blepharisma, Dileptus and Euplotes. In Euplotes species, in which the molecular structures have been determined, pheromones form species-specific families of structurally homologous helical, cysteine-rich, highly-stable proteins. Being structurally homologous, they can bind cells in competition with one another, raising interesting functional analogies with the families of growth factors and cytokines that regulate cell differentiation and development in higher organisms. In addition to inducing conjugation by binding cells in heterologous fashion, Euplotes pheromones act also as autocrine growth factors by binding to, and promoting the vegetative reproduction of the same cells from which they originate. This autocrine activity is most likely primary, providing a concrete example of how the original function of a molecule can be obscured during evolution by the acquisition of a new one.

  5. Low level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans

    EPA Science Inventory

    Background: The effects of low level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known, however much less is known about the inflammatory and immuno-modulatory effects oflow level ozone in the airways. Techniques such as induced sputum and flo...

  6. 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...

  7. 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

  8. Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

    PubMed

    Favia, Maria; Mancini, Maria T; Bezzerri, Valentino; Guerra, Lorenzo; Laselva, Onofrio; Abbattiscianni, Anna C; Debellis, Lucantonio; Reshkin, Stephan J; Gambari, Roberto; Cabrini, Giulio; Casavola, Valeria

    2014-07-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

  9. An evolutionary balance: conservation vs innovation in ciliate membrane trafficking.

    PubMed

    Guerrier, Sabrice; Plattner, Helmut; Richardson, Elisabeth; Dacks, Joel B; Turkewitz, Aaron P

    2017-01-01

    As most of eukaryotic diversity lies in single-celled protists, they represent unique opportunities to ask questions about the balance of conservation and innovation in cell biological features. Among free-living protists the ciliates offer ease of culturing, a rich array of experimental approaches, and versatile molecular tools, particularly in Tetrahymena thermophila and Paramecium tetraurelia. These attributes have been exploited by researchers to analyze a wealth of cellular structures in these large and complex cells. This mini-review focuses on 3 aspects of ciliate membrane dynamics, all linked with endolysosomal trafficking. First is nutrition based on phagocytosis and maturation of food vacuoles. Secondly, we discuss regulated exocytosis from vesicles that have features of both dense core secretory granules but also lysosome-related organelles. The third topic is the targeting, breakdown and resorption of parental nuclei in mating partners. For all 3 phenomena, it is clear that elements of the canonical membrane-trafficking system have been retained and in some cases repurposed. In addition, there is evidence that recently evolved, lineage-specific proteins provide determinants in these pathways.

  10. Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

    SciTech Connect

    Jeong, Young-Il; Kim, Seung Hyun; Ju, Jung Won; Cho, Shin Hyeong; Lee, Won Ja; Park, Jin Wook; Park, Yeong-Min; Lee, Sang Eun

    2011-04-22

    Highlights: {yields} Treatment with Clonorchis sinensis-derived total protein attenuates OVA-induced airway inflammation and AHR to methacholine. {yields} Induction of CD4{sup +}CD25{sup +}Foxp3{sup +} T cells and IL-10 along with suppression of splenocyte proliferation by C. sinensis-derived total protein. {yields} C. sinensis-derived total protein interferes with the expression of co-stimulatory molecules in DCs. -- Abstract: Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naive T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical

  11. Act together—implications of symbioses in aquatic ciliates

    PubMed Central

    Dziallas, Claudia; Allgaier, Martin; Monaghan, Michael T.; Grossart, Hans-Peter

    2012-01-01

    Mutual interactions in the form of symbioses can increase the fitness of organisms and provide them with the capacity to occupy new ecological niches. The formation of obligate symbioses allows for rapid evolution of new life forms including multitrophic consortia. Microbes are important components of many known endosymbioses and their short generation times and strong potential for genetic exchange may be important drivers of speciation. Hosts provide endo- and ectosymbionts with stable, nutrient-rich environments, and protection from grazers. This is of particular importance in aquatic ecosystems, which are often highly variable, harsh, and nutrient-deficient habitats. It is therefore not surprising that symbioses are widespread in both marine and freshwater environments. Symbioses in aquatic ciliates are good model systems for exploring symbiont-host interactions. Many ciliate species are globally distributed and have been intensively studied in the context of plastid evolution. Their relatively large cell size offers an ideal habitat for numerous microorganisms with different functional traits including commensalism and parasitism. Phagocytosis facilitates the formation of symbiotic relationships, particularly since some ingested microorganisms can escape the digestion. For example, photoautotrophic algae and methanogens represent endosymbionts that greatly extend the biogeochemical functions of their hosts. Consequently, symbiotic relationships between protists and prokaryotes are widespread and often result in new ecological functions of the symbiotic communities. This enables ciliates to thrive under a wide range of environmental conditions including ultraoligotrophic or anoxic habitats. We summarize the current understanding of this exciting research topic to identify the many areas in which knowledge is lacking and to stimulate future research by providing an overview on new methodologies and by formulating a number of emerging questions in this field

  12. RSV-specific airway resident memory CD8+ T cells and differential disease severity after experimental human infection.

    PubMed

    Jozwik, Agnieszka; Habibi, Maximillian S; Paras, Allan; Zhu, Jie; Guvenel, Aleks; Dhariwal, Jaideep; Almond, Mark; Wong, Ernie H C; Sykes, Annemarie; Maybeno, Matthew; Del Rosario, Jerico; Trujillo-Torralbo, Maria-Belen; Mallia, Patrick; Sidney, John; Peters, Bjoern; Kon, Onn Min; Sette, Alessandro; Johnston, Sebastian L; Openshaw, Peter J; Chiu, Christopher

    2015-12-21

    In animal models, resident memory CD8+ T (Trm) cells assist in respiratory virus elimination but their importance in man has not been determined. Here, using experimental human respiratory syncytial virus (RSV) infection, we investigate systemic and local virus-specific CD8+ T-cell responses in adult volunteers. Having defined the immunodominance hierarchy, we analyse phenotype and function longitudinally in blood and by serial bronchoscopy. Despite rapid clinical recovery, we note surprisingly extensive lower airway inflammation with persistent viral antigen and cellular infiltrates. Pulmonary virus-specific CD8+ T cells display a CD69+CD103+ Trm phenotype and accumulate to strikingly high frequencies into convalescence without continued proliferation. While these have a more highly differentiated phenotype, they express fewer cytotoxicity markers than in blood. Nevertheless, their abundance before infection correlates with reduced symptoms and viral load, implying that CD8+ Trm cells in the human lung can confer protection against severe respiratory viral disease when humoral immunity is overcome.

