Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface.

Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.

Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells.

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

Cyclophilin D Contributes to Airway Epithelial Mitochondrial Damage in Chronic Obstructive Pulmonary Disease.

INTRODUCTION: Airway epithelial mitochondrial injury is an important pathogenesis of chronic obstructive pulmonary disease (COPD). Cyclophilin D (CypD) is a component of mitochondrial permeability transition pore and related to mitochondrial damage. However, the role of CypD in airway epithelial mitochondrial injury and COPD pathogenesis remains unclear. METHODS: CypD expression in human airway epithelium was determined by immunohistochemistry, and mitochondrial structure of airway epithelial cell was observed under the transmission electron microscopy. The expression of CypD signaling pathway in cigarette smoke extract (CSE)-treated airway epithelial cells was measured by real-time PCR and Western-blot. CSE-induced damage of airway epithelial cell and mitochondria was further studied. RESULTS: Immunohistochemistry and transmission electron microscopy analysis revealed that CypD expression in airway epithelium was significantly increased associated with notable airway epithelial mitochondrial structure damage in the patients with COPD. The mRNA and protein expression of CypD was significantly increased in concentration- and time-dependent manners when airway epithelial cells were treated with CSE. CypD siRNA pretreatment significantly suppressed the increases of CypD and Bax expression, and reduced the decline of Bcl-2 expression in 7.5% CSE-treated airway epithelial cells. Furthermore, CypD silencing significantly attenuated mitochondrial damage and cell apoptosis, and increased cell viability when airway epithelial cells were stimulated with 7.5% CSE. CONCLUSION: These data suggest that CypD signaling pathway is involved in the pathogenesis of COPD and provide a potential therapeutic target for COPD.

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

Chitinase 3-like 1 is involved in the induction of IL-8 expression by double-stranded RNA in airway epithelial cells.

Viral respiratory infection causes inflammatory lung disease. Chitinase 3-like 1 (CHI3L1) contributes to airway inflammation, but its role in human airway epithelial cells following viral infection is unclear. Thus, we investigated whether CHI3L1 regulates inflammatory responses caused by viral infections in airway epithelial cells. Human bronchial epithelial cells, BEAS-2B, were stimulated with a synthetic analog of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly(I:C)). To confirm the specific role of CHI3L1, CHI3L1 was knocked down in BEAS-2B cells using shRNA lentivirus. The expression of CHI3L1 and proinflammatory cytokines such as IL-8 and phosphorylation of mitogen-activated protein kinase (MAPK) pathways were analyzed. In addition to poly(I:C), BEAS-2B cells were infected with the human respiratory syncytial virus (RSV) A2 strain, and CHI3L1 and IL-8 expression was analyzed. Stimulating the cells with poly(I:C) increased CHI3L1 and IL-8 expression, whereas IL-8 expression was abrogated in CHI3L1 knockdown BEAS-2B cells. Poly(I:C) stimulation of BEAS-2B cells resulted in phosphorylation of MAPK pathways, and inhibition of MAPK pathways significantly abolished IL-8 secretion. Phosphorylation of MAPK pathways was diminished in CHI3L1 knockdown BEAS-2B cells. Infection with RSV increased CHI3L1 and IL-8 expression. IL-8 expression induced by RSV infection was abrogated in CHI3L1 knockdown cells. In conclusion, CHI3L1 may be involved in IL-8 secretion by regulating MAPK pathways during respiratory viral infections in airway epithelial cells.

Propylene glycol, a component of electronic cigarette liquid, damages epithelial cells in human small airways.

