Human Bronchial Cells Research Articles

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

Malignant transformation of human cells is associated with their re-programming which results in uncontrolled proliferation and in the same time biochemical activation of immunosuppressive pathways which form cancer immune evasion machinery. However, there is no conceptual understanding of whether immune evasion machinery pathways and expression of immune checkpoint proteins form a part of the process of malignant transformation or if they are triggered by T lymphocytes and natural killers (NK) attempting to attack cells which are undergoing or already underwent malignant transformation. To address this fundamental question, we performed experimental malignant transformation of BEAS-2B human bronchial epithelium cells and RC-124 non-malignant human kidney epithelial cells using bracken extracts containing carcinogenic alkaloid called ptaquiloside. This transformation led to a significant upregulation of cell proliferation velocity and in the same time led to a significant upregulation in expression of key immune checkpoint proteins – galectin-9, programmed death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO1). Their increased expression levels were in line with upregulation of the levels and activities of HIF-1 transcription complex and transforming growth factor beta type 1 (TGF-β)-Smad3 signalling pathway. When co-cultured with T cells, transformed epithelial cells displayed much higher and more efficient immune evasion activity compared to original non-transformed cells. Therefore, this work resolved a very important scientific and clinical question and suggested that cancer immune evasion machinery is activated during malignant transformation of human cells regardless the presence of immune cells in microenvironment.

HtrA3: a promising prognostic biomarker and therapeutic target for head and neck squamous cell carcinoma.

The dysregulation of the human high-temperature requirement A (HtrA) family of serine proteases is associated with many malignancies. However, there are few reports on HtrAs in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the expression, prognostic value, and biological functions of HtrAs in HNSCC. The RNA-sequencing data and clinical data of HNSCC were downloaded from The Cancer Genome Atlas (TCGA) database. The GSE30784 and GSE31056 datasets from the Gene Expression Omnibus (GEO) database were used for further verification. This study explored the differential expression of HtrAs and assessed their potential impact on the prognosis of HNSCC patients using a survival module. Correlations between clinical characteristics and HtrA expression levels were then explored using a Wilcoxon rank sum test. A Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed using "clusterProfile" in the R software. A Pearson/Spearman correlation test was applied to analyze the relationship between HtrAs and immune infiltration level/checkpoint genes. Validation of HtrA expression levels were carried out by RT-PCR and western blot in human squamous carcinoma cell lines (Fadu and Cal-27) and human non-tumorigenic bronchial epithelium cells (BEAS-2B). Finally, through cell transfection, CCK-8, Ki-67 immunofluorescence, and flow cytometry assays, the effect of HtrA3 knockdown on the malignant biological behavior of HNSCC cells was explored. The gene expression levels of HtrAs were significantly upregulated and associated with patient age, TNM stage, clinical stage, and TP53 mutation status in the TCGA-HNSCC cohort. High expressions of HtrA1/3 were associated with shorter overall survival, shorter progress-free interval, and lower disease-specific survival in HNSCC. A nomogram for HtrAs was constructed and validated. HtrA-related genes were significantly enriched in the immune response and cell apoptosis pathway. In addition, the expression of HtrAs showed significant correlations with B cells, M cells, DC cell infiltration, and immune infiltration checkpoint (CD276, TNFRSF14). Validation of HtrA expression was carried out by RT-PCR and western blot. Results of in vitro experiments indicated that HtrA3 gene knockdown inhibits the proliferation of FaDu and Cal-27 cells while concurrently promoting apoptosis. HtrA3 shows significant potential as both a prognostic marker and a promising therapeutic target for HNSCC, highlighting its relevance and importance in future research and potential clinical applications.

Hypermethylation of the ADIRF promoter regulates its expression level and is involved in NNK-induced malignant transformation of lung bronchial epithelial cells.

The carcinogenic mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a well-known tobacco carcinogen, has not been fully elucidated in epigenetic studies. 5-Methylcytosine (5mC) modification plays a major role in epigenetic regulation. In this study, the 5mC level increased in both BEAS-2B human bronchial epithelium cells treated with 100mg/L NNK for 24h and NNK-induced malignant-transformed BEAS-2B cells (2B-NNK cells), suggesting that 5mC modification is associated with the malignant transformation mechanism of NNK. Using a combination of Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq), RNA sequencing (RNA-seq), and bioinformatics analysis of data from the Genomic Data Commons database, we found that the Adipogenesis regulatory factor (ADIRF) promoter region was abnormally hypermethylated, yielding low ADIRF mRNA expression, and that ADIRF overexpression could inhibit the proliferation, migration, and invasion of 2B-NNK cells. This finding suggests that ADIRF plays a tumor suppressor role in the NNK-induced malignant transformation of cells. Subsequently, using 5-Aza-2'-deoxycytidine (5-Aza-2'-dC) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Catalytically Dead Cas9 (dCas9 system), we verified that the demethylation of the ADIRF promoter region in 2B-NNK cells inhibited the proliferation, migration, and invasion ability of the cells and increased their apoptosis ability. These results suggest that abnormal 5mC modification of the ADIRF promoter plays a positive regulatory role in the pathogenesis of NNK-induced lung cancer. This study offers a new experimental basis for the epigenetic mechanism of NNK-induced lung cancer.

Raman imaging and chemometric methods in human normal bronchial and cancer lung cells: Raman biomarkers of lipid reprogramming

This paper presents an approach to study biochemical changes in human normal bronchial cells (BEpiC) and human cancer lung cells (A549) by Raman spectroscopy and Raman imaging combined with chemometric methods. Based on Raman spectra and Raman imaging combined with chemometric methods we have proved that peaks at 845 cm−1, 2845 cm−1, 2936 cm−1, 1444 cm−1, 750 cm−1, 1126 cm−1, 1584 cm−1, can be treated as Raman biomarkers probing phosphorylation, lipid reprogramming, oxidative phosphorylation and changes in cholesterol and cytochrome in normal and cancer cells. Raman analysis of the bands at 845 cm−1, 2845 cm−1, 1444 cm−1, and 1126 cm−1 in human cancer lung cells and human normal bronchial cells demonstrate enhanced phosphorylation and triglycerides de novo synthesis, reduced levels of cholesterol and cytochrome c in cancer cells. The sensitivity is equal to 100% (nucleus), 87.5% (mitochondria), 100% (endoplasmic reticulum), 87.5% (lipid droplets), 87.5% (cytoplasm), 87.5% (cell membrane) for A549 cell line and 83.3% (nucleus), 100% (mitochondria), 83.3% (endoplasmic reticulum), 100% (lipid droplets), 100% (cytoplasm), 83.3% (cell membrane) for BEpiC. The values of specificity for cross-validation equal 93.4% (nucleus), 85.5% (mitochondria), 89.5% (endoplasmic reticulum), 90.8% (lipid droplets), 61.8% (cytoplasm), 94.7% (cell membrane) for A549 cell line and 88.5% (nucleus), 85.9% (mitochondria), 85.9% (endoplasmic reticulum), 97.4% (lipid droplets), 75.6% (cytoplasm), 92.3% (cell membrane) for BEpiC. We have confirmed that Raman spectroscopy methods combined with PLS-DA are useful tools to monitor changes in human cancer lung cells and human normal bronchial cells.

3D model of the bronchial epithelial barrier to study repeated exposure to xenobiotics: Application to silver nanoparticles

There is still a lack of in vitro human models to evaluate the chronic toxicity of drugs and environmental pollutants. Here, we used a 3D model of the human bronchial epithelium to assess repeated exposures to xenobiotics. The Calu-3 human bronchial cell line was exposed to silver nanoparticles (AgNP) 5 times during 12 days, at the air-liquid interface, to mimic single and repeated exposure to inhaled particles. Repeated exposures induced a stronger induction of the metal stress response and a steady oxidative stress over time. A sustained translocation of silver was observed after each exposure without any loss of the epithelial barrier integrity. The proteomic analysis of the mucus revealed changes in the secreted protein profiles associated with the epithelial immune response after repeated exposures only. These results demonstrate that advanced in vitro models are efficient to investigate the adaptive response of human cells submitted to repeated xenobiotic exposures.

Control of Mitochondrial Electron Transport Chain Flux and Apoptosis by Retinoic Acid: Raman Imaging In Vitro Human Bronchial and Lung Cancerous Cells.