  13. γ-Secretase Inhibitor Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation

    PubMed Central

    Zhang, Weixi; Zhang, Xueya; Sheng, Anqun; Weng, Cuiye; Zhu, Tingting; Zhao, Wei; Li, Changchong

    2015-01-01

    T helper 17 (Th17) cells play an important role in the pathogenesis of allergic asthma. Th17 cell differentiation requires Notch signaling. γ-Secretase inhibitor (GSI) blocks Notch signaling; thus, it may be considered as a potential treatment for allergic asthma. The aim of this study was to evaluate the effect of GSI on Th17 cell differentiation in a mouse model of allergic asthma. OVA was used to induce mouse asthma model in the presence and absence of GSI. GSI ameliorated the development of OVA-induced asthma, including suppressing airway inflammation responses and reducing the severity of clinical signs. GSI also significantly suppressed Th17-cell responses in spleen and reduced IL-17 levels in serum. These findings suggest that GSI directly regulates Th17 responses through a Notch signaling-dependent pathway in mouse model of allergic asthma, supporting the notion that GSI is a potential therapeutic agent for the treatment of allergic asthma. PMID:26339131

  14. Schistosome-induced pulmonary B cells inhibit allergic airway inflammation and display a reduced Th2-driving function.

    PubMed

    van der Vlugt, L E; Obieglo, K; Ozir-Fazalalikhan, A; Sparwasser, T; Haeberlein, S; Smits, H H

    2017-04-04

    Chronic schistosome infections protect against allergic airway inflammation (AAI) via the induction of IL-10-producing splenic regulatory B (Breg) cells. Previous experiments have demonstrated that schistosome-induced pulmonary B cells can also reduce AAI, but act independently of IL-10. We have now further characterized the phenotype and inhibitory activity of these protective pulmonary B cells. We excluded a role for regulatory T (Treg) cell induction as putative AAI-protective mechanisms. Schistosome-induced B cells showed increased CD86 expression and reduced cytokine expression in response to Toll-like receptor (TLR) ligands compared with control B cells. To investigate the consequences for T cell activation we cultured ovalbumin (OVA)-pulsed, schistosome-induced B cells with OVA-specific transgenic T cells and observed less Th2 cytokine expression and T cell proliferation compared with control conditions. This suppressive effect was preserved even under optimal T cell stimulation by anti-CD3/28. Blocking of the inhibitory cytokines IL-10 or TGF-β only marginally restored Th2 cytokine induction. These data suggest that schistosome-induced pulmonary B cells are impaired in their capacity to produce cytokines to TLR ligands and to induce Th2 cytokine responses independent of their antigen-presenting function. These findings underline the presence of distinct B cell subsets with different stimulatory or inhibitory properties even if induced by the same type of helminth.

  15. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  16. 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.

  17. 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

  18. PKC-DEPENDENT REGULATION OF Kv7.5 CHANNELS BY THE BRONCHOCONSTRICTOR HISTAMINE IN HUMAN AIRWAY SMOOTH MUSCLE CELLS.

    PubMed

    Haick, Jennifer M; Brueggemann, Lioubov I; Cribbs, Leanne L; Denning, Mitchell F; Schwartz, Jeffrey; Byron, Kenneth L

    2017-03-10

    Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, though the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

  19. H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal K(ATP) channel.

    PubMed

    Fitzgerald, Robert; DeSantiago, Breann; Lee, Danielle Y; Yang, Guangdong; Kim, Jae Yeon; Foster, D Brian; Chan-Li, Yee; Horton, Maureen R; Panettieri, Reynold A; Wang, Rui; An, Steven S

    2014-03-28

    Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)-the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1-10mM). GYY4137 slowly released appreciable levels of H2S in the range of 10-275 μM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

  20. 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.

  1. Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles.

    PubMed

    Wang, Zongxing; Wu, Hung-jen; Fine, Daniel; Schmulen, Jeffrey; Hu, Ye; Godin, Biana; Zhang, John X J; Liu, Xuewu

    2013-08-07

    We fabricated a microfluidic device consisting of ciliated micropillars, forming a porous silicon nanowire-on-micropillar structure. We demonstrated that the prototype device can preferentially trap exosome-like lipid vesicles, while simultaneously filtering out proteins and cell debris. Trapped lipid vesicles can be recovered intact by dissolving the porous nanowires in PBS buffer.

  2. 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...

  3. 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...

  4. Trends on the distribution of ciliates in the open Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Gómez, Fernando

    2007-09-01

    The distribution of planktonic ciliates, aloricate (naked) and loricate (tintinnid) was investigated in the open waters of the Oyashio and Kuroshio Currents, Philippine, Sulu, Celebes and South China Seas, and the western and central equatorial Pacific. The abundance of nauplii and post-naupliar copepods as potential predators was estimated. In average, the tintinnids represented 10-20% of the abundance of aloricate ciliates (50-200 cells L -1). One hundred and two species of 37 genera of tintinnids were recorded. As a general trend, the highest species richness was found in moderate oligotrophic waters. Photographic records of some taxa of interest and unidentified specimens were reported. In the subarctic waters of the Oyashio Current, a few tintinnid species showed high abundance fluctuations that may be controlled by the copepods. During the summer the species of Parafavella with longer loricae predominated in parallel to the increase of the copepodite abundance. In warm open waters, the success of a ciliate species could depend on its anti-grazing strategy. Eutintinnus apertus attached to a spine-bearing diatom was the most ubiquitous species. Tintinnids may be subjected to lower predation pressure than aloricate ciliates. The increase of the lorica length or the association with diatoms may be an anti-grazing strategy, which determines the success of one tintinnid taxon versus other congeneric species. Although the aloricate ciliates would be less affected than tintinnids by the reduction of food availability under oligotrophic conditions, the ciliate populations as a general trend seem to be controlled by the predators (top-down) rather than by the availability of food resources (bottom-up).