BACKGROUND: Electronic cigarettes (e-cigarettes) are used worldwide as a substitute for conventional cigarettes. Although they are primarily intended to support smoking cessation, e-cigarettes have been identified as a gateway to smoking habits for young people. Multiple recent reports have described the health effects of inhaling e-cigarettes. E-cigarette liquid (e-liquid) is mainly composed of propylene glycol (PG) and glycerol (Gly), and the aerosol generated by these devices primarily contains these two components. Thus, this study aimed to evaluate the effects of PG and Gly on human small airway epithelial cells (SAECs). METHODS: SAECs were exposed to PG or Gly, and cell proliferation, cell viability, lactate dehydrogenase (LDH) release, DNA damage, cell cycle, and apoptosis were evaluated. Additionally, SAECs derived from chronic obstructive pulmonary disease (COPD) patients (COPD-SAECs) were investigated. RESULTS: Exposure of SAECs to PG significantly inhibited proliferation (1%, PG, p = 0.021; 2-4% PG, p < 0.0001) and decreased cell viability (1-4% PG, p < 0.0001) in a concentration-dependent manner. Gly elicited similar effects but to a reduced degree as compared to the same concentration of PG. PG also increased LDH release in a concentration-dependent manner (3% PG, p = 0.0055; 4% PG, p < 0.0001), whereas Gly did not show a significant effect on LDH release. SAECs exposed to 4% PG contained more cells that were positive for phosphorylated histone H2AX (p < 0.0001), a marker of DNA damage, and an increased proportion of cells in the G1 phase (p < 0.0001) and increased p21 expression (p = 0.0005). Moreover, caspase 3/7-activated cells and cleaved poly (ADP-ribose) polymerase 1 expression were increased in SAECs exposed to 4% PG (p = 0.0054). Furthermore, comparing COPD-SAECs to SAECs without COPD in PG exposure, cell proliferation, cell viability, DNA damage and apoptosis were significantly greater in COPD-SAECs. CONCLUSION: PG damaged SAECs more than Gly. In addition, COPD-SAECs were more susceptible to PG than SAECs without COPD. Usage of e-cigarettes may be harmful to the respiratory system, especially in patients with COPD.

ITGB4 deficiency induces DNA damage by downregulating HDAC1 in airway epithelial cells under stress stimulation.

BACKGROUND: DNA damage in airway epithelia under exogenous disruptors can trigger various pulmonary diseases. Integrin beta 4 (ITGB4) is a structural adhesion molecule, which is indicated to regulate the process of DNA damage in airway epithelia for its unique long cytoplasmic domain subunit. METHODS: The expression level of ITGB4 and the degree of DNA damage were observed in the house dust mite (HDM)-stressed model and ozone-challenged model, respectively. Besides, ITGB4 conditional knockout mice and ITGB4-deficient airway epithelial cells were constructed to observe the influence of ITGB4 deficiency on DNA damage. Furthermore, the influence of ITGB4 deficiency on HDAC1 expression in airway epithelia was determined under stress stimulation. Finally, corresponding intervention strategies were carried out to verify the involvement of the ITGB4-mediated HDAC1 pathway in DNA damage of airway epithelial cells. RESULTS: HDM stress and ozone challenge reduced the expression of ITGB4, which is accompanied by the increased expression of 8-oxoG and γ-H2AX both in vivo and in vitro. Moreover, ITGB4 deficiency in airway epithelia aggravates the degree of DNA damage under HDM stimulation and ozone stress, respectively. Furthermore, ITGB4 deficiency downregulated the expression of HDAC1 during DNA damage, and restoring HDAC1 can reverse the enhanced DNA damage in airway epithelial cells after exogenous stress. CONCLUSIONS: This study confirmed the involvement of ITGB4 in the regulation of DNA damage through mediating HDAC1 in airway epithelial cells under exogenous stress. These results supply some useful insights into the mechanism of DNA damage in airway epithelial cells, which would provide possible targets for early prediction and intervention of pulmonary diseases.

Effective-component compatibility of Bufei Yishen formula III ameliorated COPD by improving airway epithelial cell senescence by promoting mitophagy via the NRF2/PINK1 pathway.

