Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

COVID-19 Plasma Extracellular Vesicles Increase the Density of Lipid Rafts in Human Small Airway Epithelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the causative agent of the COVID-19 disease. COVID-19 viral infection can affect many cell types, including epithelial cells of the lungs and airways. Extracellular vesicles (EVs) are released by virtually all cell types, and their packaged cargo allows for intercellular communication, cell differentiation, and signal transduction. Cargo from virus-infected cells may include virally derived metabolites, miRNAs, nucleic acids, and proteins. We hypothesized that COVID-19 plasma EVs can induce the formation of signaling platforms known as lipid rafts after uptake by normal human small airway epithelial cells (SAECs). Circulating EVs from patients with or without COVID-19 were characterized by nanoparticle tracking analysis, Western blotting using specific antibodies, and transmission electron microscopy. Primary cultures of normal human small airway epithelial cells were challenged with EVs from the two patient groups, and lipid raft formation was measured by fluorescence microscopy and assessed by sucrose density gradient analysis. Collectively, our data suggest that circulating EVs from COVID-19-infected patients can induce the formation of lipid rafts in normal human small airway epithelial cells. These results suggest the need for future studies aimed at investigating whether the increased density of lipid rafts in these cells promotes viral entry and alteration of specific signaling pathways in the recipient cells.

Transcriptomic analysis identifies dysregulated genes and functional networks in human small airway epithelial cells exposed to ambient PM2.5.

Cellular models exhibiting human physiological features of pseudostratified columnar epithelia, provide a more realistic approach for elucidating detailed mechanisms underlying PM2.5-induced pulmonary toxicity. In this study, we characterized the barrier and mucociliary functions of differentiated human small airway epithelial cells (SAECs), cultured at the air-liquid interface (ALI). Due to the presence of mucociliary protection, particle internalization was reduced, with a concomitant decrease in cytotoxicity in differentiated S-ALI cells, as compared to conventional submerged SAEC cultures. After 24-hour exposure to PM2.5 surrogates, 117 up-regulated genes and 156 down-regulated genes were detected in S-ALI cells, through transcriptomic analysis using the Affymetrix Clariom™ S Human Array. Transcription-level changes in >60 signaling pathways, were revealed by functional annotation of the 273 differentially expressed genes, using the PANTHER Gene List Analysis. These pathways are involved in multiple cellular processes, that include inflammation and apoptosis. Exposure to urban PM2.5 led to complex responses in airway epithelia, including a net induction of downstream pro-inflammatory and pro-apoptotic responses. Collectively, this study highlights the importance of using the more advanced ALI model rather than the undifferentiated submerged model, to avoid over-assessment of inhaled particle toxicity in human. The results of our study also suggest that reduction of ambient PM2.5 concentrations would have a protective effect on respiratory health in humans.

Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Cigarette smoke extract alters genome-wide profiles of circular RNAs and mRNAs in primary human small airway epithelial cells.

As a novel kind of non-coding RNA, circular RNAs (circRNAs) were involved in various biological processes. However, the role of circRNAs in the developmental process of chronic obstructive pulmonary disease (COPD) is still unclear. In the present study, by using a cell model of COPD in primary human small airway epithelial cells (HSAECs) treated with or without cigarette smoke extract (CSE), we uncovered 4,379 previously unknown circRNAs in human cells and 903 smoke-specific circRNAs, with the help of RNA-sequencing and bioinformatic analysis. Moreover, 3,872 up- and 4,425 down-regulated mRNAs were also identified under CSE stimulation. Furthermore, a putative circRNA-microRNA-mRNA network was constructed for in-depth mechanism exploration, which indicated that differentially expressed circRNAs could influence expression of some key genes that participate in response to pentose phosphate pathway, ATP-binding cassette (ABC) transporters, glycosaminoglycan biosynthesis pathway and cancer-related pathways. Our research indicated that cigarette smoke had an influence on the biogenesis of circRNAs and mRNAs. CircRNAs might be involved in the response to CSE in COPD through the circRNA-mediated ceRNA networks.