  5. The bacterivory of interstitial ciliates in association with bacterial biomass and production in the hyporheic zone of a lowland stream.

    PubMed

    Königs, Sascha; Cleven, Ernst-Josef

    2007-07-01

    Rates of bacteria ingestion by interstitial ciliates were estimated and compared to bacterial biomass and production. Investigation was carried out in the hyporheic zone of a lowland stream. FISH was applied to quantitatively determine bacteria within the ciliate's food vacuoles. To estimate bacteria ingestion rates using FISH, we had to strike a new path. When numbers of bacteria in the food vacuoles remains constant with time (bacterial digestion and ingestion are at equilibrium), ingestion rate can be estimated based on the digestion time and the average number of bacteria per cell. Ciliate community was predominantly composed of bacterivorous ciliates. FISH-signals deriving from ingested bacteria were detected in Cinetochilum margaritaceum, 'other small scuticociliates', Pleuronema spp., and Vorticella spp. Ingestion rates for these taxa were 78, 150, 86, and 38 bacteria ind(-1) h(-1), respectively. The grazing impacts on bacterial biomass and carbon production were calculated based on these ingestion rates. Ciliate grazing caused a decrease in bacterial biomass of 0.024% day(-1) and in bacterial carbon production of 1.60%. These findings suggest that interstitial ciliate grazing impact on bacteria biomass and production was too low to represent an important link in the carbon flow of the hyporheic zone under study.

  6. Ciliates and their picophytoplankton-feeding activity in a high-altitude warm-monomictic saline lake.

    PubMed

    Pestová, Dana; Macek, Miroslav; Elena Martínez Pérez, María

    2008-02-01

    The impact of feeding on autotrophic picoplankton (APP) on the ciliate composition of the assemblage was surveyed monthly along a depth gradient in the maar crater, athalassohaline, warm monomictic Lake Alchichica (Puebla, Mexico) from June 2003 to December 2005. Numbers of APP were evaluated from their autofluorescence. DAPI staining and the Fluorescently Labeled Bacteria technique were employed to count ciliates and estimate their feeding rates. A total of 38 taxa of ciliates have been identified using Quantitative Protargol Staining. Peritrichs followed by minute spirotrichs (particularly Halteria grandinella) often numerically dominated the ciliate assemblage and emerged as the most efficient APP feeders. A maximum of 54 ciliate cells ml(-1) was observed in the surface layer at the end of the mixing period, during the development of diatoms (Cyclotella alchichicana), the cyanobacterial bloom (Nodularia sp.) and its decay. Vorticellids (Pelagovorticella natans, Vorticella sp.) had the highest APP uptake (median 130 APP cil(-1) h(-1)). Mixotrophic Euplotes cf. daidaleos were important APP grazers near the oxycline. Scuticociliates (Cyclidium glaucoma, Uronema nigricans and an anaerobic cf. Isocyclidium globossum), were numerically dominant within the hypolimnetic assemblages and did not ingest APP. Generally, APP were not an important food source for the majority of the ciliate assemblage, being positively selected by a few species during the APP decay in aerobic and microaerobic conditions.

  7. 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}.

  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. 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. Preventative Effect of an Herbal Preparation (HemoHIM) on Development of Airway Inflammation in Mice via Modulation of Th1/2 Cells Differentiation

    PubMed Central

    Kim, Jong-Jin; Cho, Hyun Wook; Park, Hae-Ran; Jung, Uhee; Jo, Sung-Kee; Yee, Sung-Tae

    2013-01-01

    HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4+ T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4+ T cells displayed increased Th1 (IFN-γ+ cell) as well as decreased Th2 (IL-4+ cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance. PMID:23844220

  11. Preventative effect of an herbal preparation (HemoHIM) on development of airway inflammation in mice via modulation of Th1/2 cells differentiation.

    PubMed

    Kim, Jong-Jin; Cho, Hyun Wook; Park, Hae-Ran; Jung, Uhee; Jo, Sung-Kee; Yee, Sung-Tae

    2013-01-01

    HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4(+) T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4(+) T cells displayed increased Th1 (IFN-γ(+) cell) as well as decreased Th2 (IL-4(+) cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance.

  12. 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.

  13. 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

  14. Chronic inhalation of biomass smoke is associated with DNA damage in airway cells: involvement of particulate pollutants and benzene.

    PubMed

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

    2013-04-01

    This study examined whether indoor air pollution from biomass fuel burning induces DNA damage in airway cells. For this, sputum cells were collected from 56 premenopausal rural women who cooked with biomass (wood, dung, crop residues) and 49 age-matched controls who cooked with cleaner liquefied petroleum gas. The levels of particulate matters with diameters of less than 10 and 2.5 µm (PM(10) and PM(2.5)) in indoor air were measured using a real-time aerosol monitor. Benzene exposure was monitored by measuring trans,trans-muconic acid (t,t-MA) in urine by HPLC-UV. DNA damage was examined by alkaline comet assay in sputum cells. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in sputum cells were measured by flow cytometry and spectrophotometry, respectively. Compared with controls, biomass users had 4 times higher tail percentage DNA, 37% more comet tail length and 5 times more Olive tail moment (p < 0.001) in inflammatory and epithelial cells in sputum, suggesting extensive DNA damage. In addition, women who cooked with biomass had 6 times higher levels of urinary t,t-MA and 2-fold higher levels of ROS generation concomitant with 28% depletion of SOD. Indoor air of biomass-using households had 2-4 times more PM(10) and PM(2.5) than that of controls. After controlling potential confounders, positive association was found between DNA damage parameters, particulate pollution, urinary t,t-MA and ROS. Thus, long-term exposure to biomass smoke induces DNA damage in airway cells and the effect was probably mediated, at least in part, by oxidative stress generated by inhaled particulate matter and benzene.

  15. Dysfunction of the non-neuronal cholinergic system in the airways and blood cells of patients with cystic fibrosis.

    PubMed

    Wessler, Ignaz; Bittinger, Fernando; Kamin, Wolfgang; Zepp, Fred; Meyer, Eckhard; Schad, Arno; Kirkpatrick, Charles James

    2007-05-30

    The non-neuronal cholinergic system is widely expressed in human airways, skin and immune cells. Choline acetyltransferase (ChAT), acetylcholine and nicotine/muscarine receptors are demonstrated in epithelial surface cells, submucosal glands, airway smooth muscle fibres and immune cells. Moreover, acetylcholine is involved in the regulation of cell functions like proliferation, differentiation, migration, organization of the cytoskeleton, cell-cell contact, secretion and transport of ions and water. Cystic fibrosis (CF), the most frequent genetic disorder, is known to be caused by a mutation of the CF-gene coding for the cystic fibrosis transmembrane regulator protein (CFTR). CFTR represents a regulating transport protein for ion channels and processes involving endo- and exocytosis. Despite the identification of the genetic mutation knowledge of the underlying cellular pathways is limited. In the present experiments the cholinergic system was investigated in the peripheral blood and in the lung of CF patients undergoing lung transplantation (n=7). Acetylcholine content in bronchi and lung parenchyma of CF was reduced by 70% compared to controls (tumor-free tissue obtained from patients with lung tumor; n=13). In contrast, ChAT activity was elevated to some extent (p>0.05) in CF, and esterase activity did not differ from control. Acetylcholine content extracted from peripheral leucocytes (30 ml) was also reduced by 70% in CF (n=13) compared to healthy volunteers (n=9). Double labelling experiments with anti-CF antibodies and anti-ChAT antibodies showed a co-localization in peripheral lymphocytes, giving first evidence that CFTR may be linked with the intracellular storage/transport of non-neuronal acetylcholine. It is concluded that the non-neuronal cholinergic system is involved in the pathogenesis of CF. A reduced content of non-neuronal acetylcholine could contribute to the deleterious changes of epithelial ion and water movements in CF, because acetylcholine

  16. Prenatal secondhand cigarette smoke promotes Th2 polarization and impairs goblet cell differentiation and airway mucus formation.

    PubMed

    Singh, Shashi P; Gundavarapu, Sravanthi; Peña-Philippides, Juan C; Rir-Sima-ah, Jules; Mishra, Neerad C; Wilder, Julie A; Langley, Raymond J; Smith, Kevin R; Sopori, Mohan L

    2011-11-01

    Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.

  17. Streptomycin treatment alters the intestinal microbiome, pulmonary T cell profile and airway hyperresponsiveness in a cystic fibrosis mouse model.

    PubMed

    Bazett, Mark; Bergeron, Marie-Eve; Haston, Christina K

    2016-01-12

    Cystic fibrosis transmembrane conductance regulator deficient mouse models develop phenotypes of relevance to clinical cystic fibrosis (CF) including airway hyperresponsiveness, small intestinal bacterial overgrowth and an altered intestinal microbiome. As dysbiosis of the intestinal microbiota has been recognized as an important contributor to many systemic diseases, herein we investigated whether altering the intestinal microbiome of BALB/c Cftr(tm1UNC) mice and wild-type littermates, through treatment with the antibiotic streptomycin, affects the CF lung, intestinal and bone disease. We demonstrate that streptomycin treatment reduced the intestinal bacterial overgrowth in Cftr(tm1UNC) mice and altered the intestinal microbiome similarly in Cftr(tm1UNC) and wild-type mice, principally by affecting Lactobacillus levels. Airway hyperresponsiveness of Cftr(tm1UNC) mice was ameliorated with streptomycin, and correlated with Lactobacillus abundance in the intestine. Additionally, streptomycin treated Cftr(tm1UNC) and wild-type mice displayed an increased percentage of pulmonary and mesenteric lymph node Th17, CD8 + IL-17+ and CD8 + IFNγ+ lymphocytes, while the CF-specific increase in respiratory IL-17 producing γδ T cells was decreased in streptomycin treated Cftr(tm1UNC) mice. Bone disease and intestinal phenotypes were not affected by streptomycin treatment. The airway hyperresponsiveness and lymphocyte profile of BALB/c Cftr(tm1UNC) mice were affected by streptomycin treatment, revealing a potential intestinal microbiome influence on lung response in BALB/c Cftr(tm1UNC) mice.

  18. IL-25 promotes Th2 immunity responses in airway inflammation of asthmatic mice via activation of dendritic cells.

    PubMed

    Hongjia, Li; Caiqing, Zhang; Degan, Lu; Fen, Liu; Chao, Wang; Jinxiang, Wu; Liang, Dong