BACKGROUND: Effective-component compatibility of Bufei Yishen formula III (ECC-BYF III) demonstrates positive effects on stable chronic obstructive pulmonary disease (COPD). PURPOSE: To investigate the mechanisms of ECC-BYF III on COPD rats from the aspect of airway epithelial cell senescence. METHODS: COPD model rats (Sprague-Dawley rat) were treated with ECC-BYF III for 8 weeks, and the efficacy was evaluated. Cigarette smoke extract (CSE)-induced senescence model of airway epithelial cells was treated with ECC-BYF III, and related enzymes and proteins involved in oxidative stress and mitophagy were detected. RESULTS: ECC-BYF III markedly rescued pulmonary function and histopathological changes, which might be associated with the amelioration of lung senescence, including the reduction of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-9 levels, increase of the level in total superoxide dismutase (T-SOD), and decease in the p21 level in the airways. Furthermore, ECC-BYF III suppressed p16 and p21 expressions and senescence-associated ß-galactosidase (SA-ß-Gal) in CSE-induced airway epithelial cells. Moreover, ECC-BYF III upregulated mitophagy-related proteins, including the co-localizations of TOM20 and LC3B, PINK1 and PARK2, and improved mitochondrial function by upregulating mitochondrial mitofusin (MFN)2 and reducing dynamin-related protein 1 (DRP1) expression. ECC-BYF III enhanced the activities of T-SOD and GSH-PX by up-regulating NRF2, thus inhibiting oxidative stress. After intervention with NRF2 inhibitor, the regulation effects of ECC-BYF III on oxidative stress, mitophagy and senescence in airway epithelial cells were significantly suppressed. CONCLUSIONS: ECC-BYF III exerts beneficial effects on COPD rats by ameliorating airway epithelial cell senescence, which is mediated by inhibiting oxidative stress and subsequently enhancing mitophagy through the activation of NRF2 signaling.

Clara cell 16 KDa protein mitigates house dust mite-induced airway inflammation and damage via regulating airway epithelial cell apoptosis in a manner dependent on HMGB1-mediated signaling inhibition.

BACKGROUND: House dust mite (HDM) inhalation can cause airway epithelial damage which is implicated in the process of airway inflammation in asthma. High mobility group box 1 (HMGB1) is critically required for cellular damage and apoptosis as an important endogenous danger signal. Recently, Clara cell 16KDa protein (CC16) has been identified to exert anti-inflammatory and immunomodulatory influence in various injury-related diseases model. However, little is known about its ability to protect against airway epithelial injury in allergic asthma. This study was aimed to clarify the protective roles of CC16 on airway epithelia in HDM-induced asthma and the regulation of HMGB1 by CC16. METHODS: Mice were sensitized and challenged by HDM extract and administrated intranasally with CC16 (5 µg/g or 10 µg/g) or saline in the challenged period. The BEAS-2B human airway epithelial cell line were cultured with CC16 or the control vehicle and then exposed to HDM. Knockdown or overexpression of HMGB1 was induced by cell transfection or intratracheal injection of recombinant adenovirus. RESULTS: CC16 treatment decreased airway inflammation and histological damage of airway epithelium dose-dependently in HDM-induced asthma model. Airway epithelia apoptosis upon HDM stimulation was noticeably abrogated by CC16 in vivo and in vitro. In addition, upregulation of HMGB1 expression and its related signaling were also detected under HDM conditions, while silencing HMGB1 significantly inhibited the apoptosis of BEAS-2B cells. Furthermore, the activity of HMGB1-mediated signaling was restrained after CC16 treatment whereas HMGB1 overexpression abolished the protective effect of CC16 on HDM-induced airway epithelia apoptosis. CONCLUSIONS: Our data confirm that CC16 attenuates HDM-mediated airway inflammation and damage via suppressing airway epithelial cell apoptosis in a HMGB1-dependent manner, suggesting the role of CC16 as a potential protective option for HDM-induced asthma.

Increased epithelial galectin-13 expression associates with eosinophilic airway inflammation in asthma.

