Phenotypic Responses of Differentiated Asthmatic Human Airway Epithelial Cultures to Rhinovirus

ObjectivesHuman airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.MethodsAir-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.ConclusionsALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.     

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.     

Human Metapneumovirus Glycoprotein G Disrupts Mitochondrial Signaling in Airway Epithelial Cells

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.     

Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H₂O₂ and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H₂O₂ formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.     

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

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

Regulation of TLR2 Expression and Function in Human Airway Epithelial Cells

Toll-like receptor (TLR1–6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM₃CSK₄. Pretreatment with anti-TLR2 blocking antibody inhibited the PAM₃CSK₄-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM₃CSK₄), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.     

Human Rhinovirus Induced Cytokine/Chemokine Responses in Human Airway Epithelial and Immune Cells

Infections with human rhinovirus (HRV) are commonly associated with acute upper and lower respiratory tract disease and asthma exacerbations. The role that HRVs play in these diseases suggests it is important to understand host-specific or virus-specific factors that contribute to pathogenesis. Since species A HRVs are often associated with more serious HRV disease than species B HRVs, differences in immune responses they induce should inform disease pathogenesis. To identify species differences in induced responses, we evaluated 3 species A viruses, HRV 25, 31 and 36 and 3 species B viruses, HRV 4, 35 and 48 by exposing human PBMCs to HRV infected Calu-3 cells. To evaluate the potential effect of memory induced by previous HRV infection on study responses, we tested cord blood mononuclear cells that should be HRV naïve. There were HRV-associated increases (significant increase compared to mock-infected cells) for one or more HRVs for IP-10 and IL-15 that was unaffected by addition of PBMCs, for MIP-1α, MIP-1β, IFN-α, and HGF only with addition of PBMCs, and for ENA-78 only without addition of PBMCs. All three species B HRVs induced higher levels, compared to A HRVs, of MIP-1α and MIP-1β with PBMCs and ENA-78 without PBMCs. In contrast, addition of CBMCs had less effect and did not induce MIP-1α, MIP-1β, or IFN-α nor block ENA-78 production. Addition of CBMCs did, however, increase IP-10 levels for HRV 35 and HRV 36 infection. The presence of an effect with PBMCs and no effect with CBMCs for some responses suggest differences between the two types of cells possibly because of the presence of HRV memory responses in PBMCs and not CBMCs or limited response capacity for the immature CBMCs relative to PBMCs. Thus, our results indicate that different HRV strains can induce different patterns of cytokines and chemokines; some of these differences may be due to differences in memory responses induced by past HRV infections, and other differences related to virus factors that can inform disease pathogenesis.     

LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser⁷³⁷ in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl⁻ secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser⁷³⁷ mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl⁻ channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl⁻ channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.     

Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63⁺ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.     

气道上皮细胞在哮喘中的作用

随着现代医学的发展,人们对支气管哮喘发病机制的研究有了进一步发展.支气管哮喘(简称哮喘)是一种由多种细胞,多种细胞因子参与形成的慢性气道炎症性疾病.支气管上皮细胞是气道结构细胞,它是抵抗外界损伤因素的第一道防线,当吸人性刺激物质时,首先激化支气管上皮细胞并破坏支气管上皮细胞的正常结构和生理功能,在应激状态下的上皮细胞通过分泌炎性介质与自身细胞或其他气道结构细胞、炎性细胞、抗原递呈细胞等相互作用,积极参与哮喘的气道慢性炎症发生与发展进程.因此气道上皮损伤是影响哮喘发生发展的重要因素,阐明维持气道上皮正常结构和功能的分子机制是目前防治哮喘的重要课题.本文综述气道上皮在哮喘发生发展中的作用及相关机制研究进展.     

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

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.     

