Human Bronchial Epithelial Cell Line Research Articles

Long Noncoding RNA PSMB8-AS1 Mediates the Tobacco-Carcinogen-Induced Transformation of a Human Bronchial Epithelial Cell Line by Regulating Cell Cycle.

Lung cancer is the main cause of cancer deaths around the world. Nitrosamine 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific carcinogen of lung cancer. Abundant evidence implicates long noncoding RNAs (lncRNAs) in tumorigenesis. Yet, the effects and mechanisms of lncRNAs in NNK-induced carcinogenesis are still unclear. In this study, we discovered that NNK-induced transformed Beas-2B cells (Beas-2B-NNK) showed increased cell migration and proliferation while decreasing rates of apoptosis. RNA sequencing and differentially expressed lncRNAs analyses showed that lncRNA PSMB8-AS1 was obviously upregulated. Interestingly, silencing the lncRNA PSMB8-AS1 in Beas-2B-NNK cells reduced cell proliferation and migration and produced cell cycle arrest in the G2/M phase along with a decrease in CDK1 expression. Conclusively, our results demonstrate that lncRNA PSMB8-AS1 could promote the malignant characteristics of Beas-2B-NNK cells by regulating CDK1 and affecting the cell cycle, suggesting that it may supply a new prospective epigenetic mechanism for lung cancer.

TRIM47 promotes HDM-induced bronchial epithelial pyroptosis by regulating NEMO ubiquitination to activate NF-κB/NLRP3 signaling.

Asthma is an inflammatory disease. Airway epithelial cell pyroptosis and cytokine secretion promote asthma progression. Tripartite motif 47 (TRIM47)belongs to the E3 ubiquitin ligase family and is associated with apoptosis and inflammation in a range of diseases. However, the role of TRIM47 in asthma has not been explored. In this study, the human bronchial epithelial cell line BEAS-2B was treated with house dust mite (HDM) and TRIM47 expression was detected by RT-qPCR and Western blot. After transfection with TRIM47 interfering and overexpressing plasmids, the synthesis and secretion of cytokines, as well as pyroptosis-related indicators, were examined.Nuclear factor kappa-B (NF-κB)pathway proteins and nod-like receptor protein 3 (NLRP3)inflammasome were measured to explore the mechanism of TRIM47 action. In addition, the effect of TRIM47 on the level of NF-κB essential modulator (NEMO) ubiquitination was detected by an immunoprecipitation assay. The results showed that TRIM47 was upregulated in HDM-induced BEAS-2B cells and that TRIM47 mediated HDM-induced BEAS-2B cell pyroptosis and cytokine secretion. Mechanistically, TRIM47 promoted the K63-linked ubiquitination of NEMO and facilitated NF-κB/NLRP3 pathway activation. In conclusion, TRIM47 may promote cytokine secretion mediating inflammation and pyroptosis in bronchial epithelial cells by activating the NF-κB/NLRP3 pathway. Therefore, TRIM47 may be a potential therapeutic target for HDM-induced asthma.

Polyhydroxylated Sirostanol Saponins from the Rhizomes of Paris dulongensis.

Three new polyhydroxylated spirostanol steroidal saponins, dulongenosides B-D (2-4), along with 14 known compounds, dulongenoside A (1), padelaoside B (5), parisyunnanoside G (6), polyphyllin D (7), ophiopogonin C' (8), formosanin C (9), dioscin (10), paris saponin VII (11), paris H (12), parisyunnanoside I (13), protodioscin (14), proprotogracillin (15), crustecdysone (16), and stigmasterol-3-O-β-d-glucopyranoside (17), were isolated from the rhizomes of Paris dulongensis (Melanthiaceae). Their chemical structures were elucidated based on extensive analyses of NMR and MS data and acidic hydrolyses. The isolates were evaluated for their cytotoxicity to five human cancer cell lines (HL-60, SW480, MDA-MB-231, A549, and A549/Taxol) and the normal human bronchial epithelial cell line BEAS-2B by the MTS test. Compounds 7-12 and 14 showed cytotoxic activity, with IC50 values ranging from 0.20 to 4.35 μM. Proprogracillin selectively inhibited A549(IC50 = 0.58 μM) and A549/Taxol (IC50 = 0.74 μM) cells, with no significant cytotoxic activity against HL-60, SW480, MDA-MB-231, or BEAS-2B cells, with IC50 values greater than 40 μM.

BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

Bile acid metabolites enhance expression of cathelicidin antimicrobial peptide in airway epithelium through activation of the TGR5-ERK1/2 pathway

Signals for the maintenance of epithelial homeostasis are provided in part by commensal bacteria metabolites, that promote tissue homeostasis in the gut and remote organs as microbiota metabolites enter the bloodstream. In our study, we investigated the effects of bile acid metabolites, 3-oxolithocholic acid (3-oxoLCA), alloisolithocholic acid (AILCA) and isolithocholic acid (ILCA) produced from lithocholic acid (LCA) by microbiota, on the regulation of innate immune responses connected to the expression of host defense peptide cathelicidin in lung epithelial cells. The bile acid metabolites enhanced expression of cathelicidin at low concentrations in human bronchial epithelial cell line BCi-NS1.1 and primary bronchial/tracheal cells (HBEpC), indicating physiological relevance for modulation of innate immunity in airway epithelium by bile acid metabolites. Our study concentrated on deciphering signaling pathways regulating expression of human cathelicidin, revealing that LCA and 3-oxoLCA activate the surface G protein-coupled bile acid receptor 1 (TGR5, Takeda-G-protein-receptor-5)—extracellular signal-regulated kinase (ERK1/2) cascade, rather than the nuclear receptors, aryl hydrocarbon receptor, farnesoid X receptor and vitamin D3 receptor in bronchial epithelium. Overall, our study provides new insights into the modulation of innate immune responses by microbiota bile acid metabolites in the gut-lung axis, highlighting the differences in epithelial responses between different tissues.

German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 invivo.

In vitro EVALUATION of THE EFFECTS of POTENTIAL GSK-3β INHIBITORS TERBUTALINE and ORCIPRENALINE

Lung cancer is a type of cancer that is mostly diagnosed at an advanced stage and has a short survival time despite standard chemotherapy and targeted therapies. Terbutaline and Orciprenaline are bronchodilator agent that are potent and selective β2 receptor agonists. The purpose of this study was to investigate by continuous monitoring to assay the effects of Terbutaline and Orciprenaline on human lung carcinoma cell line. Cells were treated 1, 10, 100, 200, 400 µM concentrations with Terbutaline/Orciprenaline. MTT assay and xCELLigence real-time cell analyzer were used to determine these compounds effect on cell viability. The cell index was monitored continuously by visualizing the impedance of the E-plate wells. Because of GSK3β roles in a diverse range of cellular processes like metabolism, cell proliferation, differentiation and survival and its key position at several signaling pathways, GSK3β inhibition was investigated of these molecules. It has been determined that Terbutaline and Orciprenaline inhibits GSK-3β.
 This is the first known study to demonstrate the effects of Terbutaline and Orciprenaline on A549 and the human bronchial epithelial cell line Beas-2b in real time. In this study, it was detected that Terbutaline and especially Orciprenaline may have potential therapeutic effects in lung carcinoma.

A Polyclonal Antibody against a Burkholderia cenocepacia OmpA-like Protein Strongly Impairs Pseudomonas aeruginosa and B. multivorans Virulence.