The cullin4A is up-regulated in chronic obstructive pulmonary disease patient and contributes to epithelial-mesenchymal transition in small airway epithelium.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with high morbidity and mortality. The most important pathophysiological change of COPD is airway obstruction. Airway obstruction can cause airflow restriction and obstructive ventilation dysfunction. Currently, many studies have shown that there is EMT phenomenon in the process of airway remodeling of COPD. Cullin4A (CUL4A) is an E3 ubiquitin ligase that interacts with other factors to form the E3 complex. Studies have shown that CLU4A is associated with EMT in non-small cell lung cancer and other cancers. However, its relationship with EMT in COPD has not been reported systematically. In this study, we detected the expression of CUL4A in lung epithelium of COPD patients. In addition, the regulatory effect and mechanism of CUL4A on EMT in COPD were clarified in small airway epithelial cells. METHODS: The expression of CUL4A was assessed by immunohistochemistry in lung epithelium specimens from smokers, non-smokers and patients with chronic obstructive pulmonary disease. The role of CUL4A on cigarette smoke extract (CSE)-induced epithelial-mesenchymal transition (EMT) in human small airway epithelial cells (HSAEpiCs) was assessed by silencing or overexpression CUL4A in vitro. Cigarette smoke is recognized as a high-risk factor in the induction of COPD, and its damage to the airway involves airway damage, airway inflammation and airway remodeling. RESULTS: The results shown that CUL4A expression in small airway epithelium was significantly increased in patients with COPD. We also observed a significant negative association between CUL4A and FEV1%, a useful clinical marker for the diagnosis and evaluation of COPD severity, in small airway epithelial cells. In vitro, CSE-induced EMT is associated with high expression of CUL4A, and targeted silencing of CUL4A with shRNA inhibits CSE-induced EMT in human small airway epithelial cells. CONCLUSIONS: Our results showed that CUL4A was overexpressed in lung epithelium of COPD patients, and CUL4A could regulate EMT of human small airway epithelium, which revealed a new mechanism of remodeling of small airway epithelium of COPD patients.

Lung Dendritic Cells Drive Natural Killer Cytotoxicity in Chronic Obstructive Pulmonary Disease via IL-15Rα.

RATIONALE: Lung natural killer cells (NKs) kill a greater percentage of autologous lung parenchymal cells in chronic obstructive pulmonary disease (COPD) than in nonobstructed smokers. To become cytotoxic, NKs require priming, typically by dendritic cells (DCs), but whether priming occurs in the lungs in COPD is unknown. METHODS: We used lung tissue and in some cases peripheral blood from patients undergoing clinically indicated resections to determine in vitro killing of CD326+ lung epithelial cells by isolated lung CD56+ NKs. We also measured the cytotoxicity of unprimed blood NKs after preincubation with lung DCs. To investigate mechanisms of DC-mediated priming, we used murine models of COPD induced by cigarette smoke (CS) exposure or by polymeric immunoglobulin receptor (pIgR) deficiency, and blocked IL-15Rα (IL-15 receptor α subunit) trans-presentation by genetic and antibody approaches. RESULTS: Human lung NKs killed isolated autologous lung epithelial cells; cytotoxicity was increased (P = 0.0001) in COPD, relative to smokers without obstruction. Similarly, increased lung NK cytotoxicity compared with control subjects was observed in CS-exposed mice and pIgR-/- mice. Blood NKs both from smokers without obstruction and subjects with COPD showed minimal epithelial cell killing, but in COPD, preincubation with lung DCs increased cytotoxicity. NKs were primed by CS-exposed murine DCs in vitro and in vivo. Inhibiting IL-15Rα trans-presentation eliminated NK priming both by murine CS-exposed DCs and by lung DCs from subjects with COPD. CONCLUSIONS: Heightened NK cytotoxicity against lung epithelial cells in COPD results primarily from lung DC-mediated priming via IL-15 trans-presentation on IL-15Rα. Future studies are required to test whether increased NK cytotoxicity contributes to COPD pathogenesis.

SOCS-1 ameliorates smoke inhalation-induced acute lung injury through inhibition of ASK-1 activity and DISC formation.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of apoptosis and pro-inflammatory cytokine signaling, two major contributors to the pathogenesis of ALI. We have found that SOCS-1 protects lung epithelial cells from smoke-induced apoptosis through two mechanisms. One is that SOCS-1 enhances degradation of ASK-1 and diminishes cleavage of pro-caspase-3 to repress smoke-triggered apoptosis in lung epithelial cells. The other is that SOCS-1 represses smoke-triggered DISC formation through altering TRADD-caspase-8 interaction rather than TNFR-1-TRADD interaction or TNFR-1-TRAF-2 interaction. In conclusion, SOCS-1 relieves smoke inhalation-induced lung injury by repressing ASK-1 and DISC-mediated epithelium apoptosis.

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

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.

Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

Cystathionine-y-lyase (CSE) is a critical enzyme for hydrogen sulfide (H2S) biosynthesis and plays a key role in respiratory syncytial virus (RSV) pathogenesis. The transcription factor NRF2 is the master regulator of cytoprotective and antioxidant gene expression, and is degraded during RSV infection. While some evidence supports the role of NRF2 in CSE gene transcription, its role in CSE expression in airway epithelial cells is not known. Here, we show that RSV infection decreased CSE expression and activity in primary small airway epithelial (SAE) cells, while treatment with tert-butylhydroquinone (tBHQ), an NRF2 inducer, led to an increase of both. Using reporter gene assays, we identified an NRF2 response element required for the NRF2 inducible expression of the CSE promoter. Electrophoretic mobility shift assays demonstrated inducible specific NRF2 binding to the DNA probe corresponding to the putative CSE promoter NRF2 binding sequence. Using chromatin immunoprecipitation assays, we found a 50% reduction in NRF2 binding to the endogenous CSE proximal promoter in SAE cells infected with RSV, and increased binding in cells stimulated with tBHQ. Our results support the hypothesis that NRF2 regulates CSE gene transcription in airway epithelial cells, and that RSV-induced NRF2 degradation likely accounts for the observed reduced CSE expression and activity.

Broad-Spectrum In Vitro Antiviral Activity of ODBG-P-RVn: An Orally-Available, Lipid-Modified Monophosphate Prodrug of Remdesivir Parent Nucleoside (GS-441524).

The necessity for intravenous administration of remdesivir confines its utility for treatment of coronavirus disease 2019 (COVID-19) to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524), against viruses that cause diseases of human public health concern, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had activity nearly equivalent to that of remdesivir in primary-like human small airway epithelial cells. Our results warrant in vivo efficacy evaluation of ODBG-P-RVn. IMPORTANCE While remdesivir remains one of the few drugs approved by the FDA to treat coronavirus disease 2019 (COVID-19), its intravenous route of administration limits its use to hospital settings. Optimizing the stability and absorption of remdesivir may lead to a more accessible and clinically potent therapeutic. Here, we describe an orally available lipid-modified version of remdesivir with activity nearly equivalent to that of remdesivir against emerging viruses that cause significant disease, including Ebola and Nipah viruses. Our work highlights the importance of such modifications to optimize drug delivery to relevant and appropriate human tissues that are most affected by such diseases.

Nicotine-Free e-Cigarette Vapor Exposure Stimulates IL6 and Mucin Production in Human Primary Small Airway Epithelial Cells.

PURPOSE: Electronic cigarettes (e-cigs) are relatively new devices that allow the user to inhale a heated and aerosolized solution. At present, little is known about their health effects in the human lung, particularly in the small airways (<2 mm in diameter), a key site of airway obstruction and destruction in chronic obstructive pulmonary disease and other acute and chronic lung conditions. The aim of this study was to investigate the effect of e-cigarettes on human distal airway inflammation and remodeling. METHODS: We isolated primary small airway epithelial cells from donor lungs without known lung disease. Small airway epithelial cells were cultured at air-liquid interface and exposed to 15 puffs vapor obtained by heating a commercially available e-cigarette solution (e-vapor) with or without nicotine. After 24 hrs of e-vapor exposure, basolateral and apical media as well as cell lysates were collected to measure the pleiotropic cytokine interleukin 6 (IL6) and MUC5AC, one of the major components in mucus. RESULTS: Unlike the nicotine-containing e-vapor, nicotine-free e-vapor significantly increased the amount of IL6, which was coupled with increased levels of intracellular MUC5AC protein. Importantly, a neutralizing IL6 antibody (vs an IgG isotype control) significantly inhibited the production of MUC5AC induced by nicotine-free e-vapor. CONCLUSION: Our results suggest that human small airway epithelial cells exposed to nicotine-free e-vapor increase the inflammatory response and mucin production, which may contribute to distal lung airflow limitation and airway obstruction.

Acquisition of Cancer Stem Cell-like Properties in Human Small Airway Epithelial Cells after a Long-term Exposure to Carbon Nanomaterials.