BACKGROUND: Airway eosinophilic inflammation is a central feature in asthma which is mainly driven by type 2 response. The expression of galectin-13 was up-regulated in a parasitic infection model which is also characterized by type 2 immune response. We hypothesized that galectin-13 may be involved in airway eosinophilic inflammation in asthma. OBJECTIVE: To unveil the role of galectin-13 in asthma airway inflammation. METHODS: We measured galectin-13 expressions in bronchial brushings, sputum, and plasma of asthma patients (n = 54) and healthy controls (n = 15), and analysed the correlations between galectin-13 expression and airway eosinophilia. We used human bronchial epithelial cell line 16HBE to investigate the possible mechanism by which galectin-13 participates in eosinophilic inflammation. RESULTS: The expression of galectin-13 was markedly increased in subjects with asthma compared to controls. Epithelial galectin-13 mRNA levels in asthmatic subjects were strongly correlated with eosinophilic airway inflammation (the percentage of sputum eosinophils, the number of eosinophils in bronchial submucosa and FeNO) and the expression of Th2 signature genes (CLCA1, POSTN and SERPINB2). Inhaled corticosteroid (ICS) treatment reduced plasma galectin-13 levels, and baseline plasma galectin-13 levels reflect the response to ICS treatment. In cultured 16HBE cells, knockdown of galectin-13 suppressed IL-13-stimulated MCP-1 and eotaxin-1 expression by inhibiting the activation of EGFR and ERK. CONCLUSIONS & CLINICAL RELEVANCE: Galectin-13 is a novel marker for airway eosinophilia in asthma, and may contribute to allergic airway eosinophilic inflammation by up-regulating the expression of MCP-1 and eotaxin-1. Plasma galectin-13 levels may be useful for predicting responses to ICS treatment.

Activation of NLRP3 inflammasome by lymphocytic microparticles via TLR4 pathway contributes to airway inflammation.

The presence of elevated T lymphocytic microparticles (TLMPs) during respiratory illness is associated with airway and lung inflammation and epithelial injuries. Although inflammasome and IL-1ß signaling are crucial in airway inflammation, little was known about their regulatory mechanism. We hypothesized that TLMPs trigger inflammasome activation and IL-1ß production in bronchial and alveolar epithelial cells to induce airway and lung inflammation. In this study, TLMPs induced IL-1ß and IL-18 secretion through NLRP3 inflammasome activation and upregulated TLR4 mRNA and protein expression in alveolar (A549) and human airway epithelial (16HBE) cells. Pretreatment with CLI-095, a specific inhibitor of TLR4 signaling, dramatically diminished the TLMP-induced release of IL-1ß and IL-18 by inhibiting the formation of NLRP3/ASC/pro-caspase-1 inflammasome in a dose-dependent manner. The TLMP-induced autophagy inhibition in epithelial cells was dependent on the PI3K/Akt signaling pathway, which significantly increased NLRP3 expression and enhanced TLMP-induced inflammation. TLR4, IL-1ß, and IL-18 proteins harbored in TLMPs were nonessential for the pro-inflammatory effect. In conclusion, TLMPs induce bronchial and alveolar epithelial cell secretion of IL-1ß and IL-18 cytokines by activating the TLR4 and PI3K/Akt signaling pathways and inhibiting autophagy. These effects lead to NLRP3 inflammasome formation and accumulation. TLMPs may be regarded as deleterious markers of airway and lung damage in respiratory diseases.

Apolipoprotein E Signals via TLR4 to Induce CXCL5 Secretion by Asthmatic Airway Epithelial Cells.