The multiple functions of cytochrome c (cyt c) and their regulation in life and death decisions of the mammalian cell go beyond respiration, apoptosis, ROS scavenging, and oxidation of cardiolipine. It has become increasingly evident that cyt c is involved in the propagation of mitogenic signals. It has been proposed that the mitogenic signals occur via the PKCδ-retinoic acid signal complex comprising the protein kinase Cδ, the adapter protein Src homologous collagen homolog (p66Shc), and cyt c. We showed the importance of retinoic acid in regulating cellular processes monitored by the Raman bands of cyt c. To understand the role of retinoids in regulating redox status of cyt c, we recorded the Raman spectra and images of cells receiving redox stimuli by retinoic acid at in vitro cell cultures. For these purposes, we incubated bronchial normal epithelial lung (BEpC) and lung cancer cells (A549) with retinoic acid at concentrations of 1, 10, and 50 µM for 24 and 48 h of incubations. The new role of retinoic acid in a change of the redox status of iron ion in the heme group of cyt c from oxidized Fe3+ to reduced Fe2+ form may have serious consequences on ATPase effectiveness and aborting the activation of the conventional mitochondrial signaling protein-dependent pathways, lack of triggering programmed cell death through apoptosis, and lack of cytokine induction. To explain the effect of retinoids on the redox status of cyt c in the electron transfer chain, we used the quantum chemistry models of retinoid biology. It has been proposed that retinol catalyzes resonance energy transfer (RET) reactions in cyt c. The paper suggests that RET is pivotally important for mitochondrial energy homeostasis by controlling oxidative phosphorylation by switching between activation and inactivation of glycolysis and regulation of electron flux in the electron transport chain. The key role in this process is played by protein kinase C δ (PKCδ), which triggers a signal to the pyruvate dehydrogenase complex. The PKCδ-retinoic acid complex reversibly (at normal physiological conditions) or irreversibly (cancer) responds to the redox potential of cyt c that changes with the electron transfer chain flux.

Role of Chemical Reduction and Formulation of Graphene Oxide on Its Cytotoxicity towards Human Epithelial Bronchial Cells.

Graphene-based materials may pose a potential risk for human health due to occupational exposure, mainly by inhalation. This study was carried out on bronchial epithelial 16HBE14o- cells to evaluate the role of chemical reduction and formulation of graphene oxide (GO) on its cytotoxic potential. To this end, the effects of GO were compared to its chemically reduced form (rGO) and its stable water dispersion (wdGO), by means of cell viability reduction, reactive oxygen species (ROS) generation, pro-inflammatory mediators release and genotoxicity. These materials induced a concentration-dependent cell viability reduction with the following potency rank: rGO > GO >> wdGO. After 24 h exposure, rGO reduced cell viability with an EC50 of 4.8 μg/mL (eight-fold lower than that of GO) and was the most potent material in inducing ROS generation, in contrast to wdGO. Cytokines release and genotoxicity (DNA damage and micronucleus induction) appeared low for all the materials, with wdGO showing the lowest effect, especially for the former. These results suggest a key role for GO reduction in increasing GO cytotoxic potential, probably due to material structure alterations resulting from the reduction process. In contrast, GO formulated in a stable dispersion seems to be the lowest cytotoxic material, presumably due to its lower cellular internalization and damaging capacity.

Can oestrogenic activity in air contribute to the overall body burden of endocrine disruptors?

Endocrine disruptors (EDCs) are emerging contaminants that are harmful to health. Human exposure occurs mainly through ingestion or dermal contact, but inhalation could be an additional exposure route; therefore, this study was conducted to evaluate the oestrogenic activity of airborne particulate matter (PM). Outdoor PM was collected for a year in five Italian sites and extracted with organic solvents (four seasonal extracts/site). The oestrogenic activity was assessed using a gene reporter assay (MELN), and the risk to human health through inhalation was quantified using the results. Moreover, extracts were analysed to assess cytotoxicity (WST-1 and LDH assays) on human bronchial cells (BEAS-2B). The extracts induced a significant cytotoxicity and oestrogenic activity. Oestrogenic activity showed a seasonal trend and was correlated with concentrations of benzo(a)pyrene and toxic equivalency factor. Although a low inhalation cancer risk was found, this study confirmed that oestrogenic activity in air could contribute to overall health risks due to EDC exposure.

P13-049-23 N-Acetyltransferase 1 Knockout in Immortalized Human Bronchial Cells Results in a Reduction of Cellular Growth

P13-049-23 N-Acetyltransferase 1 Knockout in Immortalized Human Bronchial Cells Results in a Reduction of Cellular Growth

Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells.

It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations.

