Olfactory receptors impact pathophysiological processes of lung diseases in bronchial epithelial cells.

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are limited. Bronchial epithelial cells are key in the pathogenesis by releasing the central proinflammatory cytokine interleukine-8 (IL-8). Olfactory receptors (ORs) are expressed in various cell types. This study examined the drug target potential of ORs by investigating their impact on associated pathophysiological processes in lung epithelial cells. METHODS: Experiments were performed in the A549 cell line and in primary human bronchial epithelial cells. OR expression was investigated using RT-PCR, Western blot, and immunocytochemical staining. OR-mediated effects were analyzed by measuring 1) intracellular calcium concentration via calcium imaging, 2) cAMP concentration by luminescence-based assays, 3) wound healing by scratch assays, 4) proliferation by MTS-based assays, 5) cellular vitality by Annexin V/PI-based FACS staining, and 6) the secretion of IL-8 in culture supernatants by ELISA. RESULTS: By screening 100 potential OR agonists, we identified two, Brahmanol and Cinnamaldehyde, that increased intracellular calcium concentrations. The mRNA and proteins of the corresponding receptors OR2AT4 and OR2J3 were detected. Stimulation of OR2J3 with Cinnamaldehyde reduced 1) IL-8 in the absence and presence of bacterial and viral pathogen-associated molecular patterns (PAMPs), 2) proliferation, and 3) wound healing but increased cAMP. In contrast, stimulation of OR2AT4 by Brahmanol increased wound healing but did not affect cAMP and proliferation. Both ORs did not influence cell vitality. CONCLUSION: ORs might be promising drug target candidates for lung diseases with non-type 2 inflammation. Their stimulation might reduce inflammation or prevent tissue remodeling by promoting wound healing.

Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells.

Airway epithelial cells form a physical barrier against inhaled pathogens and coordinate innate immune responses in the lungs. Bronchial cells in people with cystic fibrosis (pwCF) are colonized by Pseudomonas aeruginosa because of the accumulation of mucus in the lower airways and an altered immune response. This leads to chronic inflammation, lung tissue damage, and accelerated decline in lung function. Thus, identifying the molecular factors involved in the host response in the airways is crucial for developing new therapeutic strategies. The septin (SEPT) cytoskeleton is involved in tissue barrier integrity and anti-infective responses. SEPT7 is critical for maintaining SEPT complexes and for sensing pathogenic microbes. In the lungs, SEPT7 may be involved in the epithelial barrier resistance to infection; however, its role in cystic fibrosis (CF) P. aeruginosa infection is unknown. This study aimed to investigate the role of SEPT7 in controlling P. aeruginosa infection in bronchial epithelial cells, particularly in CF. The study findings showed that SEPT7 encages P. aeruginosa in bronchial epithelial cells and its inhibition downregulates the expression of other SEPTs. In addition, P. aeruginosa does not regulate SEPT7 expression. Finally, we found that inhibiting SEPT7 expression in bronchial epithelial cells (BEAS-2B 16HBE14o- and primary cells) resulted in higher levels of internalized P. aeruginosa and decreased IL-6 production during infection, suggesting a crucial role of SEPT7 in the host response against this bacterium. However, these effects were not observed in the CF cells (16HBE14o-/F508del and primary cells) which may explain the persistence of infection in pwCF. The study findings suggest the modification of SEPT7 expression as a potential approach for the anti-infective control of P. aeruginosa, particularly in CF.

Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium.

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

Membrane lipid composition of bronchial epithelial cells influences antiviral responses during rhinovirus infection.

Lipids and their mediators have important regulatory functions in many cellular processes, including the innate antiviral response. The aim of this study was to compare the lipid membrane composition of in vitro differentiated primary bronchial epithelial cells (PBECs) with ex vivo bronchial brushings and to establish whether any changes in the lipid membrane composition affect antiviral defense of cells from donors without and with severe asthma. Using mass spectrometry, we showed that the lipid membrane of in vitro differentiated PBECs was deprived of polyunsaturated fatty acids (PUFAs) compared to ex vivo bronchial brushings. Supplementation of the culture medium with arachidonic acid (AA) increased the PUFA-content to more closely match the ex vivo membrane profile. Rhinovirus (RV16) infection of AA-supplemented cultures from healthy donors resulted in significantly reduced viral replication while release of inflammatory mediators and prostaglandin E2 (PGE2) was significantly increased. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthases, suppressed RV16-induced PGE2 release and significantly reduced CXCL-8/IL-8 release from AA-supplemented cultures indicating a link between PGE2 and CXCL8/IL-8 release. In contrast, in AA-supplemented cultures from severe asthmatic donors, viral replication was enhanced whereas PTGS2 expression and PGE2 release were unchanged and CXCL8/IL-8 was significantly reduced in response to RV16 infection. While the PTGS2/COX-2 pathway is initially pro-inflammatory, its downstream products can promote symptom resolution. Thus, reduced PGE2 release during an RV-induced severe asthma exacerbation may lead to prolonged symptoms and slower recovery. Our data highlight the importance of reflecting the in vivo lipid profile in in vitro cell cultures for mechanistic studies.