Cancer stem cells (CSCs) are a key driver of tumor formation and metastasis, but how they are affected by nanomaterials is largely unknown. The present study investigated the effects of different carbon-based nanomaterials (CNMs) on neoplastic and CSC-like transformation of human small airway epithelial cells and determined the underlying mechanisms. Using a physiologically relevant exposure model (long-term/low-dose) with system validation using a human carcinogen, asbestos, we demonstrated that single-walled carbon nanotubes, multi-walled carbon nanotubes, ultrafine carbon black, and crocidolite asbestos induced particle-specific anchorage-independent colony formation, DNA-strand break, and p53 downregulation, indicating genotoxicity and carcinogenic potential of CNMs. The chronic CNM-exposed cells exhibited CSC-like properties as indicated by 3D spheroid formation, anoikis resistance, and CSC markers expression. Mechanistic studies revealed specific self-renewal and epithelial-mesenchymal transition (EMT)-related transcription factors that are involved in the cellular transformation process. Pathway analysis of gene signaling networks supports the role of SOX2 and SNAI1 signaling in CNM-mediated transformation. These findings support the potential carcinogenicity of high aspect ratio CNMs and identified molecular targets and signaling pathways that may contribute to the disease development.

MMP-2 and MMP-9 mediate cigarette smoke extract-induced epithelial-mesenchymal transition in airway epithelial cells via EGFR/Akt/GSK3ß/ß-catenin pathway: Amelioration by fisetin.

Matrix metalloproteinases (MMPs) have been implicated in EMT but their role in the regulation of cigarette smoke-induced EMT in airway epithelium is not clear. We have therefore investigated the potential role of MMP-2 and -9 in cigarette smoke extract (CSE) induced EMT using A549 lung epithelial cells and human small airway epithelial cells (SAEC). The cells were treated with different concentration of CSE, and MTT and trypan blue assays, acridine orange-ethidium bromide assay, gelatin zymography, Western blotting, immunofluorescence studies, Boyden-chamber assay, wound healing assay and air-liquid interface (ALI) culture were used to assess different cellular and molecular changes associated with EMT. The results depict that CSE increased the cytotoxicity along with a concurrent increase in the expression and activity of MMP-2 and -9. CSE further altered EMT markers like E-cadherin, N-cadherin, vimentin, and the molecular modulators of EMT such as ß-catenin and pGSK-3ß. Further, CSE also upregulated EGFR, AKT, and ERK1/2 in airway epithelial cells. SB-3CT, a known inhibitor of MMP-2 and -9, altered and reversed the expression of markers of EMT and kinases, validating the role of MMP-2 and -9 in CSE-induced EMT. Fisetin, a plant-derived bioflavonoid, also reversed the expression of EMT markers and molecular regulators in a similar fashion as SB-3CT. In summary, this study highlights the role of MMP-2 and -9 in CSE-induced EMT and curate its molecular cascade through EGFR/AKT/ERK/ß-catenin axis, which could be restored by MMP-2 and -9 inhibitor and fisetin. Fisetin is hitherto unknown to modulate CSE-induced MMPs activity in airway epithelial cells, and our study suggests its potential role as a therapeutic approach in CSE-induced EMT in lung epithelial cells.

Low concentrations of clarithromycin upregulate cellular antioxidant enzymes and phosphorylation of extracellular signal-regulated kinase in human small airway epithelial cells.

BACKGROUND: It is well known that low-dose, long-term macrolide therapy is effective against chronic inflammatory airway diseases. Oxidative stress is considered to be a key pathogenesis factor in those diseases. However, the mechanism of action of low-dose, long-term macrolide therapy remains unclear. We have reported that clarithromycin (CAM), which is a representative macrolide antibiotic, could inhibit hydrogen peroxide (H2O2)-induced reduction of the glutathione (GSH)/glutathione disulfide (GSSG) ratio in human small airway epithelial cells (SAECs), via the maintenance of GSH levels through an effect on γ-glutamylcysteine synthetase (γ-GCS) expression. In this study, we examined the influence of CAM against H2O2-induced activities of cellular antioxidant enzymes and phosphorylated extracellular signal regulatory kinase (p-ERK) using SAECs, the main cells involved in chronic airway inflammatory diseases. METHODS: SAECs were pretreated with CAM (1, 5, and 10 µM) for 72 h, and subsequently exposed to H2O2 (100 µM) for 0.5-2 h. Levels of GSH and GSSG, and activities of glutathione peroxidase (GPx)-1, glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), heme oxygenase (HO)-1 and p-ERK were assayed. mRNA expressions of GPx-1 and HO-1 were measured using the real-time reverse transcription polymerase chain reaction (RT-PCR). Tukey's multiple comparison test was used for analysis of statistical significance. RESULTS: Pretreatment with low-dose (1 and 5 µM) CAM for 72 h inhibited H2O2-induced reductions of GPx-1, GR, SOD, CAT and HO-1 activities, and mRNA expressions of GPx-1 and HO-1, and improved the GSH/GSSG ratio. However, these alterations were not observed after pretreatment with high-dose (10 µM) CAM, which suppressed phosphorylation of cell proliferation-associated ERK to cause a significant (p < 0.01) decrease in cell viability. CONCLUSIONS: CAM is efficacious against deterioration of cellular antioxidant enzyme activity caused by oxidative stress under low-dose, long-term treatment conditions. On the other hand, pretreatment with high-dose CAM suppressed phosphorylation of cell proliferation-associated ERK and decreased cell viability. The present study may provide additional evidence as to why low-dose, long-term administration of macrolides is effective for treating chronic inflammatory airway diseases.