    2014-08-01

    Allergic asthma occurs as a consequence of inappropriate immunologic inflammation to allergens and characterized by Th2 adaptive immune response. Recent studies indicated that interleukin (IL)-25, a member of the IL-17 cytokine family, had been implicated in inducing Th2 cell-dependent inflammation in airway epithelium and IL-25-deficient mice exhibit impaired Th2 immunity responses; however, how these cytokines influence innate immune responses remains poorly understood. In this study, we used ovalbumin (OVA) sensitization and challenge to induce the murine asthmatic model and confirmed by histological analysis of lung tissues and serum levels of total and OVA-specific immunoglobulin (Ig)-E. The expression of IL-25 was detected by quantitative real-time PCR and immunohistochemistry, respectively, and the dendritic cells (DCs) activation was detected by levels of CD80 and CD86 in bronchoalveolar lavage fluid (BALF) by flow cytometry. The mice sensitized and challenged with OVA showed high expression of IL-25 in both mRNA and protein levels in lungs. We detected the expression of CD80 and CD86 in BALF was also increased. A tight correlation between IL-25 mRNA and other Th2 cells producing cytokines such as IL-4, IL-5, and IL-13 in BALF was identified. Furthermore, when the asthmatic mice were treated with inhaled corticosteroids, the inflammatory cells infiltration and the inflammatory cytokines secretion were significantly decreased. In this study, we show that IL-25 promoted the accumulation of co-stimulatory molecules of CD80 and CD86 on DCs and then induced the differentiation of prime naive CD4(+) T cells to become proinflammatory Th2 cells and promoted Th2 cytokine responses in OVA-induced airway inflammation. The ability of IL-25 to promote the activation and differentiation of DCs population was identified as a link between the IL-17 cytokine family and the innate immune response and suggested a previously unrecognized innate immune pathway that promotes Th2

  19. 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

  20. Simulation of micro-organisms swimming near ciliated surfaces

    NASA Astrophysics Data System (ADS)

    Shum, Henry; Tripathi, Anurag; Yeomans, Julia; Balazs, Anna

    2012-11-01

    Ciliated tissues can be found lining the respiratory tract and Fallopian tubes in mammals. The main function of the cilia is to sweep objects such as the ovum, dirt or bacteria in a directed manner. The self-cleaning action of these tissues would be a desirable property for surfaces that are continually submerged and prone to biofouling. We therefore investigate the effect of artificially driven cilia on swimming organisms. In this study we use a 3-D immersed boundary approach, with the fluid flow solved by the lattice Boltzmann method and the immersed objects modeled as elastic structures. Two types of objects are considered: (i) cilia, which are driven by an external field, and (ii) bacteria, which are self-motile and propelled by a rotating helical flagellum. Placing a bacterial cell in the vicinity of a surface covered by an array of actuated cilia yields a rich system to explore. Of particular interest is the possibility of guiding the motion of bacteria towards, along or away from the ciliated surface.

  1. Cryptophyte farming by symbiotic ciliate host detected in situ

    PubMed Central

    Qiu, Dajun; Huang, Liangmin; Lin, Senjie

    2016-01-01

    Protist–alga symbiosis is widespread in the ocean, but its characteristics and function in situ remain largely unexplored. Here we report the symbiosis of the ciliate Mesodinium rubrum with cryptophyte cells during a red-tide bloom in Long Island Sound. In contrast to the current notion that Mesodinium retains cryptophyte chloroplasts or organelles, our multiapproach analyses reveal that in this bloom the endosymbiotic Teleaulax amphioxeia cells were intact and expressing genes of membrane transporters, nucleus-to-cytoplasm RNA transporters, and all major metabolic pathways. Among the most highly expressed were ammonium transporters in both organisms, indicating cooperative acquisition of ammonium as a major N nutrient, and genes for photosynthesis and cell division in the cryptophyte, showing active population proliferation of the endosymbiont. We posit this as a “Mesodinium-farming-Teleaulax” relationship, a model of protist–alga symbiosis worth further investigation by metatranscriptomic technology. PMID:27791006

  2. 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.

  3. Pulmonary neuroendocrine cells and neuroepithelial bodies in sudden infant death syndrome: potential markers of airway chemoreceptor dysfunction.

    PubMed

    Cutz, Ernest; Perrin, Donald G; Pan, Jie; Haas, Elisabeth A; Krous, Henry F

    2007-01-01

    Pulmonary neuroendocrine cells (PNEC), including neuroepithelial bodies (NEB), are amine- and peptide (for example, bombesin)-producing cells that function as hypoxia/hypercapnia-sensitive chemoreceptors that could be involved in the pathophysiology of sudden infant death syndrome (SIDS). We assessed morphometrically the frequency and size of PNEC/NEB in lungs of infants who died of SIDS (n = 21) and compared them to an equal number PNEC/NEB in lungs of age-matched control infants who died of accidental death or homicide, with all cases obtained from the San Diego SIDS/SUDC Research Project database. As a marker for PNEC/NEB we used an antibody against chromogranin A (CGA), and computer-assisted morphometric analysis was employed to determine the relative frequency of PNEC per airway epithelial area (% immunostained area, %IMS), the size of NEB, the number of nuclei/NEB, and the size of the NEB cells. The lungs of SIDS infants showed significantly greater %IMS of airway epithelium (2.72 +/- 0.28 [standard error of the mean, SEM] versus 1.88 +/- 0.24; P < 0.05) and larger NEB (1557 +/- 153 microm(2) versus 1151 +/- 106 microm(2); P < 0.05) compared to control infants. The size of NEB cells was also significantly increased in SIDS cases compared to the controls (180 +/- 6.39 microm(2) versus 157 +/- 8.0 microm(2); P < 0.05), indicating the presence of hypertrophy in addition to hyperplasia. Our findings support previous studies demonstrating hyperplasia of PNEC/NEB in lungs of infants who died of SIDS. These changes could be secondary to chronic hypoxia and/or could be attributable to maturational delay. Morphometric assessment and/or measurement of the secretory products of these cells (for example, CGA, bombesin) could provide a potential biological marker for SIDS.

  4. 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.

  5. Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

    PubMed

    Zhao, Limin; Sullivan, Michelle N; Chase, Marlee; Gonzales, Albert L; Earley, Scott

    2014-06-01

    Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

  6. An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells

    PubMed Central

    Massoud, Amir Hossein; Charbonnier, Louis-Marie; Lopez, David; Pellegrini, Matteo; Phipatanakul, Wanda; Chatila, Talal A

    2016-01-01

    Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the interleukin-4 receptor alpha chain (IL4RA R576) promotes conversion of induced Treg (iTreg) cells towards a T helper 17 (TH17) cell fate. This skewing is mediated by the recruitment by IL-4Rα-R576 of the growth factor receptor-bound protein 2 (GRB2) adaptor protein, which drives IL-17 expression by activating a pathway involving extracellular signal-regulated kinase, IL-6 and STAT3. Treg cell-specific deletion of Il6ra or Rorc, but not Il4 or Il13, prevented exacerbated airway inflammation in Il4raR576 mice. Furthermore, treatment of Il4raR576 mice with a neutralizing anti-IL-6 antibody prevented iTreg cell reprogramming into TH17-like cells and protected against severe airway inflammation. These findings identify a novel mechanism for the development of mixed TH2-TH17 cell inflammation in genetically prone individuals, and point to interventions that stabilize iTreg cells as potentially effective therapeutic strategies. PMID:27479084

  7. 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.

