Response Of Bronchial Epithelial Cells Research Articles

Exposure to PM2.5 modulate the pro-inflammatory and interferon responses against influenza virus infection in a human 3D bronchial epithelium model

Epidemiological studies showed a positive association between exposure to PM2.5 and the severity of influenza virus infection. However, the mechanisms by which PM2.5 can disrupt antiviral defence are still unclear. From this perspective, the objective of this study was to evaluate the effects of PM2.5 on antiviral signalling in the respiratory epithelium using the bronchial Calu-3 cell line grown at the air-liquid interface. Pre-exposure to PM2.5 before infection with the influenza virus was investigated, as well as a co-exposure. Although a physical interaction between the virus and the particles seems possible, no effect of PM2.5 on viral replication was observed during co-exposure, although a downregulation of IFN-β release was associated to PM2.5 exposure. However, pre-exposure slightly increased the viral nucleoprotein production and the pro-inflammatory response. Conversely, the level of the myxovirus resistance protein A (MxA), an interferon-stimulated gene (ISG) induced by IFN-β, was reduced. Therefore, these results suggest that pre-exposure to PM2.5 could alter the antiviral response of bronchial epithelial cells, increasing their susceptibility to viral infection.

STAT3-Mediated Ferroptosis is Involved in Sepsis-Associated Acute Respiratory Distress Syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) poses a grave danger to life, resulting from sepsis-induced multi-organ failure. Although ferroptosis, a form of iron-dependent lipid peroxidative cell death, has been associated with sepsis-induced ARDS, the specific mechanisms are not fully understood. In this study, we utilized WGCNA, PPI, friends analysis, and six machine learning techniques (Lasso, SVM, RFB, XGBoost, AdaBoost, and LightGBM) to pinpoint STAT3 as a potential diagnostic marker. A significant increase in monocyte and neutrophil levels was observed in patients with sepsis-induced ARDS, as revealed by immune infiltration analyses, when compared to controls. Moreover, there was a positive correlation between STAT3 expression and the level of infiltration. Single-cell analysis uncovered a notable disparity in B-cell expression between sepsis and sepsis-induced ARDS. Furthermore, in vitro experiments using LPS-treated human bronchial epithelial cells (BEAS-2B) and THP1 cells demonstrated a significant increase in STAT3 phosphorylation expression. Additionally, the inhibition of STAT3 phosphorylation by Stattic effectively prevented LPS-induced ferroptosis in both BEAS-2B and THP1 cells. This indicates that the activation of STAT3 phosphorylation promotes ferroptosis in human bronchial epithelial cells in response to LPS. In summary, this research has discovered and confirmed STAT3 as a potential biomarker for the diagnosis and treatment of sepsis-induced ARDS.

Long non-coding RNA SNHG4 aggravates cigarette smoke-induced COPD by regulating miR-144-3p/EZH2 axis

ObjectiveThe purpose of this study was to explore the expression level of SNHG4 in patients with COPD and its diagnostic value in COPD, to probe the biological function of SNHG4 in COPD at the cellular level, and to reveal the interaction between SNHG4 and miR-144-3p/EZH2 axis.MethodsThe serum levels of SNHG4, miR-144-3p and EZH2 in healthy people and patients with COPD were detected by RT-qPCR. The diagnostic value of SNHG4 in COPD was evaluated by ROC curve. Pearson method was chosen to estimate the correlation between SNHG4 and clinical indicators in patients with COPD. Cigarette smoke extract (CSE) was obtained, and Beas-2B cells were exposed with 2% CSE to establish an inflammatory cell model of COPD in vitro. MTT assay was used to detect cell viability, flow cytometry was used to evaluate cell apoptosis, and ELISA was performed to detect inflammatory cytokines. Dual-luciferase reporting assay was carried out to verify the targeting of lncRNA-miRNA or miRNA-mRNA.Results(1) The expression of SNHG4 is decreased in patients with COPD, and the expression level in acute exacerbation COPD was lower than that in stable COPD. SNHG4 demonstrated high diagnostic accuracy in distinguishing between stable and acute exacerbation COPD. (2) The expression of SNHG4 was decreased in CSE-induced Beas-2B cells, and overexpression of SNHG4 was beneficial to alleviate CSE-induced apoptosis and inflammation. (3) The expression of miR-144-3p is up-regulated in patients with COPD and CSE-induced Beas-2B cells. MiR-144-3p has a targeting relationship with SNHG4, which is negatively regulated by SNHG4. Overexpression of miR-144-3p could counteract the beneficial effects of increased SNHG4 on CSE-induced cells. (4) The expression of EZH2 is reduced in patients with COPD and CSE-induced Beas-2B cells. Bioinformatics analysis and luciferase reporter gene confirmed that EZH2 is the downstream target gene of miR-144-3p and is negatively regulated by miR-144-3p.ConclusionThe expression of SNHG4 decreased in patients with COPD, and it may promote the progression of COPD by inhibiting the viability, promoting apoptosis and inflammatory response of bronchial epithelial cells via regulating the miR-144-3p/EZH2 axis.