A Three-Dimensional Human Tissue-Engineered Lung Model to Study Influenza A Infection.

Influenza A virus (IAV) claims ∼250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (two-dimensional [2D] cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineered Lung Model (3D-HTLM), we describe the 3D culture of primary human small airway epithelial cells (HSAEpCs) and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2. The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.

Long-term treatment of clarithromycin at a low concentration improves hydrogen peroxide-induced oxidant/antioxidant imbalance in human small airway epithelial cells by increasing Nrf2 mRNA expression.

BACKGROUND: Clarithromycin (CAM), a representative macrolide antibiotic, has been used widely at low doses for long-term therapy of chronic inflammatory airway diseases. Anti-inflammatory effects of macrolide antibiotics were first discovered in clinical practice. Although oxidative stress is known as a key pathogenesis factor in chronic airway inflammatory diseases, the mechanism of action of low-dose, long-term CAM therapy remains unclear. We aimed to examine the cytoprotective action of CAM against hydrogen peroxide (H2O2)-induced cell dysfunction, focusing on CAM dose and treatment duration, and using human small airway epithelial cells (SAECs), the main cells involved in chronic airway inflammatory diseases. METHODS: SAECs were pretreated with CAM (1, 5 or 10 µM) for 24, 48 or 72 h, and were subsequently exposed to H2O2 for 0.5-4 h. Levels of interleukin (IL)-8, glutathione (GSH) and glutathione disulfide (GSSG), and the activities of nuclear factor (NF)-κB and γ-glutamylcysteine synthetase (γ-GCS) were assayed using specific methods. IL-8 mRNA and NF erythroid 2-related factor 2 (Nrf2) mRNA expression were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). Tukey's multiple comparison test was used for analysis of statistical significance. RESULTS: Pretreatment with low-dose (1 or 5 µM), long-term (72 h) CAM inhibited H2O2-induced IL-8 levels, NF-κB activity, and IL-8 mRNA expression, and improved the GSH/GSSG ratio via the maintenance of γ-GCS expression levels. Similar to its enhancing effect on the GSH/GSSG ratio, pretreatment with low-dose CAM for 72 h significantly increased Nrf2 mRNA expression (p < 0.01 and p < 0.05). In contrast, these alterations were not observed after pretreatment with high-dose (10 µM) or short-term (24 and 48 h) CAM. CONCLUSIONS: CAM is efficacious against cell dysfunction caused by oxidative stress under low-dose, long-term treatment conditions. This effect depended on the suppression of NF-κB activation and improvement of the H2O2-induced oxidant/antioxidant imbalance that is achieved by increasing Nrf2 mRNA expression in SAECs. The present study may provide the first evidence of why low-dose, long-term administration of macrolides is effective for treating chronic inflammatory airway diseases.

A Phenotypic High-Throughput Screen with RSV-Infected Primary Human Small Airway Epithelial Cells (SAECs).

Respiratory syncytial virus (RSV) is a commonly occurring pathogen that can cause severe disease in children, the elderly, and immunocompromised individuals with a large, unmet clinical need. We developed a high-throughput, primary cell-based antiviral RSV assay to enable identification of small molecules using cytopathic effect (CPE) as a phenotypic end point. To provide increased biological relevance, we developed our assay with primary human small airway epithelial cells (SAECs), which originate from known sites of RSV infection and replication instead of a more traditional immortalized cell line. Using purchased low-passage cells, cost-effective large-scale culture methods were developed to provide assay-ready frozen SAECs. A high-throughput screening campaign using the GSK Screening Collection was performed. The screen was executed in 384-well plates over a 12-week period with an average Z' of 0.5. The screen yielded 17 post-entry hits with activity in the primary cells, which were not active in immortalized cells. Potencies for this class of compounds were equal between the primary and immortalize cell lines. For entry inhibitors, the number was much lower, with increased potency observed in immortalized cells. This is the first known use of frozen primary human cells for an RSV high-throughput screening phenotypic campaign.