  8. Activation of Store-Operated Calcium Entry in Airway Smooth Muscle Cells: Insight from a Mathematical Model

    PubMed Central

    Croisier, Huguette; Tan, Xiahui; Perez-Zoghbi, Jose F.; Sanderson, Michael J.; Sneyd, James; Brook, Bindi S.

    2013-01-01

    Intracellular dynamics of airway smooth muscle cells (ASMC) mediate ASMC contraction and proliferation, and thus play a key role in airway hyper-responsiveness (AHR) and remodelling in asthma. We evaluate the importance of store-operated entry (SOCE) in these dynamics by constructing a mathematical model of ASMC signaling based on experimental data from lung slices. The model confirms that SOCE is elicited upon sufficient depletion of the sarcoplasmic reticulum (SR), while receptor-operated entry (ROCE) is inhibited in such conditions. It also shows that SOCE can sustain agonist-induced oscillations in the absence of other influx. SOCE up-regulation may thus contribute to AHR by increasing the oscillation frequency that in turn regulates ASMC contraction. The model also provides an explanation for the failure of the SERCA pump blocker CPA to clamp the cytosolic of ASMC in lung slices, by showing that CPA is unable to maintain the SR empty of . This prediction is confirmed by experimental data from mouse lung slices, and strongly suggests that CPA only partially inhibits SERCA in ASMC. PMID:23936056

  9. 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.

  10. Adalimumab ameliorates OVA-induced airway inflammation in mice: Role of CD4(+) CD25(+) FOXP3(+) regulatory T-cells.

    PubMed

    Elsakkar, Mohamed G; Sharaki, Olla A; Abdallah, Dina M; Mostafa, Dalia K; Shekondali, Fadia T

    2016-09-05

    Asthma is a chronic inflammatory heterogeneous disorder initiated by a dysregulated immune response which drives disease development in susceptible individuals. Though T helper 2 (TH2) biased responses are usually linked to eosinophilic asthma, other Th cell subsets induce neutrophilic airway inflammation which provokes the most severe asthmatic phenotypes. A growing evidence highlights the role of T regulatory (Treg) cells in damping abnormal Th responses and thus inhibiting allergy and asthma. Therefore, strategies to induce or augment Treg cells hold promise for treatment and prevention of allergic airway inflammation. Recently, the link between Tumor necrosis factor-α (TNF-α) and Treg has been uncovered, and TNF-α antagonists are increasingly used in many autoimmune diseases. Yet, their benefits in allergic airway inflammation is not clarified. We investigated the effect of Adalimumab, a TNF-α antagonist, on Ovalbumin (OVA)-induced allergic airway inflammation in CD1 mice and explored its impact on Treg cells. Our results showed that Adalimumab treatment attenuated the OVA-induced increase in serum IgE, TH2 and TH1 derived inflammatory cytokines (IL-4 and IFN-γ, respectively) in bronchoalveolar lavage (BAL) fluid, suppressed recruitment of inflammatory cells in BAL fluid and lung, and inhibited BAL fluid neutrophilia. It also ameliorated goblet cell metaplasia and bronchial fibrosis. Splenocytes flow cytometry revealed increased percentage of CD4(+) CD25(+) FOXP3(+) Treg cells by Adalimumab that was associated with increase in their suppressive activity as shown by elevated BAL fluid IL-10. We conclude that the beneficial effects of Adalimumab in this CD1 neutrophilic model of allergic airway inflammation are attributed to augmentation of Treg cell number and activity.

  11. 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.

  12. Broncho-Vaxom Attenuates Allergic Airway Inflammation by Restoring GSK3β-Related T Regulatory Cell Insufficiency

    PubMed Central

    Zhong, Hua; Yu, Dehong; Zeng, Xianping; Deng, Mengxia; Sun, Yueqi; Wen, Weiping; Li, Huabin

    2014-01-01

    Background Oral administration of bacterial extracts (eg, Broncho-Vaxom (BV)) has been proposed to attenuate asthma through modulating Treg cells. However, the underlying mechanism has not been fully characterized. This study sought to assess the effects of oral administration of BV on GSK-3β expression and Treg cells in ovalbumin (OVA)-induced asthmatic mice models. Method Asthmatic mice models were established with OVA challenge and treated with oral administration of BV. Next, infiltration of inflammatory cells including eosinophil and neutrophils, mucous metaplasia, levels of Th1/Th2/Treg-typed cytokines and expression of GSK3β and Foxp3 were examined in asthmatic mice models by histological analysis, Bio-Plex and western blot, respectively. Moreover, the frequencies of Treg cells were evaluated in cultured splenocytes by flow cytometry in the presence of BV or GSK3β siRNA interference. Results We found significant decrease of infiltrated inflammatory cells in bronchoalveolar lavage fluid (BALF) in asthmatic mice models after oral administration of BV. Oral administration of BV was shown to significantly suppress mucus metaplasia, Th2-typed cytokine levels and GSK3β expression while increasing Foxp3 production in asthmatic mice models. Moreover, BV significantly enhanced GSK3β-related expansion of Treg cells in cultured spleen cells in vitro. Conclusion Our findings provide evidence that oral administration of BV is capable of attenuating airway inflammation in asthmatic mice models, which may be associated with GSK3β-related expansion of Treg cells. PMID:24667347

  13. 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

  14. 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

  15. 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

  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. Evaluation of double formalin--Lugol's fixation in assessing number and biomass of ciliates: an example of estimations at mesoscale in NE Atlantic.