LncRNA PVT1 induces apoptosis and inflammatory response of bronchial epithelial cells by regulating miR-30b-5p/BCL2L11 axis in COPD

BackgroundChronic obstructive pulmonary disease (COPD) is a serious health burden worldwide with high mortality. LncRNA plasmacytoma variant translocation 1 (PVT1) has been illustrated to serve as a biomarker for COPD progression. Nonetheless, its specific functions and mechanisms in COPD are unclarified.MethodsCigarette smoke extract (CSE) was utilized to stimulate 16HBE cells, and cigarette smoke combining with lipopolysaccharide (LPS) was employed to induce COPD in rats. Western blotting and RT-qPCR were utilized for measuring protein and RNA levels. Flow cytometry was implemented for detecting cell apoptosis. Concentrations of inflammatory factors TNF-α and IFN-γ were examined using ELISA. Luciferase reporter assay was utilized for verifying the interaction between molecules. Hematoxylin–eosin staining was performed for histological analysis of rat lung tissues.ResultsPVT1 was highly expressed in CSE-stimulated 16HBE cells and the lungs of COPD rats. PVT1 depletion restored the viability, restrained apoptosis and hindered inflammatory cytokine production in 16HBE cells under CSE treatment and alleviated pathological damages in COPD rats. PVT1 bound to miR-30b-5p and miR-30b-5p targeted BCL2 like 11 (BCL2L11). Overexpressing BCL2L11 offset the above effects mediated by PVT1 in CSE-triggered 16HBE cells.ConclusionPVT1 enhances apoptosis and inflammation of 16HBE cells under CSE stimulation by modulating miR-30b-5p/BCL2L11 axis.

Recent Clinical Isolates of Enterovirus D68 Have Increased Replication and Induce Enhanced Epithelial Immune Response Compared to the Prototype Fermon Strain.

In 2014, enterovirus D68 (EV-D68), previously associated primarily with mild respiratory illness, caused a large outbreak of severe respiratory illness and, in rare instances, paralysis. We compared the viral binding and replication of eight recent EV-D68 clinical isolates collected both before and during the 2014 outbreak and the prototype Fermon strain from 1962 in cultured HeLa cells and differentiated human primary bronchial epithelial cells (BEC) to understand the possible reasons for the change in virus pathogenicity. We selected pairs of closely related isolates from the same phylogenetic clade that were associated with severe vs. asymptomatic infections. We found no significant differences in binding or replication in HeLa cell cultures between the recent clinical isolates. However, in HeLa cells, Fermon had significantly greater binding (2-3 logs) and virus progeny yields (2-4 logs) but a similar level of replication (1.5-2 log increase in viral RNA from 2 h to 24 h post infection) compared to recent isolates. In differentiated BECs, Fermon and the recent EV-D68 isolates had similar levels of binding; however, the recent isolates produced 1.5-2-log higher virus progeny yields than Fermon due to increased replication. Interestingly, no significant differences in replication were identified between the pairs of genetically close recent EV-D68 clinical isolates despite the observed differences in associated disease severity. We then utilized RNA-seq to define the transcriptional responses in BECs infected with four recent EV-D68 isolates, representing major phylogenetic clades, and the Fermon strain. All the tested clinical isolates induced similar responses in BECs; however, numerous upregulated genes in antiviral and pro-inflammatory response pathways were identified when comparing the response to clinical isolates versus Fermon. These results indicate that the recent emergence in severe EV-D68 cases could be explained by an increased replication efficiency and enhanced inflammatory response induced by newly emerged clinical isolates; however, host factors are likely the main determinants of illness severity.