Despite advances in therapies, bacterial chronic respiratory infections persist as life-threatening to patients suffering from cystic fibrosis (CF). Pseudomonas aeruginosa and bacteria of the Burkholderia cepacia complex are among the most difficult of these infections to treat, due to factors like their resistance to multiple antibiotics and ability to form biofilms. The lack of effective antimicrobial strategies prompted our search for alternative immunotherapies that can effectively control and reduce those infections among CF patients. Previous work from our group showed that the anti-BCAL2645 goat polyclonal antibody strongly inhibited Burkholderia cenocepacia to adhere and invade cultured epithelial cells. In this work, we showed that the polyclonal antibody anti-BCAL2645 also strongly inhibited the ability of P. aeruginosa to form biofilms, and to adhere and invade the human bronchial epithelial cell line CFBE41o-. The polyclonal antibody also inhibited, to a lesser extent, the ability of B. multivorans to adhere and invade the human bronchial epithelial cell line CFBE41o. We also show that the ability of B. cenocepacia, P. aeruginosa and B. multivorans to kill larvae of the Galleria mellonella model of infection was impaired when bacteria were incubated with the anti-BCAL2645 antibody prior to the infection. Our findings show that an antibody against BCAL2645 possesses a significant potential for the development of new immunotherapies against these three important bacterial species capable of causing devastating and often lethal infections among CF patients.

HDAC6-selective inhibitor CAY10603 ameliorates cigarette smoke-induced small airway remodeling by regulating epithelial barrier dysfunction and reversing

BackgroundSmall airway remodelling is a vital characteristic of chronic obstructive pulmonary disease (COPD), which is mainly caused by epithelial barrier dysfunction and epithelial-mesenchymal transition (EMT). Recent studies have indicated that histone deacetylase 6 (HDAC6) plays an important role in the dysregulation of epithelial function. In this study, we investigated the therapeutic effects and underlying mechanisms of an inhibitor with high selectivity for HDAC6 in COPD.MethodsCigarette smoke (CS) exposure was used to establish a CS-induced COPD mouse model. CAY10603 at doses of 2.5 and 10 mg/kg was injected intraperitoneally on alternate days. The protective effects of CAY10603 against CS-induced emphysema, epithelial barrier function and small airway remodeling were evaluated using hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, immunohistochemical staining, and western blot. The human lung bronchial epithelial cell line (HBE) was used to elucidate the underlying molecular mechanism of action of CAY10603.ResultsHDAC6 levels in the lung homogenates of CS-exposed mice were higher than that those in control mice. Compared to the CS group, the mean linear intercept (MLI) of the CAY10603 treatment group decreased and the mean alveolar number (MAN)increased. Collagen deposition was reduced in groups treated with CAY10603. The expression of α-SMA was markedly upregulated in the CS group, which was reversed by CAY10603 treatment. Conversely, E-cadherin expression in the CS group was further downregulated, which was reversed by CAY10603 treatment. CAY10603 affects the tight junction protein expression of ZO-1 and occludin. ZO-1 and occludin expression were markedly downregulated in the CS group. After CAY10603treatment, the protein expression level of ZO-1 and occludin increased significantly. In HBE cells, Cigarette smoke extract (CSE) increased HDAC6 levels. CAY10603 significantly attenuated the release of TGF-β1 induced by CSE. CAY10603 significantly increased the E-cadherin levels in TGF-β1 treated HBE cells, while concurrently attenuated α-SMA expression. This effect was achieved through the suppression of Smad2 and Smad3 phosphorylation. CAY10603 also inhibited TGF-β1 induced cell migration.ConclusionsThese findings suggested that CAY10603 inhibited CS induced small airway remodelling by regulating epithelial barrier dysfunction and reversing EMT via the TGF-β1/Smad2/3 signalling pathway.

Decoding the Link Between XPC and Lung Cancer Susceptibility: A Study of Cigarette Smoke-Induced DNA Damage in the Setting of XPC Deficiency