Phospholipase C-activating Plasma Membrane Receptors and Calcium Signaling in Immortalized Human Airway Epithelial Cells

Mechanical clearance of inhaled dust particles and microorganisms is an important part of the innate defense mechanisms of mammalian airways. Airway epithelia are composed of various cell types with different degrees of cell polarity. Serous cells regulate composition and volume of luminal periciliary fluid and mucus. Autocrine, paracrine, or neuronal messengers determine the secretory and reabsorptive rates of electrolytes and water via cAMP-or inositol triphosphate/calcium-mediated intracellular signals. Comparison of the expression of calcium-mobilizing receptor types (G protein-coupled-, growth factor-, and cytokine receptors) in two types of human immortalized airway epithelial cells (S9, 16HBE14o-) revealed that receptor populations were qualitatively and quantitatively different in the two cell types. Sustained calcium signals were elicited by activation of purinergic receptors in 16HBE14o-cells or muscarinic acetylcholine or histamine receptors in S9 cells. These G protein-coupled receptors mobilized calcium from intracellular stores and activated capacitative calcium influx. The experimental cells may represent different types of original airway epithelial cells and seem to be suited as model cells to study cell signaling and protein expression during interaction with pathogens or their secretory products (e.g., virulence factors).     

Electronic Cigarette Liquid Increases Inflammation and Virus Infection in Primary Human Airway Epithelial Cells

Background/Objective

The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection.

Methodology/Main Results

We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.     

Normal CFTR Inhibits Epidermal Growth Factor Receptor-Dependent Pro-Inflammatory Chemokine Production in Human Airway Epithelial Cells

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis, a disease characterized by exaggerated airway epithelial production of the neutrophil chemokine interleukin (IL)-8, which results in exuberant neutrophilic inflammation. Because activation of an epidermal growth factor receptor (EGFR) signaling cascade induces airway epithelial IL-8 production, we hypothesized that normal CFTR suppresses EGFR-dependent IL-8 production and that loss of CFTR at the surface exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade. We examined this hypothesis in human airway epithelial (NCI-H292) cells and in normal human bronchial epithelial (NHBE) cells containing normal CFTR treated with a CFTR-selective inhibitor (CFTR-172), and in human airway epithelial (IB3) cells containing mutant CFTR versus isogenic (C38) cells containing wild-type CFTR. In NCI-H292 cells, CFTR-172 induced IL-8 production EGFR-dependently. Pretreatment with an EGFR neutralizing antibody or the metalloprotease TACE inhibitor TAPI-1, or TACE siRNA knockdown prevented CFTR-172-induced EGFR phosphorylation (EGFR-P) and IL-8 production, implicating TACE-dependent EGFR pro-ligand cleavage in these responses. Pretreatment with neutralizing antibodies to IL-1R or to IL-1alpha, but not to IL-1beta, markedly suppressed CFTR-172-induced EGFR-P and IL-8 production, suggesting that binding of IL-1alpha to IL-1R stimulates a TACE-EGFR-IL-8 cascade. Similarly, in NHBE cells, CFTR-172 increased IL-8 production EGFR-, TACE-, and IL-1alpha/IL-1R-dependently. In IB3 cells, constitutive IL-8 production was markedly increased compared to C38 cells. EGFR-P was increased in IB3 cells compared to C38 cells, and exaggerated IL-8 production in the IB3 cells was EGFR-dependent. Activation of TACE and binding of IL-1alpha to IL-1R contributed to EGFR-P and IL-8 production in IB3 cells but not in C38 cells. Thus, we conclude that normal CFTR suppresses airway epithelial IL-8 production that occurs via a stimulatory EGFR cascade, and that loss of normal CFTR activity exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade.     

Dexamethasone Inhibits Repair of Human Airway Epithelial Cells Mediated by Glucocorticoid-Induced Leucine Zipper (GILZ)

Background

Glucocorticoids (GCs) are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ) is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK)-extracellular-signal-regulated kinase (ERK) signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX) is mediated by GILZ.

Methods

We tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays.

Results

DEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway), proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced.

Conclusions

The inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs.