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.     

Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.     

IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.     

b型流感嗜血杆菌结合疫苗促进人气道上皮细胞hBD-2表达

目的:检验b型流感嗜血杆菌结合疫苗在体外能否刺激人气道上皮细胞hBD-2表达,并探讨人工诱导防御素表达的方法.方法:体外分离、培养原代人气道上皮细胞.b型流感嗜血杆菌疫苗刺激原代人气道上皮细胞后,用RT-PCR和ELISA法检测培养上清液中hBD-2蛋白的表达.收集刺激后的细胞培养上清液进行抑菌实验.结果:浓度＞1 μg/ml的b型流感嗜血杆菌结合疫苗刺激原代人气道上皮细胞12h后,可诱导hBD-2 mRNA的表达,与对照组相比较有显著差异(P＜0.01),无剂量依赖性,同时细胞培养上清液中hBD-2蛋白的表达上调.1μg/mlb型流感嗜血杆菌结合疫苗浓缩后的细胞培养上清液具有抑菌作用.结论:一定浓度(1μg/mL)以上的b型流感嗜血杆菌结合疫苗作用于人原代气道上皮细胞可诱导hBD-2的表达,为人工方法刺激机体产生人β防御素-2,对抗病原菌感染提供新的策略.     

KCa3.1 K+ Channel Expression and Function in Human Bronchial Epithelial Cells

The K_Ca3.1 K⁺ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the K_Ca3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed K_Ca3.1 mRNA and protein, and robust K_Ca3.1 ion currents. K_Ca3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and K_Ca3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective K_Ca3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by K_Ca3.1 blockade. Treatment with K_Ca3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT.     

Arsenite-Induced Pseudo-Hypoxia Results in Loss of Anchorage-Dependent Growth in BEAS-2B Pulmonary Epithelial Cells

Epidemiology studies have established a strong link between lung cancer and arsenic exposure. Currently, the role of disturbed cellular energy metabolism in carcinogenesis is a focus of scientific interest. Hypoxia inducible factor-1 alpha (HIF-1A) is a key regulator of energy metabolism, and it has been found to accumulate during arsenite exposure under oxygen-replete conditions. We modeled arsenic-exposed human pulmonary epithelial cells in vitro with BEAS-2B, a non-malignant lung epithelial cell line. Constant exposure to 1 µM arsenite (As) resulted in the early loss of anchorage-dependent growth, measured by soft agar colony formation, beginning at 6 weeks of exposure. This arsenite exposure resulted in HIF-1A accumulation and increased glycolysis, similar to the physiologic response to hypoxia, but in this case under oxygen-replete conditions. This “pseudo-hypoxia” response was necessary for the maximal acquisition of anchorage-independent growth in arsenite-exposed BEAS-2B. The HIF-1A accumulation and induction in glycolysis was sustained throughout a 52 week course of arsenite exposure in BEAS-2B. There was a time-dependent increase in anchorage-independent growth during the exposure to arsenite. When HIF-1A expression was stably suppressed, arsenite-induced glycolysis was abrogated, and the anchorage-independent growth was reduced. These findings establish that arsenite exerts a hypoxia-mimetic effect, which plays an important role in the subsequent gain of malignancy-associated phenotypes.     

Expression of an Antigen Associated with Basal Bodies of Human Ciliated Epithelial Cells

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000–45,000Mr protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9+0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.     

Global Gene Expression Analysis of Human Bronchial Epithelial Cells Treated with Tobacco Condensates

Gene expression patterns were assessed in normal human bronchial epithelial (NHBE) cells exposed to cigarette smoke condensates (CSC) from commercial cigarettes in order to develop a better understanding of the genomic impact of tobacco exposure, and to define biomarkers that can potentially discriminate tobacco-related effects and outcomes in a clinical setting.

NHBE cells were treated with CSCs from two American brands for up to 12 hours in the presence of S9 microsomal fraction from Aroclor 1254-treated rats. High-density oligonucleotide microarrays coupled with a novel statistical analysis that relies on statistical significance levels rather than arbitrary fold-change differences was used to identify genes that undergo expression alterations upon treatment.

Expression patterns of approximately 3700 genes were altered after CSC treatments. While a majority of these genes were affected by both CSCs, each condensate also affected a unique subset of ~1000 genes. An unexpected finding was that S9, required for metabolizing procarcinogens in CSCs to carcinogenic metabolites, also altered the expression of approximately 1700 genes.

Exposure of NHBE cells to different CSCs alters the expression of a large set of genes that affect a common set of biological pathways including those relevant to carcinogenesis. Identification of CSC-affected genes and underlying biological processes may generate an atlas of molecular events that includes biomarkers of tobacco exposure and disease status in smokers. Finally, the finding that S9 affects the expression of a number of genes may have implications for various toxicogenetic assays currently used by regulatory agencies to evaluate harmful effects in exposed humans.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Perilla frutescens Leaf Extract Inhibits Mite Major Allergen Der p 2-induced Gene Expression of Pro-Allergic and Pro-Inflammatory Cytokines in Human Bronchial Epithelial Cell BEAS-2B

Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens.     

Loss of PI(4,5)P2 5-Phosphatase A Contributes to Resistance of Human Melanoma Cells to RAF/MEK Inhibitors

Past studies have shown that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase (PIB5PA), is commonly downregulated or lost in melanomas, which contributes to elevated activation of phosphatidylinositol 3-kinase (PI3K)/Akt in melanoma cells. In this report, we provide evidence that PIB5PA deficiency plays a role in resistance of melanoma cells to RAF/mitogen-activated protein kinase kinase (MEK) inhibitors. Ectopic expression of PIB5PA enhanced apoptosis induced by the RAF inhibitor PLX4720 in BRAF^V600E and by the MEK inhibitor U0126 in both BRAF^V600E and wild-type BRAF melanoma cells. This was due to inhibition of PI3K/Akt, as co-introduction of an active form of Akt (myr-Akt) abolished the effect of overexpression of PIB5PA on apoptosis induced by PLX4720 or U0126. While overexpression of PIB5PA triggered activation of Bad and down-regulation of Mcl-1, knockdown of Bad or overexpression of Mcl-1 recapitulated, at least in part, the effect of myr-Akt, suggesting that regulation of Bad and Mcl-1 is involved in PIB5PA-mediated sensitization of melanoma cells to the inhibitors. The role of PIB5PA deficiency in BRAF inhibitor resistance was confirmed by knockdown of PIB5PA, which led to increased growth of BRAF^V600E melanoma cells selected for resistance to PLX4720. Consistent with its role in vitro, overexpression of PIB5PA and the MEK inhibitor selumetinib cooperatively inhibited melanoma tumor growth in a xenograft model. Taken together, these results identify loss of PIB5PA as a novel resistance mechanism of melanoma to RAF/MEK inhibitors and suggest that restoration of PIB5PA may be a useful strategy to improve the therapeutic efficacy of the inhibitors in the treatment of melanoma.     

Cytopathogenesis of Sendai Virus in Well-Differentiated Primary Pediatric Bronchial Epithelial Cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.Sendai virus (SeV) is a nonsegmented negative-strand RNA virus of the Paramyxoviridae family. Recombinant SeV (rSeV) has been extensively studied as a vector for vaccines, cancer immunotherapy, and gene therapy (14, 22, 34, 41, 43). SeV is virulent in rodents, but despite extensive antigenic and genetic similarity to human parainfluenza virus type 1 (hPIV1), it is not known to cause disease in humans (33). Interest in rSeV as a vector is exemplified by the fact that (i) its genome can easily be manipulated to stably express heterologous genes (9), (ii) it does not undergo homologous recombination, (iii) cell transduction is independent of the cell cycle, (iv) there is no DNA phase during replication and hence no possibility of cell transformation, and (v) its cell or tissue tropisms and replication competency may be modulated by reverse genetics and appropriate rescue systems (5, 8).Much of the research on rSeV as a vector involves monolayer cells and animal models and employs both replication-competent and transmission-incompetent viruses. In view of its respiratory tract tropism, particular attention was paid to its use as a gene therapy vector for lung diseases such as cystic fibrosis (CF) (2, 13, 14, 43). Indeed, early studies demonstrated its capacity to efficiently overcome a series of extra- and intracellular barriers in the respiratory tract, such as the glycocalyx, mucus layer, mucociliary clearance, and cell membranes (13, 14, 43). However, in vivo studies demonstrated that rSeV-mediated gene transduction was transient (lasting ∼7 days) and that repeated administration was inefficient (16). The reasons for this transient transduction remain unclear.In contrast, the capacity to efficiently and transiently transduce host cells is of considerable interest from a vaccine vector viewpoint. Indeed, promising rSeV-vectored vaccine candidates have been described for other respiratory viruses, such as respiratory syncytial virus (RSV), hPIV1, hPIV2, hPIV3, and systemic viruses, such as HIV-1 (22, 40, 44). Despite its considerable promise as a viral vector, little is known about how rSeV interacts with human airway epithelial cells (HAE).To address this, we established an ex vivo/in vitro model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs) in air-liquid interface (ALI) cultures. The pediatric origin of the primary bronchial cells allowed us to investigate SeV-host interactions in a pediatric context. The need for CF gene therapy or respiratory virus vaccines for infants or children emphasizes the relevance of this ex vivo/in vitro pediatric model. Using rSeV expressing enhanced GFP (rSeV/eGFP), we comprehensively investigated the consequences of SeV infection in these cultures, including the types of cells infected, virus growth kinetics, cytopathic effects (CPE), and inflammatory responses. Our data provided novel insights into the interaction of SeV with pediatric airway epithelium and the limitations and/or advantages of its use as a vector.     

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to th...