    PubMed

    Karayanni, Hera; Christaki, Urania; Van Wambeke, France; Dalby, Andrew P

    2004-03-01

    Ciliated protozoa are potential grazers of primary and bacterial production and act as intermediaries between picoplankton and copepods and other large suspension feeders. Accurate determination of ciliate abundance and feeding mode is crucial in oceanic carbon budget estimations. However, the impact of different fixatives on the abundance and cell volume of ciliates has been investigated in only a few studies using either laboratory cultures or natural populations. Lugol's solution and formalin are the most commonly used fixatives for the preservation of ciliates samples. In the present study, the aim was to compare 0.4% Lugol's solution and 2% borated-formalin fixation and evaluate the need of counting duplicate samples each using a different fixative. For this, a large number of samples (n = 110) from the NE Atlantic was analyzed in the frame of POMME program (Multidisciplinary Mesoscale Ocean Program). We established a statistically significant relationship (p < 0.0001) between Lugol's and formalin fixed samples for both abundance (r2 = 0.50) and biomass (r2 = 0.76) of aloricate ciliates which showed that counts were higher in Lugol's solution by a factor of 2 and a non-taxon specific cell-loss in formalin. However, loricate ciliate abundance in our samples which were represented primarily by Tintinnus spp. did not show any difference between the two treatments. Abundance and biomass of mixotrophic ciliates (chloroplast-bearing cells) were for various reasons underestimated in both treatments. Our results show that unique fixation by formalin may severely underestimate ciliates abundance and biomass although their population may not alter. For this reason, Lugol's solution is best for the estimation of their abundance and biomass. However, for counts of mixotrophs and the evaluation of the ecological role of ciliates in carbon flux, double fixation is essential. Compromises regarding the fixatives have lead to severe underestimations of mixotrophs in studies

  18. Respiratory virus infection up-regulates TRPV1, TRPA1 and ASICS3 receptors on airway cells.

    PubMed

    Omar, Shadia; Clarke, Rebecca; Abdullah, Haniah; Brady, Clare; Corry, John; Winter, Hanagh; Touzelet, Olivier; Power, Ultan F; Lundy, Fionnuala; McGarvey, Lorcan P A; Cosby, S Louise

    2017-01-01

    Receptors implicated in cough hypersensitivity are transient receptor potential vanilloid 1 (TRPV1), transient receptor potential cation channel, Subfamily A, Member 1 (TRPA1) and acid sensing ion channel receptor 3 (ASIC3). Respiratory viruses, such as respiratory syncytial virus (RSV) and measles virus (MV) may interact directly and/or indirectly with these receptors on sensory nerves and epithelial cells in the airways. We used in vitro models of sensory neurones (SHSY5Y or differentiated IMR-32 cells) and human bronchial epithelium (BEAS-2B cells) as well as primary human bronchial epithelial cells (PBEC) to study the effect of MV and RSV infection on receptor expression. Receptor mRNA and protein levels were examined by qPCR and flow cytometry, respectively, following infection or treatment with UV inactivated virus, virus-induced soluble factors or pelleted virus. Concentrations of a range of cytokines in resultant BEAS-2B and PBEC supernatants were determined by ELISA. Up-regulation of TRPV1, TRPA1 and ASICS3 expression occurred by 12 hours post-infection in each cell type. This was independent of replicating virus, within the same cell, as virus-induced soluble factors alone were sufficient to increase channel expression. IL-8 and IL-6 increased in infected cell supernatants. Antibodies against these factors inhibited TRP receptor up-regulation. Capsazepine treatment inhibited virus induced up-regulation of TRPV1 indicating that these receptors are targets for treating virus-induced cough.

  19. Respiratory virus infection up-regulates TRPV1, TRPA1 and ASICS3 receptors on airway cells

    PubMed Central

    Omar, Shadia; Clarke, Rebecca; Abdullah, Haniah; Brady, Clare; Corry, John; Winter, Hanagh; Touzelet, Olivier; Power, Ultan F.; Lundy, Fionnuala; McGarvey, Lorcan P. A.

    2017-01-01

    Receptors implicated in cough hypersensitivity are transient receptor potential vanilloid 1 (TRPV1), transient receptor potential cation channel, Subfamily A, Member 1 (TRPA1) and acid sensing ion channel receptor 3 (ASIC3). Respiratory viruses, such as respiratory syncytial virus (RSV) and measles virus (MV) may interact directly and/or indirectly with these receptors on sensory nerves and epithelial cells in the airways. We used in vitro models of sensory neurones (SHSY5Y or differentiated IMR-32 cells) and human bronchial epithelium (BEAS-2B cells) as well as primary human bronchial epithelial cells (PBEC) to study the effect of MV and RSV infection on receptor expression. Receptor mRNA and protein levels were examined by qPCR and flow cytometry, respectively, following infection or treatment with UV inactivated virus, virus-induced soluble factors or pelleted virus. Concentrations of a range of cytokines in resultant BEAS-2B and PBEC supernatants were determined by ELISA. Up-regulation of TRPV1, TRPA1 and ASICS3 expression occurred by 12 hours post-infection in each cell type. This was independent of replicating virus, within the same cell, as virus-induced soluble factors alone were sufficient to increase channel expression. IL-8 and IL-6 increased in infected cell supernatants. Antibodies against these factors inhibited TRP receptor up-regulation. Capsazepine treatment inhibited virus induced up-regulation of TRPV1 indicating that these receptors are targets for treating virus-induced cough. PMID:28187208

  20. Ciliated Protozoa in marine pollution studies. A conspectus

    SciTech Connect

    Parker, J.G.

    1983-04-01

    The ecological role of the marine ciliates is discussed with reference to the interaction of this group with contaminants. While the ciliates may be of some value in routine screening of potential toxins, simple laboratory bioassays are unlikely to be of predictive value in assessing threats to the marine environment. There is some evidence that ciliates may take part in the transfer and transformation of potential pollutants. Further field and laboratory studies are required in order to identify and quantify the role of ciliates in these processes.

  1. Biodiversity patterns of soil ciliates along salinity gradients.

    PubMed

    Zhao, Feng; Xu, Kuidong

    2016-04-01

    We evaluated ciliate diversity in saline soils with a salinity range from 6.5 to 65 psu by the morphological method of the Ludox-quantitative protargol stain (QPS) and the molecular techniques of ciliate-specific clone library and denaturing gradient gel electrophoresis. No active ciliates could be detected with the Ludox-QPS method, while high molecular diversity of ciliates was found. The highest ciliate molecular diversity was obtained from the soil at salinity of 8.9 psu, moderate diversity was found at salinity of 6.5 psu, and the diversity sharply decreased at salinity of 50.5 psu. By contrast, the number of ciliate classes clearly decreased with increasing soil salinity: six, five, four and two classes from sites with salinity of 6.5 psu, 8.9 psu, 29.5 psu and 50.5 psu, respectively. Ciliate diversity pattern is different from that of bacteria, whose diversity is also high in extremely saline environments. Meanwhile, the composition of ciliate community was significantly different along salinity gradient. Colpodea and Oligohymenophorea were diverse in soils at salinity less than 29.5 psu, while absent in soils with salinity above 50.5 psu. BIOENV analysis indicated soil salinity and water content were the main factors regulating the distribution of ciliates in saline soils.

  2. Inhibition of Human Metapneumovirus Binding to Heparan Sulfate Blocks Infection in Human Lung Cells and Airway Tissues

    PubMed Central

    Klimyte, Edita M.; Smith, Stacy E.; Oreste, Pasqua; Lembo, David

    2016-01-01

    ABSTRACT Human metapneumovirus (HMPV), a recently discovered paramyxovirus, infects nearly 100% of the world population and causes severe respiratory disease in infants, the elderly, and immunocompromised patients. We previously showed that HMPV binds heparan sulfate proteoglycans (HSPGs) and that HMPV binding requires only the viral fusion (F) protein. To characterize the features of this interaction critical for HMPV binding and the role of this interaction in infection in relevant models, we utilized sulfated polysaccharides, heparan sulfate mimetics, and occluding compounds. Iota-carrageenan demonstrated potent anti-HMPV activity by inhibiting binding to lung cells mediated by the F protein. Furthermore, analysis of a minilibrary of variably sulfated derivatives of Escherichia coli K5 polysaccharide mimicking the HS structure revealed that the highly O-sulfated K5 polysaccharides inhibited HMPV infection, identifying a potential feature of HS critical for HMPV binding. The peptide dendrimer SB105-A10, which binds HS, reduced binding and infection in an F-dependent manner, suggesting that occlusion of HS at the target cell surface is sufficient to prevent infection. HMPV infection was also inhibited by these compounds during apical infection of polarized airway tissues, suggesting that these interactions take place during HMPV infection in a physiologically relevant model. These results reveal key features of the interaction between HMPV and HS, supporting the hypothesis that apical HS in the airway serves as a binding factor during infection, and HS modulating compounds may serve as a platform for potential antiviral development. IMPORTANCE Human metapneumovirus (HMPV) is a paramyxovirus that causes respiratory disease worldwide. It has been previously shown that HMPV requires binding to heparan sulfate on the surfaces of target cells for attachment and infection. In this study, we characterize the key features of this binding interaction using heparan sulfate

  3. Divergent pro-inflammatory profile of human dendritic cells in response to commensal and pathogenic bacteria associated with the airway microbiota.

    PubMed

    Larsen, Jeppe Madura; Steen-Jensen, Daniel Bisgaard; Laursen, Janne Marie; Søndergaard, Jonas Nørskov; Musavian, Hanieh Sadat; Butt, Tariq Mahmood; Brix, Susanne

    2012-01-01

    Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties of individual bacterial species are unknown. In this study, we compared the immune stimulatory capacity on human monocyte-derived dendritic cells (DCs) of selected airway commensal and pathogenic bacteria predominantly associated with lungs of asthma or COPD patients (pathogenic Haemophillus spp. and Moraxella spp.), healthy lungs (commensal Prevotella spp.) or both (commensal Veillonella spp. and Actinomyces spp.). All bacteria were found to induce activation of DCs as demonstrated by similar induction of CD83, CD40 and CD86 surface expression. However, asthma and COPD-associated pathogenic bacteria provoked a 3-5 fold higher production of IL-23, IL-12p70 and IL-10 cytokines compared to the commensal bacteria. Based on the differential cytokine production profiles, the studied airway bacteria could be segregated into three groups (Haemophilus spp. and Moraxella spp. vs. Prevotella spp. and Veillonella spp. vs. Actinomyces spp.) reflecting their pro-inflammatory effects on DCs. Co-culture experiments found that Prevotella spp. were able to reduce Haemophillus influenzae-induced IL-12p70 in DCs, whereas no effect was observed on IL-23 and IL-10 production. This study demonstrates intrinsic differences in DC stimulating properties of bacteria associated with the airway microbiota.

  4. 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...

  5. 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

  6. Airway smooth muscle growth in asthma: proliferation, hypertrophy, and migration.

    PubMed

    Bentley, J Kelley; Hershenson, Marc B

    2008-01-01

    Increased airway smooth muscle mass is present in fatal and non-fatal asthma. However, little information is available regarding the cellular mechanism (i.e., hyperplasia vs. hypertrophy). Even less information exists regarding the functional consequences of airway smooth muscle remodeling. It would appear that increased airway smooth muscle mass would tend to increase airway narrowing and airflow obstruction. However, the precise effects of increased airway smooth muscle mass on airway narrowing are not known. This review will consider the evidence for airway smooth muscle cell proliferation and hypertrophy in asthma, potential functional effects, and biochemical mechanisms.

  7. 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

  8. 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