Bronchial epithelial cell transcriptional responses to inhaled corticosteroids dictate severe asthmatic outcomes

Inhaled corticosteroids (CSs) are the backbone of asthma treatment, improving quality of life, exacerbation rates, and mortality. Although effective for most, a subset of patients with asthma experience CS-resistant disease despite receiving high-dose medication. We sought to investigate the transcriptomic response of bronchial epithelial cells (BECs) to inhaled CSs. Independent component analysis was performed on datasets, detailing the transcriptional response of BECs to CS treatment. The expression of these CS-response components was examined in 2 patient cohorts and investigated in relation to clinical parameters. Supervised learning was used to predict BEC CS responses using peripheral blood gene expression. We identified a signature of CS response that was closely correlated with CS use in patients with asthma. Participants could be separated on the basis of CS-response genes into groups with high and low signature expression. Patients with low expression of CS-response genes, particularly those with a severe asthma diagnosis, showed worse lung function and quality of life. These individuals demonstrated enrichment for T-lymphocyte infiltration in endobronchial brushings. Supervised machine learning identified a 7-gene signature from peripheral blood that reliably identified patients with poor CS-response expression in BECs. Loss of CS transcriptional responses within bronchial epithelium was related to impaired lung function and poor quality of life, particularly in patients with severe asthma. These individuals were identified using minimally invasive blood sampling, suggesting these findings may enable earlier triage to alternative treatments.

Differences in Treatment Response in Bronchial Epithelial Cells from Idiopathic Pulmonary Fibrosis (IPF) Patients: A First Step towards Personalized Medicine?

Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 (p = 0.09) and IL-8 secretion (p = 0.014) were increased in IPF-derived HBE cells and significantly reduced by saracatinib. Finally, saracatinib significantly decreased TGF-β gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.

Peanut lipids influence the response of bronchial epithelial cells to the peanut allergens Ara h 1 and Ara h 2 by decreasing barrier permeability.

Background: Peanut-allergic individuals react upon their first known ingestion of peanuts, suggesting sensitization occurs through non-oral exposure. Increasing evidence suggests that the respiratory tract is a probable site for sensitization to environmental peanuts. However, the response of the bronchial epithelium to peanut allergens has never been explored. Furthermore, food matrix-derived lipids play an important role in allergic sensitization. Objective: To contribute to a better understanding of the mechanisms of allergic sensitization to peanuts via inhalation, by exploring the direct effect of the major peanut allergens Ara h 1 and Ara h 2 and peanut lipids on bronchial epithelial cells. Methods: Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with peanut allergens and/or peanut lipids (PNL). Barrier integrity, transport of allergens across the monolayers, and release of mediators were monitored. Results: Ara h 1 and Ara h 2 impacted the barrier integrity of the 16HBE14o- bronchial epithelial cells and crossed the epithelial barrier. Ara h 1 also induced the release of pro-inflammatory mediators. PNL improved the barrier function of the cell monolayers, decreased paracellular permeability and reduced the amount of allergens crossing the epithelial layer. Conclusion: Our study provides evidence of the transport of Ara h 1 and Ara h 2 across the airway epithelium, of the induction of a pro-inflammatory milieu, and identifies an important role for PNL in controlling the amount of allergens that can cross the epithelial barrier. These, all together, contribute to a better understanding of the effects of peanuts exposure on the respiratory tract.