The primary function of APOE (apolipoprotein E) is to mediate the transport of cholesterol- and lipid-containing lipoprotein particles into cells by receptor-mediated endocytosis. APOE also has pro- and antiinflammatory effects, which are both context and concentration dependent. For example, Apoe-/- mice exhibit enhanced airway remodeling and hyperreactivity in experimental asthma, whereas increased APOE levels in lung epithelial lining fluid induce IL-1ß secretion from human asthmatic alveolar macrophages. However, APOE-mediated airway epithelial cell inflammatory responses and signaling pathways have not been defined. Here, RNA sequencing of human asthmatic bronchial brushing cells stimulated with APOE identified increased expression of mRNA transcripts encoding multiple proinflammatory genes, including CXCL5 (C-X-C motif chemokine ligand 5), an epithelial-derived chemokine that promotes neutrophil activation and chemotaxis. We subsequently characterized the APOE signaling pathway that induces CXCL5 secretion by human asthmatic small airway epithelial cells (SAECs). Neutralizing antibodies directed against TLR4 (Toll-like receptor 4), but not TLR2, attenuated APOE-mediated CXCL5 secretion by human asthmatic SAECs. Inhibition of TAK1 (transforming growth factor-ß-activated kinase 1), IκKß (inhibitor of nuclear factor κ B kinase subunit ß), TPL2 (tumor progression locus 2), and JNK (c-Jun N-terminal kinase), but not p38 MAPK (mitogen-activated protein kinase) or MEK1/2 (MAPK kinase 1/2), attenuated APOE-mediated CXCL5 secretion. The roles of TAK1, IκKß, TPL2, and JNK in APOE-mediated CXCL5 secretion were verified by RNA interference. Furthermore, RNA interference showed that after APOE stimulation, both NF-κB p65 and TPL2 were downstream of TAK1 and IκKß, whereas JNK was downstream of TPL2. In summary, elevated levels of APOE in the airway may activate a TLR4/TAK1/IκKß/NF-κB/TPL2/JNK signaling pathway that induces CXCL5 secretion by human asthmatic SAECs. These findings identify new roles for TLR4 and TPL2 in APOE-mediated proinflammatory responses in asthma.

Cell-type specific tumorigenesis with Ras oncogenes in human lung epithelial cells.

The oncogenic Ras mutation is one of the most common genomic abnormalities having the highest incidence in cancer; it has three isoforms: Hras, Kras, and Nras. Although the Ras isoforms are highly similar in the primary sequence, each mutational frequency is clearly distinct according to tissue- or cell-type. Regarding non-small-cell lung carcinoma, almost all Kras mutations have been detected in lung adenocarcinoma, whereas lung squamous cell carcinoma is extremely rare. Here, we focus on the cell-type specific tumorigenesis of mutant Ras isoforms and determine the mechanisms of oncogenic signaling outputs between lung adenocarcinoma and squamous cell carcinoma. An in vitro transformation model with mutant Ras isoforms in immortalized bronchial epithelial cells (BEC-E6E7/myc) and immortalized small airway epithelial cells (SAEC-E6E7/myc) revealed that only the HrasG12V mutation, not the KrasG12V mutation, could induce tumorigenesis in BEC-E6E7/myc. In contrast, SAEC-E6E7/myc showed high sensitivity to the KrasG12V mutation compared with the HrasG12V mutation. The transformation of BEC-E6E7/myc and SAEC-E6E7/myc with mutant Ras isoforms was confirmed by soft agar assay and migration assay. HrasG12V-expressing BEC-E6E7/myc significantly increased MAPK/ERK signaling, whereas PI3K/AKT signaling was significantly elevated in KrasG12V-expressing SAEC-E6E7/myc. These results suggest a context dependency with oncogenic Ras mutations in tumorigenesis between lung adenocarcinoma and squamous cell carcinoma.

MiR-222-3p ameliorates glucocorticoid-induced inhibition of airway epithelial cell repair through down-regulating GILZ expression.

GILZ expression is induced by glucocorticoids (GCs) and is involved in the mechanism of airway epithelial cell repair in patients with asthma. The present study aimed to investigate the role of miR-222-3p/GILZ pathway in treatment of airway epithelial cell repair by GCs. 9HTE cells were treated by 10 µmol/L dexamethasone (Dex) for 6, 12, and 24 hours (h). MiR-222-3p mimic and GILZ were used for cell transfection. Cell vitality, migration, and invasion were detected by methyl-thiazolyl tetrazolium (MTT), wound healing, and Transwell. The targeting relationship between miR-222-3p and GILZ was predicted by TargetScan and further confirmed by dual-luciferase reporter assay. The expressions of relative mRNAs or proteins were detected by Western blot and quantitative polymerase chain reaction (qPCR). The results showed that Dex treatment up-regulated the GILZ expression level but inhibited the levels of p-Raf1, p-MEK1/2, p-ERK1/2, and miR-222-3p of the cells, moreover, it also inhibited cell activity, migration, and invasion in a time-dependent manner. MiR-222-3p specifically targeted GILZ. MiR-222-3p mimic ameliorated the cell viability, migration, and invasion reduced by Dex treatment, increased the expression levels of p-Raf1 and p-MEK1/2, p-ERK1/2, and partially reversed the effects of GILZ overexpression on the above indexes. Moreover, GILZ showed the opposite effects to miR-222-3p. MiR-222-3p activated MAPK signaling pathway through inhibiting the GILZ expression, thus promoting the cell viability, migration, and invasion previously reduced by Dex.