Lung viral infection modelling in a bioengineered whole-organ

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

Transferability and reproducibility of exposed air-liquid interface co-culture lung models

BackgroundThe establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. ResultsOverall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. ConclusionThe transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

97 Transferability and Reproducibility of Exposed Air-Liquid Interface co-Culture Lung Models

Abstract The establishment of reliable and robust in vitro models for hazard assessment, moving away from animal testing, requires evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as monoculture and, to increase the relevance of the model, also as co-culture with macrophages (derived from the THP-1 monocyte cell line or from human blood monocytes). Exposure employed the VITROCELL® Cloud12 system at physiologically relevant doses. Overall, the results of the seven participating laboratories are quite similar. No effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 exposure on cell viability and barrier integrity were seen in the monoculture and co-culture models. Most laboratories showed that LPS induced cytokine release in the co-culture models, but not the monoculture model. Exposure to DQ12 or TiO2 did not increase cytokine release, probably due to too low deposited doses. Interlaboratory variability indicated acceptable variation for cell viability measurements and relatively high variation for cytokine measurements. Based on the transferability and reproducibility of the model and its air exposure we are confident that, based on detailed SOPs, performing a new round of interlaboratory comparison, and increasing the deposited doses of NMs, these models may be taken further towards a possible OECD guideline.

BMAL1 regulates MUC1 overexpression in ovalbumin-induced asthma.

Asthma often presents with a daily rhythm; however, the underlying mechanisms remain unclear. Circadian rhythm genes have been proposed to regulate inflammation and mucin expression. Here, ovalbumin (OVA)-induced mice and serum shock human bronchial epidermal cells (16HBE) were used in in vivo and in vitro models, respectively. We constructed a brain and muscle ARNT-like 1 (BMAL1) knockdown 16HBE cell line to analyze the effects of rhythmic fluctuations on mucin expression. Serum immunoglobulin E (IgE) and circadian rhythm genes in asthmatic mice showed rhythmic fluctuation amplitude. Mucin (MUC) 1 and MUC5AC expression was increased in the lung tissue of the asthmatic mice. MUC1 expression was negatively correlated with that of the circadian rhythm genes, particularly BMAL1 (r=-0.546, P=0.006). There was also a negative correlation between BMAL1 and MUC1 expression (r=-0.507, P=0.002) in the serum shock 16HBE cells. BMAL1 knockdown negated the rhythmic fluctuation amplitude of MUC1 expression and upregulated MUC1 expression in the 16HBE cells. These results indicate that the key circadian rhythm gene, BMAL1, causes periodic changes in airway MUC1 expression in OVA-induced asthmatic mice. Targeting BMAL1 to regulate periodic changes in MUC1 expression may, therefore, improve asthma treatments.

Bufei Yishen Formula Inhibits the Cell Senescence in COPD by Up-Regulating the ZNF263 and Klotho Expression.

Bufei Yishen formula (BYF) is an effective prescription for the clinical treatment of chronic obstructive pulmonary disease (COPD). However, the molecular mechanism by which it exerts its pharmacological effects remains to be explored. The human bronchial cell line BEAS-2B was treated with cigarette smoke extract (CSE). Cellular senescence markers were detected by Western blot and ELISA. Potential transcription factor of klotho was predicted using JASPAR and USCS databases. CSE induced cellular senescence with intracellular accumulation of cellular senescence biomarkers (p16, p21 and p27) and increased secretion of senescence-related secretory phenotypic (SASP) factors (IL-6, IL-8, and CCL3). In contrast, BYF treatment inhibited CSE-induced cellular senescence. CSE suppressed the transcription, expression and secretion of klotho, whereas BYF treatment rescued its transcription, expression and secretion. CSE downregulated the protein level of ZNF263, whereas BYF treatment rescued the expression of ZNF263. Furthermore, ZNF263-overexpressing BEAS-2B cells could inhibit CSE-induced cellular senescence and SASP factor secretion by upregulating the expression of klotho. This study revealed a novel pharmacological mechanism by which BYF alleviates clinical symptoms of COPD patients, and regulating ZNF263 and klotho expression may be beneficial to the treatment and prevention of COPD.