A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases

AbstractObjectivesInflammasomes induce maturation of the inflammatory cytokines IL‐1β and IL‐18, whose activity is associated with the pathophysiology of a wide range of infectious and inflammatory diseases. As validated therapeutic targets for the treatment of acute and chronic inflammatory diseases, there has been intense interest in developing small‐molecule inhibitors to target inflammasome activity and reduce disease‐associated inflammatory burden.MethodsWe examined the therapeutic potential of a novel small‐molecule inhibitor, and associated derivatives, termed ADS032 to target and reduce inflammasome‐mediated inflammation in vivo. In vitro, we characterised ADS032 function, target engagement and specificity.ResultsWe describe ADS032 as the first dual NLRP1 and NLRP3 inhibitor. ADS032 is a rapid, reversible and stable inflammasome inhibitor that directly binds both NLRP1 and NLRP3, reducing secretion and maturation of IL‐1β in human‐derived macrophages and bronchial epithelial cells in response to the activation of NLPR1 and NLRP3. ADS032 also reduced NLRP3‐induced ASC speck formation, indicative of targeting inflammasome formation. In vivo, ADS032 reduced IL‐1β and TNF‐α levels in the serum of mice challenged i.p. with LPS and reduced pulmonary inflammation in an acute model of lung silicosis. Critically, ADS032 protected mice from lethal influenza A virus challenge, displayed increased survival and reduced pulmonary inflammation.ConclusionADS032 is the first described dual inflammasome inhibitor and a potential therapeutic to treat both NLRP1‐ and NLRP3‐associated inflammatory diseases and also constitutes a novel tool that allows examination of the role of NLRP1 in human disease.

Effect of Flagellin Pre-Exposure on the Inflammatory and Antifungal Response of Bronchial Epithelial Cells to Fungal Pathogens.

Bronchial epithelial cells (BEC) play a crucial role in innate immunity against inhaled fungi. Indeed, in response to microorganisms, BEC synthesize proinflammatory cytokines involved in the recruitment of neutrophils. We have recently shown that BEC exert antifungal activity against Aspergillus fumigatus by inhibiting filament growth. In the present study, we first analyzed the inflammatory and antifungal responses of BEC infected by several fungal species such as Aspergillus spp., Scedosporium apiospermum and Candida albicans, which are frequently isolated from the sputum of people with chronic pulmonary diseases. The airways of these patients, such as people with cystic fibrosis (pwCF), are mainly colonized by P. aeruginosa and secondary by fungal pathogens. We have previously demonstrated that BEC are capable of innate immune memory, allowing them to increase their inflammatory response against A. fumigatus following a previous contact with Pseudomonas aeruginosa flagellin. To identify the impact of bacteria exposure on BEC responses to other fungal infections, we extended the analysis of BEC innate immune memory to Aspergillus spp., Scedosporium apiospermum and Candida albicans infection. Our results show that BEC are able to recognize and respond to Aspergillus spp., S. apiospermum and C. albicans infection and that the modulation of BEC responses by pre-exposure to flagellin varies according to the fungal species encountered. Deepening our knowledge of the innate immune memory of BEC should open new therapeutic avenues to modulate the inflammatory response against polymicrobial infections observed in chronic pulmonary diseases such as CF.

Gene Expression in Bronchial Epithelial Cell Responses to Vanadium Exposure.

Vanadium exposure has the adverse effect on lung function in human, whereas the detailed mechanisms of vanadium exposure-induced pulmonary toxicity are limited. Hence, the present study aimed to investigate the hub genes and signaling pathways related to sodium metavanadate (SMV)-induced pulmonary toxicity. The transcript expression profile GSE36684 downloaded from Gene Expression Omnibus contained eight human bronchial epithelial cell (HBEC) samples including five SMV-treated and three control HBEC samples. Totally 455 differentially expressed genes (DEGs) were screened, especially 201 and 254 genes were up- and down-regulated in the HBECs treated with SMV. Gene ontology analysis suggested that the DEGs were mainly involved in signal transduction, the response to drug, cell proliferation, adhesion, and migration. Pathway analysis demonstrated that the DEGs were primarily participated in NF-κB, Wnt, MAPK, and PI3K-Akt signaling pathways. Moreover, the hub genes, including ITGA5, ITGB3, ITGA2, LAMC2, MMP2, and ITGA4, might contribute to SMV-induced pulmonary toxicity. Our study improves the understanding of the molecular mechanisms by which SMV induced the pulmonary toxicity.