Isolation and characterization of WUPyV in polarized human airway epithelial cells.

BACKGROUND: Washington University polyomavirus (WUPyV) is a novel human polyomavirus detected in childwith acute respiratory infection in 2007. However, the relationship between WUPyV and respiratory diseases has yet to be established for lacking of a suitable in vitro culture system. METHODS: To isolate WUPyV with human airway epithelial (HAE) cells, the positive samples were incubated in HAE, and then the nucleic acid, VP1 protein and virions were detected using real-time PCR, immunofluorescence and electron microscopy respectively. RESULTS: The result showed that WUPyV could replicate effectively in HAE cells and virions with typical polyomavirus characteristics could be observed. Additionally, the entire genome sequence of the isolated strain (BJ0771) was obtained and phylogenetic analysis indicated that BJ0771 belongs to gene cluster I. CONCLUSIONS: Our findings demonstrated clinical WUPyV strain was successfully isolated for the first time in the world and this will help unravel the etiology and pathogenic mechanisms of WUPyV in respiratory infection diseases.

Lung function of primary cooks using LPG or biomass and the effect of particulate matter on airway epithelial barrier integrity.

BACKGROUND: Cooks exposed to biomass fuel experience increased risk of respiratory disease and mortality. We sought to characterize lung function and environmental exposures of primary cooking women using two fuel-types in southeastern India, as well as to investigate the effect of particulate matter (PM) from kitchens on human airway epithelial (HAE) cells in vitro. METHODS: We assessed pre- and post-bronchodilator lung function on 25 primary female cooks using wood biomass or liquified petroleum gas (LPG), and quantified exposures from 34 kitchens (PM2.5, PM < 40 µm, black carbon, endotoxin, and PM metal and bacterial content). We then challenged HAE cells with PM, assessing its cytotoxicity to small-airway cells (A549) and its effect on: transepithelial conductance and macromolecule permeability (NuLi cells), and antimicrobial activity (using airway surface liquid, ASL, from primary HAE cells). RESULTS: Lung function was impaired in cooks using both fuel-types. 60% of participants in both fuel-types had respiratory restriction (post bronchodilator FEV1/FVC>90). The remaining 40% in the LPG group had normal spirometry (post FEV1/FVC = 80-90), while only 10% of participants in the biomass group had normal spirometry, and the remaining biomass cooks (30%) had respiratory obstruction (post FEV1/FVC<80). Significant differences were found in environmental parameters, with biomass kitchens containing greater PM2.5, black carbon, zirconium, arsenic, iron, vanadium, and endotoxin concentrations. LPG kitchens tended to have more bacteria (p = 0.14), and LPG kitchen PM had greater sulphur concentrations (p = 0.02). In vitro, PM induced cytotoxicity in HAE A549 cells in a dose-dependent manner, however the effect was minimal and there were no differences between fuel-types. PM from homes of participants with a restrictive physiology increased electrical conductance of NuLi HAE cells (p = 0.06) and decreased macromolar permeability (p ≤ 0.05), while PM from homes of those with respiratory obstruction tended to increase electrical conductance (p = 0.20) and permeability (p = 0.07). PM from homes of participants with normal spirometry did not affect conductance or permeability. PM from all homes tended to inhibit antimicrobial activity of primary HAE cell airway surface liquid (p = 0.06). CONCLUSIONS: Biomass cooks had airway obstruction, and significantly greater concentrations of kitchen environmental contaminants than LPG kitchens. PM from homes of participants with respiratory restriction and obstruction altered airway cell barrier function, elucidating mechanisms potentially responsible for respiratory phenotypes observed in biomass cooks.