Validation of a Proteomic Signature of Lung Cancer Risk from Bronchial Specimens of Risk-Stratified Individuals.

A major challenge in lung cancer prevention and cure hinges on identifying the at-risk population that ultimately develops lung cancer. Previously, we reported proteomic alterations in the cytologically normal bronchial epithelial cells collected from the bronchial brushings of individuals at risk for lung cancer. The purpose of this study is to validate, in an independent cohort, a selected list of 55 candidate proteins associated with risk for lung cancer with sensitive targeted proteomics using selected reaction monitoring (SRM). Bronchial brushings collected from individuals at low and high risk for developing lung cancer as well as patients with lung cancer, from both a subset of the original cohort (batch 1: n = 10 per group) and an independent cohort of 149 individuals (batch 2: low risk (n = 32), high risk (n = 34), and lung cancer (n = 83)), were analyzed using multiplexed SRM assays. ALDH3A1 and AKR1B10 were found to be consistently overexpressed in the high-risk group in both batch 1 and batch 2 brushing specimens as well as in the biopsies of batch 1. Validation of highly discriminatory proteins and metabolic enzymes by SRM in a larger independent cohort supported their use to identify patients at high risk for developing lung cancer.

Maternal di-(2-ethylhexyl) phthalate exposure elicits offspring IFN-λ upregulation: Insights from birth cohort, murine model, and in vitro mechanistic analysis.

Maternal exposure to di-(2-ethylhexyl)-phthalate (DEHP), an environmental endocrine disruptor, may lead to developmental immunotoxicity in offspring. The causal relationship and underlying mechanism require further study. A subset of Taiwan Maternal and Infant Cohort Study data (n = 283) was analyzed and found a significant association between urinary DEHP metabolite levels from the third trimester of pregnancy and plasma levels of IL-28A and IL-29, named IFNλs, in cord blood. A trans-maternal murine model mimicking human DEHP exposure way showed that bone marrow-derived dendritic cells from maternal DEHP-exposed F1 offspring secreted higher IL-28A levels than control cells, indicating a potential causal relationship. Human bronchial epithelial cell lines treated with DEHP or its primary metabolite, mono-(2-ethyl-5-hexyl) phthalate (MEHP), expressed significantly higher levels of IFNλs mRNA or protein than controls. MEHP's effect on IFNλs expression was blocked by peroxisome proliferator-activated receptor α (PPARα) and PPARγ antagonists, and inhibited by a histone acetyltransferase inhibitor or a histone methyltransferase inhibitor. Chromatin immunoprecipitation assay showed that MEHP treatment promoted histone modifications at H3 and H4 proteins at the promoter regions of Il28a and Il29 genes. These results suggest maternal DEHP exposure could result in high IFNλ expression in offspring, and the health risk of early-life exposure requires further investigation.

Acute cigarette smoke exposure leads to higher viral infection in human bronchial epithelial cultures by altering interferon, glycolysis and GDF15-related pathways.

BACKGROUND: Acute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects. METHODS: Primary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air-liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and L-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, L-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge. RESULTS: CS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and L-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline. CONCLUSION: Altogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.

Mast Cell Tryptase Promotes Airway Remodeling by Inducing Anti-Apoptotic and Cell Growth Properties in Human Alveolar and Bronchial Epithelial Cells.

Bronchial and alveolar remodeling and impaired epithelial function are characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells (MCs) positive for serine proteases, tryptase and chymase, infiltrate the epithelium and alveolar parenchyma. However, little is known regarding the implication of intraepithelial MCs on the local environment, such as epithelial cell function and properties. In this study, we investigated whether MC tryptase is involved in bronchial and alveolar remodeling and the mechanisms of regulation during inflammation. Using novel holographic live cell imaging, we found that MC tryptase enhanced human bronchial and alveolar epithelial cell growth and shortened the cell division intervals. The elevated cell growth induced by tryptase remained in a pro-inflammatory state. Tryptase also increased the expression of the anti-apoptotic protein BIRC3, as well as growth factor release in epithelial cells. Thus, our data imply that the intraepithelial and alveolar MC release of tryptase may play a critical role in disturbing bronchial epithelial and alveolar homeostasis by altering cell growth-death regulation.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

An in vitro evaluation of e-vapor products: The contributions of chemical adulteration, concentration, and device power.

Homemade e-liquids and power-adjustable vaping devices may carry higher risks than commercial formulations and fixed-power devices. This study used human macrophage-like and bronchial epithelial (NHBE) cell cultures to investigate toxicity of homemade e-liquids containing propylene glycol and vegetable glycerin (PG/VG), nicotine, vitamin E acetate (VEA), medium-chain fatty acids (MCFAs), phytol, and cannabidiol (CBD). SmallAir™ organotypic epithelial cultures were exposed to aerosols generated at different power settings (10-50 W). Carbonyl levels were measured, and endpoints reflecting epithelial function (ciliary beating frequency [CBF]), integrity (transepithelial electrical resistance [TEER]), and structure (histology) were investigated. Treatment with nicotine or VEA alone or with PG/VG did not impact cell viability. CBD, phytol, and lauric acid caused cytotoxicity in both culture systems and increased lipid-laden macrophages. Exposure of SmallAir™ organotypic cultures to CBD-containing aerosols resulted in tissue injury and loss of CBF and TEER, while PG/VG alone or with nicotine or VEA did not. Aerosols generated with higher power settings had higher carbonyl concentrations. In conclusion, the presence and concentration of certain chemicals and device power may induce cytotoxicity in vitro. These results raise concerns that power-adjustable devices may generate toxic compounds and suggest that toxicity assessments should be conducted for both e-liquid formulations and their aerosols.

Assessment of Carbon Nanotubes on Barrier Function, Ciliary Beating Frequency and Cytokine Release in In Vitro Models of the Respiratory Tract.

The exposure to inhaled carbon nanotubes (CNT) may have adverse effects on workers upon chronic exposure. In order to assess the toxicity of inhaled nanoparticles in a physiologically relevant manner, an air-liquid interface culture of mono and cocultures of respiratory cells and assessment in reconstructed bronchial and alveolar tissues was used. The effect of CNT4003 reference particles applied in simulated lung fluid was studied in bronchial (Calu-3 cells, EpiAirway™ and MucilAir™ tissues) and alveolar (A549 +/-THP-1 and EpiAlveolar™ +/-THP-1) models. Cytotoxicity, transepithelial electrical resistance, interleukin 6 and 8 secretion, mucociliary clearance and ciliary beating frequency were used as readout parameters. With the exception of increased secretion of interleukin 6 in the EpiAlveolar™ tissues, no adverse effects of CNT4003 particles, applied at doses corresponding to the maximum estimated lifetime exposure of workers, in the bronchial and alveolar models were noted, suggesting no marked differences between the models. Since the doses for whole-life exposure were applied over a shorter time, it is not clear if the interleukin 6 increase in the EpiAlveolar™ tissues has physiological relevance.

[Production of innervated bronchial epithelium from a blood sample]. / Fabrication d'un épithélium bronchique innervé à partir d'une prise de sang.

Asthma is a frequent respiratory disease, with severe asthma occurring in 3 to 5% of cases. Chronic inflammation of the bronchial epithelium is essential to its pathophysiology. When activated by the bronchial environment, the peripheral sensory nervous system contributes to inflammation of the airways. However, due to a lack of reliable models, the mechanisms of action remain largely unknown. Using induced pluripotent stem cells reprogrammed from blood cells, we have set up a model of bronchial epithelium innervated by sensory neurons. This model will ensure better understanding of the mechanisms of action underlying neurogenic inflammation.

Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

Bronchial epithelial cell transcriptome shows endotype heterogeneity of asthma in patients with NSAID-exacerbated respiratory disease.

BACKGROUND: Nonsteroidal anti-inflammatory drugs-exacerbated respiratory disease (N-ERD) is currently classified as a type-2 (T2) immune-mediated disease characterized by asthma, chronic rhinosinusitis, and hypersensitivity to cyclooxygenase-1 inhibitors. OBJECTIVES: The aim of this study was to characterize immunological endotypes of N-ERD based on the gene expression profile in the bronchial epithelium. METHODS: mRNA transcriptome (mRNA-sequencing) was analyzed in bronchial brushings from patients with N-ERD (n = 22), those with nonsteroidal anti-inflammatory drug-tolerant asthma (NTA, n = 21), and control subjects (n = 11). Additionally, lipid and protein mediators were measured in bronchoalveolar lavage fluid (BALF). RESULTS: Initial analysis of the entire asthma group revealed 2 distinct gene expression signatures: "T2-high" with increased expression of T2-related genes (eg, CLCA1, CST1), and "proinflammatory" characterized by the expression of innate immunity (eg, FOSB, EGR3) and IL-17A response genes. These endotypes showed similar prevalence in N-ERD and NTA (eg, T2-high: 33% and 32%, respectively). T2-high asthma was characterized by increased expression of mast cell and eosinophil markers, goblet cell hyperplasia, and elevated LTE4 and PGD2 in BALF. Patients with a proinflammatory endotype showed mainly neutrophilic inflammation and increased innate immunity mediators in BALF. Furthermore, the proinflammatory signature was associated with a more severe course of asthma and marked airway obstruction. These signatures could be recreated in vitro by exposure of bronchial epithelial cells to IL-13 (T2-high) and IL-17A (proinflammatory). CONCLUSIONS: T2-high signature was found only in one-third of patients with N-ERD, which was similar to what was found in patients with NTA. The proinflammatory endotype, which also occurred in N-ERD, suggests a novel mechanism of severe disease developing on a non-T2 background.

Lactobacillus rhamnosus Restores Antiviral Signaling and Attenuates Cytokines Secretion from Human Bronchial Epithelial Cells Exposed to Cigarette Smoke and Infected with SARS-CoV-2.

Individuals with chronic obstructive pulmonary disease (COPD) are more susceptible to exacerbation crisis triggered by secondary lung infections due to the dysfunction of antiviral signaling, principally via suppression of IFN-γ. Although the probiotic is known for controlling pulmonary inflammation in COPD, the influence of the Lactobacillus rhamnosus (Lr) on antiviral signaling in bronchial epithelium exposed to cigarette smoke extract (CSE) and viruses, remains unknown. Thus, the present study investigated the Lr effect on the antiviral signaling and the secretion of inflammatory mediators from bronchial epithelial cells (16HBE cells) exposed to CSE and SARS-CoV-2. The 16HBE cells were cultured, treated with Lr, stimulated with CSE, and infected with SARS-CoV-2. The cellular viability was evaluated using the MTT assay and cytotoxicity measured by lactate dehydrogenase (LDH) activity. The viral load, TLR2, TLR3, TLR4, TLR7, TLR8, MAVS, MyD88, and TRIF were quantified using specific PCR. The pro-inflammatory mediators were measured by a multiplex biometric immunoassay, and angiotensin converting enzyme 2 (ACE2) activity, NF-κB, RIG-I, MAD5, and IRF3 were measured using specific ELISA kits. Lr decreased viral load, ACE2, pro-inflammatory mediators, TLR2, TLR4, NF-κB, TLR3, TLR7, and TLR8 as well as TRIF and MyD88 expression in CSE and SARS-CoV-2 -exposed 16HBE cells. Otherwise, RIG-I, MAD5, IRF3, IFN-γ, and the MAVS expression were restored in 16HBE cells exposed to CSE and SARS-CoV-2 and treated with Lr. Lr induces antiviral signaling associated to IFN-γ secreting viral sensors and attenuates cytokine storm associated to NF-κB in bronchial epithelial cells, supporting its emerging role in prevention of COPD exacerbation.

Bronchus-associated macrophages efficiently capture and present soluble inhaled antigens and are capable of local Th2 cell activation.

In allergic asthma, allergen inhalation leads to local Th2 cell activation and peribronchial inflammation. However, the mechanisms for local antigen capture and presentation remain unclear. By two-photon microscopy of the mouse lung, we established that soluble antigens in the bronchial airway lumen were efficiently captured and presented by a population of CD11c+ interstitial macrophages with high CX3CR1-GFP and MHC class II expression. We refer to these cells as Bronchus-Associated Macrophages (BAMs) based on their localization underneath the bronchial epithelium. BAMs were enriched in collagen-rich regions near some airway branchpoints, where inhaled antigens are likely to deposit. BAMs engaged in extended interactions with effector Th2 cells and promoted Th2 cytokine production. BAMs were also often in contact with dendritic cells (DCs). After exposure to inflammatory stimuli, DCs migrated to draining lymph nodes, whereas BAMs remained lung resident. We propose that BAMs act as local antigen presenting cells in the lung and also transfer antigen to DCs.

Effects of Montelukast on Arsenic-Induced Epithelial-Mesenchymal Transition and the Role of Reactive Oxygen Species Production in Human Bronchial Epithelial Cells.

Background: Epithelial-mesenchymal transition (EMT) of airway lung epithelial cells is considered a major driver of fibrosis and airway remodeling. Arsenic exposure is well known to cause the malignant transformation of cells, including those in the lung. Accumulating studies have shown that arsenic exposure is associated with chronic pulmonary diseases. However, clinical treatment for arsenic-induced pulmonary damage has not been well investigated. Materials and Methods: The therapeutic effects of montelukast and its combination with fluticasone on sodium arsenite-induced EMT changes in normal human bronchial cells were investigated. The cell migration ability was evaluated by Transwell and wound healing assays. EMT marker expression was determined by immunoblotting. Furthermore, the role of reactive oxygen species (ROS) generation in arsenic-induced EMT and the effect of montelukast on this process were determined by ROS inhibitor treatment and ROS measurement, respectively. Results: Montelukast was effective at reducing arsenic-induced cell migration and mesenchymal protein (fibronectin, MMP-2, N-cadherin, ß-catenin, and SMAD2/3) expression. Arsenic-induced ROS production was attenuated by pretreatment with montelukast. Treatment with the ROS inhibitor N-acetyl cysteine reduced arsenic-induced NF-kB phosphorylation and the mesenchymal protein expression, indicating that ROS production is critical for arsenic-induced EMT. In addition, combined treatment with montelukast and fluticasone reversed the inhibitory effects of montelukast on cell migration. The expression of fibronectin, MMP-2 induced by arsenic was further enhanced by the combination treatment compared with montelukast treatment only. Conclusion: This study demonstrated that montelukast is effective at reducing arsenic-induced EMT in human bronchial epithelial cells. Through the inhibition of arsenic-induced ROS generation and NF-kB activation, which is critical for arsenic-induced EMT, montelukast inhibited arsenic-induced cell migration and the expression of extracellular matrix proteins and several EMT-regulating transcription factors. The combination of fluticasone with montelukast reversed the inhibitory effect of montelukast on arsenic-induced EMT. This study provides therapeutic strategies and mechanisms for arsenic-induced pulmonary epithelial damage.

Inflammatory and contractile profile in LPS-challenged equine isolated bronchi: Evidence for IL-6 as a potential target against AHR in equine asthma.

BACKGROUND: Airway inflammation and airway hyperresponsiveness (AHR) are pivotal characteristics of equine asthma. Lipopolysaccharide (LPS) may have a central role in modulating airway inflammation and dysfunction. Therefore, the aim of this study was to match the inflammatory and contractile profile in LPS-challenged equine isolated bronchi to identify molecular targets potentially suitable to counteract AHR in asthmatic horses. METHODS: Equine isolated bronchi were incubated overnight with LPS (0.1-100 ng/ml). The contractile response to electrical field stimulation (EFS) and the levels of cytokines, chemokines, and neurokinin A (NKA) were quantified. The role of capsaicin sensitive-sensory nerves, neurokinin-2 (NK2) receptor, transient receptor potential vanilloid type 1 receptors (TRPV1), and epithelium were also investigated. RESULTS: LPS 1 ng/ml elicited AHR to EFS (+238.17 ± 25.20% P < 0.001 vs. control). LPS significantly (P < 0.05 vs. control) increased the levels of IL-4 (+36.08 ± 1.62%), IL-5 (+38.60 ± 3.58%), IL-6 (+33.79 ± 2.59%), IL-13 (+40.91 ± 1.93%), IL-1ß (+1650.16 ± 71.16%), IL-33 (+88.14 ± 8.93%), TGF-ß (22.29 ± 1.03%), TNF-α (+56.13 ± 4.61%), CXCL-8 (+98.49 ± 17.70%), EOTAXIN (+32.26 ± 2.27%), MCP-1 (+49.63 ± 4.59%), RANTES (+36.38 ± 2.24%), and NKA (+112.81 ± 6.42%). Capsaicin sensitive-sensory nerves, NK2 receptor, and TRPV1 were generally involved in the LPS-mediated inflammation. Epithelium removal modulated the release of IL-1ß, IL-33, and TGF-ß. Only the levels of IL-6 fitted with AHR to a wide range of EFS frequencies, an effect significantly (P < 0.05) inhibited by anti-IL-6 antibody; exogenous IL-6 induced significant (P < 0.05) AHR to EFS similar to that elicited by LPS. CONCLUSION: Targeting IL-6 with specific antibody may represent an effective strategy to treat equine asthma, especially in those animals suffering from severe forms of this disease.

Asthma Susceptibility Gene ORMDL3 Promotes Autophagy in Human Bronchial Epithelium.

The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.