Background/Objective:
 Despite the known mutagenicity of cigarette smoke, only 10-15% of smokers will develop lung cancer in their lifetimes. What determines a smoker’s susceptibility to lung cancer is poorly understood. We identify the nucleotide excision repair (NER) protein Xeroderma pigmentosum Complementation Group C (XPC) as a tumor suppressor that may contribute to lungtumorigenesis when mutated and combined with cigarette smoke. Micronuclei, which are chromosome fragments and/or lagging chromosomes separated from the main nucleus, occur in many cancers, and indicate genomic instability. We hypothesize that cigarette smoke and XPC knockdown will cause genomic instability that will activate the DNA Damage Response (DDR) and increase the frequency of micronuclei and nuclear aberrancies.
 Methods:
 A human bronchial epithelial cell line (Beas-2B), and two lung adenocarcinoma cell lines (H1299 and A549) with stable XPC lentiviral knock-down (shXPC) or control shRNA (shCtrl) were treated with cigarette smoke extract (CSE) or air control (AC). DNA Damage Response (DDR) proteins were analyzed via immunoblot (Western). Micronuclei and nuclear aberrancies were quantified through cytokinesis-block micronucleus assay (CBMT) using immunofluorescence microscopy (DAPI).
 Results:
 Both CSE and XPC knockdown independently amplify expression of phospho-ATM (pATM), a DDR protein, in H1299 cells. Nuclear aberrancies increased significantly (p<0.05) with CSE in all three cell lines. Micronucleus frequency increased significantly with CSE in H1299 and Beas-2B cells (p<0.05) and with XPC knockdown in Beas-2B cells compared to shCtrl (p<0.001).
 Scientific Impact and Implications:
 We identified a previously uncharacterized role of XPC deficiency in augmenting cigarette smoke induced chromosomal breaks manifesting as micronuclei, particularly in non-cancerous Beas-2B cells. These findings offer insight into tumorigenesis in cigarette smoking and shed light on mechanisms of continued DNA damage in cancer cells. Future research should clarify the mechanisms of micronucleus formation in human translational specimens and pinpoint additional functions of XPC beyond NER, including in replication repair.

Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses.

Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Mechanism through which the hsa-circ_0000992– hsa- miR- 936–AKT3 regulatory network promotes the PM2.5-induced inflammatory response in human bronchial epithelial cells

BackgroundStudies have shown that fine particulate matter (PM2.5) remains a significant problem in developing countries and plays a critical role in the onset and progression of respiratory illnesses. Circular RNAs (circRNAs) are involved in many pathophysiological processes,but their relationship to PM2.5 pollution is largely unexplored. ObjectivesTo elucidate the functional role of hsa_circ_0000992 in PM2.5-induced inflammation in a human bronchial epithelial cell line (16HBE) and to clarify whether the competing endogenous RNA (ceRNA) mechanism is involved in the interrelationships between hsa_circ_0000992 and hsa-miR-936 and the inflammatory signaling pathways. MethodsDetection of inflammatory factors in 16HBE cells exposed to PM2.5 by RT-qPCR and ELISA.High throughput sequencing and bioinformatics analysis methods were used to screen circRNA.The bioinformatics analysis method western blotting and dual-luciferase reporter gene system were used to verify mechanisms associated with circRNA. ResultsPM2.5 cause inflammation in the 16HBE cells. High throughput sequencing and RT-qPCR result revealed that the expression of hsa_circ_0000992 was markedly up-regulated in 16HBE exposed to PM2.5. The binding sites between hsa_circ_0000992 and hsa-miR-936 was confirmed by dual-luciferase reporter gene system.Western blotting and RT-qPCR showed that hsa_circ_0000992 can interact with hsa-miR-936 to regulate AKT serine/threonine kinase 3(AKT3),thereby activating the PI3K/AKT pathway and ultimately promoting the expression of interleukin (IL)− 1β and IL-8. ConclusionPM2.5 can induce the inflammatory response in 16HBE cells by activating the PI3K/AKT pathway. The expression of hsa_circ_0000992 increased when PM2.5 stimulated 16HBE cells,and the circRNA could then regulate the inflammatory response.Hsa_circ_0000992 regulates the hsa-miR-936/AKT3 axis through the ceRNA mechanism,thereby activating the PI3K/AKT signaling pathway,increasing the expression of cellular inflammatory factors,and promoting PM2.5-induced respiratory inflammation.

Oncostatin M's Involvement in the Pathogenesis of Chronic Rhinosinusitis: Focus on Type 1 and 2 Inflammation.

The cytokine oncostatin M (OSM) elicits pathogenic effects involving disruption of the epithelial barrier function as a part of immunological response networks. It is unclear how these integrated cytokine signals influence inflammation and other physiological processes in the pathology of chronic rhinosinusitis (CRS). We investigated the expression and distribution of OSM and OSM receptor (OSMR) in CRS patients' sinonasal specimens, and we compared the results with a panel of inflammatory cytokine levels and clinical features. We classified CRS patients as eosinophilic (ECRS, n = 36) or non-eosinophilic (non-ECRS, n = 35) based on the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis phenotypic criteria and compared their cases with those of 20 control subjects. We also examined OSM's stimulatory effects on cytokine receptor expression levels using the human bronchial epithelium cell line BEAS-2B. RT-PCR showed that the OSM mRNA levels were significantly increased in the CRS patients' ethmoid sinus mucosa. The OSM mRNA levels were positively correlated with those of TNF-α, IL-1β, IL-13, and OSMR-β. In BEAS-2B cells, OSM treatment induced significant increases in the OSMRβ, IL-1R1, and IL-13Ra mRNA levels. OSM is involved in the pathogenesis of CRS in both type 1 and type 2 inflammation, suggesting the OSM signaling pathway as a potential therapeutic target for modulating epithelial stromal interactions.

AIM2 participates in house dust mite (HDM)-induced epithelial dysfunctions and ovalbumin (OVA)-induced allergic asthma in infant mice

Objective: Allergen sensitization and high rates of concomitant allergic diseases are characteristic of severe pediatric asthma. The present study was aimed to explore the mechanism of allergic asthma via bioinformatics and experiment investigation. Methods: The GSE27011 dataset contained the expression profiles of normal and pediatric asthma white blood cells was downloaded for analyzing the different expression genes and function enrichment. The allergic asthma model in infant mice was established by ovalbumin (OVA) stimulation. The cellular model was established by house dust mite (HDM)-stimulation in human bronchial epithelial cells. The absent in melanoma 2 (AIM2) knockdown was achieved by intranasal lentivirus injection or cell infection. The bronchoalveolar lavage fluid (BALF) was collected for cell counting and ELISA assessment of cytokines. Lung tissues were collected for HE staining and immunohistochemical (IHC) staining. Real-time PCR and immunoblotting were used for the determination of key gene expressions in mouse and cell models. Results: upregulation of AIM2 gene expression was observed in pediatric asthma patients based on GSE27011 and OVA-induced infant mouse allergic asthma model. AIM2 knockdown ameliorated OVA caused elevation in airway hyper-responsiveness (AHR), elevation in cell quantities (eosinophils, neutrophils, lymphocytes), and levels of cytokines (IL-4, IL-13, TNF-α, and OVA-specific IgE) in BALF. Moreover, AIM2 knockdown relieved OVA-caused histopathological alterations in mouse lungs, up-regulation of AIM2 levels, and NOD1 and receptor-interacting protein 2 (RIP2) protein levels, as well as p65 phosphorylation. In the cell model, AIM2 knockdown partially ameliorated HDM-induced epithelial dysfunctions by promoting cell viability, down-regulating inflammatory cytokines levels, and decreasing the protein levels of AIM2, NOD1, RIP2, and phosphorylated p65. Conclusion: AIM2 participates in HDM-induced epithelial dysfunctions and OVA-induced allergic asthma progression. AIM2 could be a promising target for pediatric allergic asthma treatment regimens, which warrants further in vivo investigations.

Bei Mu Gua Lou San facilitates mucus expectoration by increasing surface area and hydration levels of airway mucus in an air-liquid-interface cell culture model of the respiratory epithelium

BackgroundBei Mu Gua Lou San (BMGLS) is an ancient formulation known for its moisturizing and expectorant properties, but the underlying mechanisms remain unknown. We investigated concentration-dependent effects of BMGLS on its rehydrating and mucus-modulating properties using an air-liquid-interface (ALI) cell culture model of the Calu-3 human bronchial epithelial cell line and primary normal human bronchial epithelial cells (NHBE), and specifically focused on quantity and composition of the two major mucosal proteins MUC5AC and MUC5B.MethodsALI cultures were treated with BMGLS at different concentrations over three weeks and evaluated by means of histology, immunostaining and electron microscopy. MUC5AC and MUC5B mRNA levels were assessed and quantified on protein level using an automated image-based approach. Additionally, expression levels of the major mucus-stimulating enzyme 15-lipoxygenase (ALOX15) were evaluated.ResultsBMGLS induced concentration-dependent morphological changes in NHBE but not Calu-3 ALI cultures that resulted in increased surface area via the formation of herein termed intra-epithelial structures (IES). While cellular rates of proliferation, apoptosis or degeneration remained unaffected, BMGLS caused swelling of mucosal granules, increased the area of secreted mucus, decreased muco-glycoprotein density, and dispensed MUC5AC. Additionally, BMGLS reduced expression levels of MUC5AC, MUC5B and the mucus-stimulating enzyme 15-lipoxygenase (ALOX15).ConclusionsOur studies suggest that BMGLS rehydrates airway mucus while stimulating mucus secretion by increasing surface areas and regulating goblet cell differentiation through modulating major mucus-stimulating pathways.

MET Inhibitor Capmatinib Radiosensitizes MET Exon 14-Mutated and MET-Amplified Non-Small Cell Lung Cancer

The objective of this study was to investigate the effects of inhibiting the MET receptor with capmatinib, a potent and clinically relevant ATP-competitive tyrosine kinase inhibitor, in combination with radiation in MET exon 14-mutated and MET-amplified non-small cell lung (NSCLC) cancer models. In vitro effects of capmatinib and radiation on cell proliferation, colony formation, MET signaling, apoptosis, and DNA damage repair were evaluated. In vivo tumor responses were assessed in cell line xenograft and patient-derived xenograft models. Immunohistochemistry was used to confirm the in vitro results. In vitro clonogenic survival assays demonstrated radiosensitization with capmatinib in both MET exon 14-mutated and MET-amplified NSCLC cell lines. No radiation-enhancing effect was observed in MET wild-type NSCLC and a human bronchial epithelial cell line. Minimal apoptosis was detected with the combination of capmatinib and radiation. Capmatinib plus radiation compared with radiation alone resulted in inhibition of DNA double-strand break repair, as measured by prolonged expression of γH2AX. In vivo, the combination of capmatinib and radiation significantly delayed tumor growth compared with vehicle control, capmatinib alone, or radiation alone. Immunohistochemistry indicated inhibition of phospho-MET and phospho-S6 and a decrease in Ki67 with inhibition of MET. Inhibition of MET with capmatinib enhances the effect of radiation in both MET exon 14-mutated and MET-amplified NSCLC models.

Knockdown of JMJD3 ameliorates cigarette smoke extract-triggered bronchial epithelial cell injury via ACSL4-dependent ferroptosis

Chronic obstructive pulmonary disease (COPD), a heterogeneity of chronic respiratory disease pattern, presents considerable prevalence and mortality. We aimed to explore the role and mechanisms of Jumonji domain-containing protein-3 (JMJD3) in COPD. The viability and JMJD3 expression in human bronchial epithelial cell line BEAS-2B were respectively assayed by CCK-8 assay and Western blot following stepwise exposure to increasing concentrations of cigarette smoke extract (CSE). After JMJD3 was silenced and acyl-CoA synthetase long-chain family member 4 (ACSL4) was overexpressed in CSE-treated BEAS-2B cells, cell viability, cytotoxicity, oxidative stress and total iron level were estimated using kits. ELISA estimated inflammatory levels. DCFH-DA probe and BODIPY 581/591 C11 probe were exposed to assess ROS production and lipid peroxidation. Western blot tested the expressions of ferroptosis-associated proteins. Besides, H3K27me3 and ACSL4 expressions were tested by Western blot and immunofluorescence staining. In CSE-induced BEAS-2B cells, JMJD3 expression was increased and deletion of JMJD3 improved cell viability, reduced LDH release, mitigated inflammation, oxidative stress and inhibited ferroptosis. Moreover, JMJD3 interference raised H3K27me3 expression whereas lessened ACSL4 expression in CSE-treated BEAS-2B cells. CSE exposure reduced the abundance of ACSL4 in H3K27me3 antibody. Further ACSL4 elevation reversed the impacts of JMJD3 silencing on the damage of CSE-induced BEAS-2B cells. Collectively, JMJD3 depletion might suppress ferroptosis mediated by ACSL4 to alleviate CSE-triggered inflammation and oxidative stress in BEAS-2B cells.

Transcriptomics profile of human bronchial epithelial cells exposed to ambient fine particles and influenza virus (H3N2)

Fine particulate matter (PM2.5) pollution remains a major threat to public health. As the physical barrier against inhaled air pollutants, airway epithelium is a primary target for PM2.5 and influenza viruses, two major environmental insults. Recent studies have shown that PM2.5 and influenza viruses may interact to aggravate airway inflammation, an essential event in the pathogenesis of diverse pulmonary diseases. Airway epithelium plays a critical role in lung health and disorders. Thus far, the mechanisms for the interactive effect of PM2.5 and the influenza virus on gene transcription of airway epithelial cells have not been fully uncovered. In this present pilot study, the transcriptome sequencing approach was introduced to identify responsive genes following individual and co-exposure to PM2.5 and influenza A (H3N2) viruses in a human bronchial epithelial cell line (BEAS-2B). Enrichment analysis revealed the function of differentially expressed genes (DEGs). Specifically, the DEGs enriched in the xenobiotic metabolism by the cytochrome P450 pathway were linked to PM2.5 exposure. In contrast, the DEGs enriched in environmental information processing and human diseases, such as viral protein interaction with cytokines and cytokine receptors and epithelial cell signaling in bacterial infection, were significantly related to H3N2 exposure. Meanwhile, co-exposure to PM2.5 and H3N2 affected G protein-coupled receptors on the cell surface. Thus, the results from this study provides insights into PM2.5- and influenza virus-induced airway inflammation and potential mechanisms.

Semaphorin4A promotes lung cancer by activation of NF-κB pathway mediated by PlexinB1.

Lung cancer (LC) is the most prevalent cancer with a poor prognosis. Semaphorin4A (Sema4A) is important in many physiological and pathological processes. This study aimed to explore the role and mechanism of Sema4A in LC. Firstly, Sema4A expression was analyzed by the available dataset and detected in human normal bronchial epithelial cell line (HBE) and LC cell line (NCI-H460). Then, LC cells were transfected with Sema4A siRNA, and the cells were stimulated by PlexinB1, PlexinB2, PlexinD1 blocking antibodies, IgG antibody, BAY 11-7082 (an inhibitor for NF-κB pathway) and Sema4A-Fc protein, alone or in combination. After transfection, PlexinB1 mRNA expression was analyzed. Next, the biological functions, including proliferative, migratory, invasive abilities and viability of the cells were detected by colony formation, scratch, Transwell and MTT assays, respectively. NF-κB, Stat3 and MAPK protein expressions were determined by western blot. Furthermore, the secretion of IL-6 in LC cells was tested by ELISA. Sema4A was highly expressed in LC tissues and cells, could activate the NF-κB pathway and upregulate PlexinB1 mRNA expression. Furthermore, we observed that Sema4A knockdown suppressed the biological functions of NCI-H460 cells, while Sema4A-Fc protein reversed the situation. However, Sema4A-induced biological functions and activation in the NF-κB pathway were inhibited by PlexinB1 blocking antibody. Consistently, Sema4A promoted IL-6 production, which was down-regulated by PlexinB1 blocking antibody and BAY 11-7082. Sema4A may facilitate LC development via the activation of the NF-κB pathway mediated by PlexinB1, suggesting that Sema4A would be a novel therapeutic target for LC treatment.