Nix/BNIP3L-dependent mitophagy accounts for airway epithelial cell injury induced by cigarette smoke.

Cigarette smoke-induced airway epithelial cell mitophagy is an important mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Mitochondrial protein Nix (also known as BNIP3L) is a selective autophagy receptor and participates in several human diseases. However, little is known about the role of Nix in airway epithelial cell injury during the development of COPD. The aim of the present study is to investigate the effects of Nix on mitophagy and mitochondrial function in airway epithelial cells exposed to cigarette smoke extract (CSE). Our present study has found that CSE could increase Nix protein expression and induce mitophagy in airway epithelial cells. And Nix siRNA significantly inhibited mitophagy and attenuated mitochondrial dysfunction and cell injury when airway epithelial cells were stimulated with 7.5% CSE. In contrast, Nix overexpression enhanced mitophagy and aggravated mitochondrial dysfunction and cell injury when airway epithelial cells were incubated with 7.5% CSE. These data suggest that Nix-dependent mitophagy promotes airway epithelial cell and mitochondria injury induced by cigarette smoke, and may be involved in the pathogenesis of COPD and other cigarette smoke-associated diseases.

Preterm birth: Born too soon for the developing airway epithelium?

Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.

The leaky lung test: a pilot study using inhaled mannitol to measure airway barrier function in asthma.

Objective: Airway epithelial barrier dysfunction is emerging as an important feature of asthma pathogenesis, but this is difficult to measure in individual subjects. We aimed to develop a noninvasive way to measure airway permeability in asthma. Methods: Healthy controls and subjects with mild asthma inhaled dry powder mannitol in a dose-escalating manner on two separate occasions, stopping at 155 mg or 315 mg. Serum mannitol levels were measured at baseline and then 30, 90, and 150 min after mannitol inhalation. Mannitol absorption was compared with measurements of airflow obstruction (FEV1) and airway inflammation (FeNO). Results: Serum mannitol levels increased in a time- and dose-dependent manner in both healthy control and subjects with asthma. There were no significant differences in mannitol absorption when comparing healthy controls and subjects with asthma. Mannitol absorption did not correlate with markers of airway obstruction or inflammation. Conclusions: Measuring serum concentrations of mannitol after inhalation challenge can potentially provide insights into airway barrier function in asthma.

Targeting Intracellular Ion Homeostasis for the Control of Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a leading cause of mortality in infants and young children. Despite the RSV disease burden, no vaccine is available, and treatment remains nonspecific. New drug candidates are needed to combat RSV. Toward this goal, we screened over 2,000 compounds to identify approved drugs with novel anti-RSV activity. Cardiac glycosides, inhibitors of the membrane-bound Na+/K+-ATPase, were identified to have anti-RSV activity. Cardiac glycosides diminished RSV infection in human epithelial type 2 cells and in primary human airway epithelial cells grown at an air-liquid interface. Digoxin, a U.S. Food and Drug Administration-approved cardiac glycoside, was also able to inhibit infection of primary nasal epithelial cells with community isolates of RSV. Our results suggest that the antiviral effects of cardiac glycosides may be dependent on changes in the intracellular Na+ and K+ composition. Consistent with this mechanism, we demonstrated that the ionophoric antibiotics salinomycin, valinomycin, and monensin inhibited RSV in human epithelial type 2 cells and primary nasal epithelial cells. Our data indicate that the K+/Na+-sensitive steps in the RSV life cycle occur within the initial 4 hours of viral infection but do not include virus binding/entry. Rather, our findings demonstrated a negative effect on the RSV transcription and/or replication process. Overall, this work suggests that targeting intracellular ion concentrations offers a novel antiviral strategy.