Distinct airway epithelial immune responses after infection with SARS-CoV-2 compared to H1N1

Children are less likely than adults to suffer severe symptoms when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), while influenza A H1N1 severity is comparable across ages except for the very young or elderly. Airway epithelial cells play a vital role in the early defence against viruses via their barrier and immune functions. We investigated viral replication and immune responses in SARS-CoV-2-infected bronchial epithelial cells from healthy paediatric (n = 6; 2.5–5.6 years old) and adult (n = 4; 47–63 years old) subjects and compared cellular responses following infection with SARS-CoV-2 or Influenza A H1N1. While infection with either virus triggered robust transcriptional interferon responses, including induction of type I (IFNB1) and type III (IFNL1) interferons, markedly lower levels of interferons and inflammatory proteins (IL-6, IL-8) were released following SARS-CoV-2 compared to H1N1 infection. Only H1N1 infection caused disruption of the epithelial layer. Interestingly, H1N1 infection resulted in sustained upregulation of SARS-CoV-2 entry factors FURIN and NRP1. We did not find any differences in the epithelial response to SARS-CoV-2 infection between paediatric and adult cells. Overall, SARS-CoV-2 had diminished potential to replicate, affect morphology and evoke immune responses in bronchial epithelial cells compared to H1N1.

Pseudomonas aeruginosa Affects Airway Epithelial Response and Barrier Function During Rhinovirus Infection

Cystic fibrosis (CF) lung disease is aggravated by recurrent and ultimately chronic bacterial infections. One of the key pathogens in adult CF lung disease is P. aeruginosa (PA). In addition to bacteria, respiratory viral infections are suggested to trigger pulmonary exacerbations in CF. To date, little is known on how chronic infections with PA influence susceptibility and response to viral infection. We investigated the interactions between PA, human rhinovirus (HRV) and the airway epithelium in a model of chronic PA infection using differentiated primary bronchial epithelial cells (pBECs) and clinical PA isolates obtained from the respiratory sample of a CF patient. Cells were repeatedly infected with either a mucoid or a non-mucoid PA isolate for 16 days to simulate chronic infection, and subsequently co-infected with HRV. Key cytokines and viral RNA were quantified by cytometric bead array, ELISA and qPCR. Proteolytic degradation of IL-6 was analyzed by Western Blots. Barrier function was assessed by permeability tests and transepithelial electric resistance measurements. Virus infection stimulated the production of inflammatory and antiviral mediators, including interleukin (IL)-6, CXCL-8, tumor necrosis factor (TNF)-α, and type I/III interferons. Co-infection with a non-mucoid PA isolate increased IL-1β protein concentrations (28.88 pg/ml vs. 6.10 pg/ml), but in contrast drastically diminished levels of IL-6 protein (53.17 pg/ml vs. 2301.33 pg/ml) compared to virus infection alone. Conditioned medium obtained from co-infections with a non-mucoid PA isolate and HRV was able to rapidly degrade recombinant IL-6 in a serine protease-dependent manner, whereas medium from individual infections or co-infections with a mucoid isolate had no such effect. After co-infection with HRV and the non-mucoid PA isolate, we detected lower mRNA levels of Forkhead box J1 (FOXJ1) and Cilia Apical Structure Protein (SNTN), markers of epithelial cell differentiation to ciliated cells. Moreover, epithelial permeability was increased and barrier function compromised compared to single infections. These data show that PA infection can influence the response of bronchial epithelial cells to viral infection. Altered innate immune responses and compromised epithelial barrier function may contribute to an aggravated course of viral infection in PA-infected airways.

Early transcriptional responses of bronchial epithelial cells to whole cigarette smoke mirror those of in-vivo exposed human bronchial mucosa

BackgroundDespite the well-known detrimental effects of cigarette smoke (CS), little is known about the complex gene expression dynamics in the early stages after exposure. This study aims to investigate early transcriptomic responses following CS exposure of airway epithelial cells in culture and compare these to those found in human CS exposure studies.MethodsPrimary bronchial epithelial cells (PBEC) were differentiated at the air–liquid interface (ALI) and exposed to whole CS. Bulk RNA-sequencing was performed at 1 h, 4 h, and 24 h hereafter, followed by differential gene expression analysis. Results were additionally compared to data retrieved from human CS studies.ResultsALI-PBEC gene expression in response to CS was most significantly changed at 4 h after exposure. Early transcriptomic changes (1 h, 4 h post CS exposure) were related to oxidative stress, xenobiotic metabolism, higher expression of immediate early genes and pro-inflammatory pathways (i.e., Nrf2, AP-1, AhR). At 24 h, ferroptosis-associated genes were significantly increased, whereas PRKN, involved in removing dysfunctional mitochondria, was downregulated. Importantly, the transcriptome dynamics of the current study mirrored in-vivo human studies of acute CS exposure, chronic smokers, and inversely mirrored smoking cessation.ConclusionThese findings show that early after CS exposure xenobiotic metabolism and pro-inflammatory pathways were activated, followed by activation of the ferroptosis-related cell death pathway. Moreover, significant overlap between these transcriptomic responses in the in-vitro model and human in-vivo studies was found, with an early response of ciliated cells. These results provide validation for the use of ALI-PBEC cultures to study the human lung epithelial response to inhaled toxicants.

Inflammatory modulation of the response of bronchial epithelial cells to lipopolysaccharide with pretreatment by montelukast

Montelukast, a leukotriene receptor antagonist, is the most prescribed nonsteroidal anti-inflammatory drug used as an add-on therapy for asthma. Besides its effect on blocking leukotriene action, montelukast has been proposed to have secondary anti-inflammatory properties. This study aimed to investigate the modulatory effect of montelukast on the expression of major genes involved in airway inflammation (TNF, IL6) and remodeling (MMP9, TGFB1) in response to lipopolysaccharide (LPS) in vitro. The expression of selected genes was measured by quantitative real-time polymerase chain reaction 0h and 24h after LPS stimulation in cells pretreated with montelukast. Montelukast was found to significantly attenuate increased TNF and IL6 gene expression, to have a mild effect on MMP9 and have no effect on TGFB1 expression upon stimulation with LPS. The results of our study indicate that patients on montelukast therapy would have an adequate response to acute microorganism-induced inflammation, so additional anti-inflammatory effects of montelukast should be better exploited.

Phenotype and severity of asthma determines bronchial epithelial immune responses to a viral mimic.

Asthma is characterised by an aggravated immune response to respiratory viral infections. This phenomenon is a clinically well-recognised driver of acute exacerbations, but how different phenotypes of asthma respond immunologically to viruses is unclear. To describe the association between different phenotypes and severity of asthma and bronchial epithelial immune responses to viral stimulation. In the Immunoreact study, healthy subjects (n=10) and 50 patients with asthma were included; 30 (60%) were atopic, and 34 (68%) were eosinophilic; 14 (28%) had severe asthma. All participants underwent bronchoscopy with collection of bronchial brushings. Bronchial epithelial cells (BECs) were expanded and stimulated with the viral replication mimic poly (I:C) (Toll-like receptor (TLR)3 agonist) in vitro. The expression of TLR3-induced pro-inflammatory and antiviral responses of BECs were analysed using reverse transcriptase quantitative PCR and multiplex ELISA and compared across asthma phenotypes and severity of disease. Patients with atopic asthma had increased induction of interleukin (IL)-4, interferon (IFN)-β, IL-6, tumour necrosis factor-α, and IL-1β after poly (I:C) stimulation compared to non-atopic patients, whereas in patients with eosinophilic asthma only IL-6 and IL-8 induction was higher than in non-eosinophilic asthma. Patients with severe asthma displayed a decreased antiviral IFN-β, and increased expression of IL-8, most pronounced in atopic and eosinophilic asthmatics. Furthermore, induction of IL-33 in response to poly (I:C) was increased in severe atopic and in severe eosinophilic asthma, but thymic stromal lymphopoietin only in severe eosinophilic asthma. The bronchial epithelial immune response to a viral mimic stimulation differs between asthma phenotypes and severities, which may be important to consider when targeting novel asthma treatments.

Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNβ or IFNλ increased PD-L1 and PD-L2, and IFNβ or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C–induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C–induced PD-L1. IC87114 enhanced poly I:C–induced gene expression of IFNβ, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C–induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C–induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus–infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

Role of scavenger receptors in silica nanoparticle-induced cytokine responses in bronchial epithelial cells

A major challenge in nanoparticle (NP) research is to elucidate how NPs activate initial targets in cells, leading to cytotoxicity and inflammation. We have previously shown that silica (Si)NPs induce pro-inflammatory responses in bronchial epithelial cells (BEAS-2B) via mechanisms involving transforming growth factor (TGF)-α release, and activation of MAP-kinase p38 and JNK besides NF-κB (p65). In the present study, the roles of scavenger receptors (SRs) in SiNP-induced cytokine responses in BEAS-2B cells were examined by siRNA silencing. Cells exposed to Si10 and Si50 (nominal sizes 10 and 50 nm) showed marked interleukin (IL)-6, CXCL8, IL-1α, IL-1β responses. Transient knockdown of SR-B1, LOX-1 and CXCL16 reduced the Si10- and Si50-induced cytokine responses, to a different magnitude dependent on the particle size, SR and cytokine. Si10-induced TGF-α responses were also markedly reduced by knockdown of SR-B1 and CXCL16. Furthermore, the role of SR-B1 in Si10-induced phosphorylations of p65 and MAP-kinases p38 and JNK were examined, and no significant reductions were observed upon knockdown of SR-B1. In conclusion, LOX-1 and CXCL16 and especially SR-B1 seem to have important roles in mediating cytokine responses and TGF-α release due to SiNP exposure in BEAS-2B cells, without a down-stream role of MAP-kinase and NF-κB.

GSK-3β Inhibitors Attenuate the PM2.5-Induced Inflammatory Response in Bronchial Epithelial Cells.

Background and PurposePM2.5-associated airway inflammation has recently been recognized as pivotal to the development of COPD. Aberrant glycogen synthase kinase (GSK)-3β signaling is linked to the inflammatory response. Therefore, we investigated the effects of GSK-3β inhibitors on the PM2.5-induced inflammatory response in bronchial epithelial cells.MethodsThe production of phosphorylated GSK-3β (p-GSK-3β) was analyzed by immunohistochemistry with PM2.5-induced mice. HBECs were treated with various inhibitors targeting GSK-3β or JNK before PM2.5 stimulation. The production of GSK-3β signaling was analyzed by Western blotting. Inflammatory cytokine production was detected by qRT–PCR and ELISA.ResultsPM2.5 exposure caused lung inflammation, upregulated serum concentrations of HMGB1 and IL-6, decreased IL-10 expression, and significantly attenuated p-GSK-3β production in mice. HBECs exposed to PM2.5 showed significantly reduced p-GSK-3β production, an increased ratio of p-JNK/JNK, increased NF-κB activation and IκB degradation, and upregulated the inflammatory cytokines HMGB1 and IL-6. Intervention with GSK-3β inhibitors TDZD-8 and SB216763 significantly suppressed PM2.5-induced outcomes. Moreover, the JNK inhibitor SP600125 also reduced the level of NF-κB phosphorylation induced by PM2.5. The differences in the levels of inflammation-related cytokines in the TDZD-8 groups were greater than those in the SB216763 groups.ConclusionInhibition of GSK-3β weakens the PM2.5-induced inflammatory response by regulating the JNK/NF-κB signaling pathway in bronchial epithelial cells.

Donor-to-donor variability of human bronchial epithelial cells in response to cigarette smoke

Donor-to-donor variability of human bronchial epithelial cells in response to cigarette smoke