Essential oil of Origanum minutiflorum exhibits anti-inflammatory and antioxidative effects in human bronchial cells and antimicrobial activity on lung pathogens

IntroductionOregano essential oil (OEO) is one of the most widely used essential oils worldwide due to its huge therapeutic benefits. Nevertheless, data on the effects of the endemic species Origanum minutiflorum, also known as wild or Turkish oregano, is scarce. On the other hand, various chronic lung diseases, characterised by persistent inflammation, oxidative stress, and common bacterial infections, do not have effective pharmacological therapy. Hence, the aim of this study is to examine the effects of wild oregano essential oil (WOEO) on human bronchial epithelial cells and lung pathogens. MethodsWe provided a detailed chemical composition of WOEO using GC-MS and GC-FID analysis. Anti-inflammatory effects of WOEO were analysed using the qRT-PCR and ELISA methods, while antioxidative properties were examined by using the dichlorofluorescein assay in BEAS-2B cells. Antibacterial activity was tested on lung pathogens by using the agar diffusion assay. ResultsThe major constituents of WOEO, analysed in this study, were carvacrol, linalool, p-cymene, γ-terpinene, and (E)-caryophyllene. We found that treatment with WOEO attenuated LPS-induced IL8 gene expression and hydrogen peroxide-induced oxidative stress in BEAS-2B cells. Moreover, WOEO showed an inhibitory effect on pathogenic bacteria Acinetobacter baumannii and highly resistant Klebsiella pneumoniae commonly seen in healthcare-associated pneumonia. ConclusionOur work presents new insights into the anti-inflammatory, antioxidative, and antimicrobial properties of WOEO which may be used as a simple and local treatment in various chronic lung diseases.

Circular RNA 0000157 depletion protects human bronchial epithelioid cells from cigarette smoke extract-induced human bronchial epithelioid cell injury through the microRNA-149-5p/bromodomain containing 4 pathway.

Circular RNA (circRNA) has been reported to regulate respiratory diseases. In the study, we aimed to elucidate the role of circ_0000157 in smoke-related chronic obstructive pulmonary disease (COPD) and the inner mechanism. COPD-like cell injury was induced by treating human bronchial epithelioid cells (16HBE) with cigarette smoke extract (CSE). The expression of circ_0000157, miR-149-5p, bromodomain containing 4 (BRD4), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blotting. Enzyme-linked immunosorbent assay was performed to detect interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels. Malondialdehyde (MDA) production was detected by a lipid peroxidation MDA assay kit. Superoxide dismutase (SOD) activity was analyzed by a SOD activity assay kit. Circ_0000157 and BRD4 expression were upregulated, while miR-149-5p expression was downregulated in the blood of smokers with COPD and CSE-induced 16HBE cells compared with control groups. CSE treatment inhibited 16HBE cell proliferation and induced cell apoptosis, inflammation, and oxidative stress; however, these effects were remitted when circ_0000157 expression was decreased. In addition, circ_0000157 acted as a miR-149-5p sponge and regulated CSE-caused 16HBE cell damage by targeting miR-149-5p. The overexpression of BRD4, a target gene of miR-149-5p, attenuated the inhibitory effects of miR-149-5p introduction on CSE-induced cell damage. Further, circ_0000157 modulated BRD4 expression by associating with miR-149-5p in CSE-treated 16HBE cells. Circ_0000157 knockdown ameliorated CSE-caused 16HBE cell damage by targeting the miR-149-5p/BRD4 pathway, providing a potential therapeutic strategy for clinic intervention in COPD.

Cyanobacteria, cyanotoxins and lipopolysaccharides in aerosols from inland freshwater bodies and their effects on human bronchial cells

Components of cyanobacterial water blooms were quantified in aerosols above agitated water surfaces of five freshwater bodies. The thoracic and respirable aerosol fraction (0.1–10 µm) was sampled using a high-volume sampler. Cyanotoxins microcystins were detected by LC-MS/MS at levels 0.3–13.5 ng/mL (water) and < 35–415 fg/m3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10–119 EU/mL (water) and 0.13–0.64 EU/m3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 104–105 cells eq./mL (water) and 101–103 cells eq./m3 (aerosol). Lipopolysaccharides isolated from bloom samples induced IL-6 and IL-8 cytokine release in human bronchial epithelial cells Beas-2B, while extracted cyanobacterial metabolites induced both pro-inflammatory and cytotoxic effects. Bloom components detected in aerosols and their bioactivities observed in upper respiratory airway epithelial cells together indicate that aerosols formed during cyanobacterial water blooms could induce respiratory irritation and inflammatory injuries, and thus present an inhalation health risk.

Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials.

For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R's to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air-liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs.