Autophagy/Mitophagy in Airway Diseases: Impact of Oxidative Stress on Epithelial Cells.

Autophagy is the key process by which the cell degrades parts of itself within the lysosomes. It maintains cell survival and homeostasis by removing molecules (particularly proteins), subcellular organelles, damaged cytoplasmic macromolecules, and by recycling the degradation products. The selective removal or degradation of mitochondria is a particular type of autophagy called mitophagy. Various forms of cellular stress (oxidative stress (OS), hypoxia, pathogen infections) affect autophagy by inducing free radicals and reactive oxygen species (ROS) formation to promote the antioxidant response. Dysfunctional mechanisms of autophagy have been found in different respiratory diseases such as chronic obstructive lung disease (COPD) and asthma, involving epithelial cells. Several existing clinically approved drugs may modulate autophagy to varying extents. However, these drugs are nonspecific and not currently utilized to manipulate autophagy in airway diseases. In this review, we provide an overview of different autophagic pathways with particular attention on the dysfunctional mechanisms of autophagy in the epithelial cells during asthma and COPD. Our aim is to further deepen and disclose the research in this direction to stimulate the develop of new and selective drugs to regulate autophagy for asthma and COPD treatment.

Overview of the Mechanisms of Oxidative Stress: Impact in Inflammation of the Airway Diseases.

Inflammation of the human lung is mediated in response to different stimuli (e.g., physical, radioactive, infective, pro-allergenic or toxic) such as cigarette smoke and environmental pollutants. They often promote an increase in inflammatory activities in the airways that manifest themselves as chronic diseases (e.g., allergic airway diseases, asthma, chronic bronchitis/chronic obstructive pulmonary disease (COPD) or even lung cancer). Increased levels of oxidative stress (OS) reduce the antioxidant defenses, affect the autophagy/mitophagy processes, and the regulatory mechanisms of cell survival, promoting inflammation in the lung. In fact, OS potentiate the inflammatory activities in the lung, favoring the progression of chronic airway diseases. OS increases the production of reactive oxygen species (ROS), including superoxide anions (O2-), hydroxyl radicals (OH) and hydrogen peroxide (H2O2), by the transformation of oxygen through enzymatic and non-enzymatic reactions. In this manner, OS reduces endogenous antioxidant defenses in both nucleated and non-nucleated cells. The production of ROS in the lung can derive from both exogenous insults (cigarette smoke or environmental pollution) and endogenous sources such as cell injury and/or activated inflammatory and structural cells. In this review, we describe the most relevant knowledge concerning the functional interrelation between the mechanisms of OS and inflammation in airway diseases.

Impact of Air Pollution in Airway Diseases: Role of the Epithelial Cells (Cell Models and Biomarkers).

Biomedical research is multidisciplinary and often uses integrated approaches performing different experimental models with complementary functions. This approach is important to understand the pathogenetic mechanisms concerning the effects of environmental pollution on human health. The biological activity of the substances is investigated at least to three levels using molecular, cellular, and human tissue models. Each of these is able to give specific answers to experimental problems. A scientific approach, using biological methods (wet lab), cell cultures (cell lines or primary), isolated organs (three-dimensional cell cultures of primary epithelial cells), and animal organisms, including the human body, aimed to understand the effects of air pollution on the onset of diseases of the respiratory system. Biological methods are divided into three complementary models: in vitro, ex vivo, and in vivo. In vitro experiments do not require the use of whole organisms (in vivo study), while ex vivo experiments use isolated organs or parts of organs. The concept of complementarity and the informatic support are useful tools to organize, analyze, and interpret experimental data, with the aim of discussing scientific notions with objectivity and rationality in biology and medicine. In this scenario, the integrated and complementary use of different experimental models is important to obtain useful and global information that allows us to identify the effect of inhaled pollutants on the incidence of respiratory diseases in the exposed population. In this review, we focused our attention on the impact of air pollution in airway diseases with a rapid and descriptive analysis on the role of epithelium and on the experimental cell models useful to study the effect of toxicants on epithelial cells.

Airway epithelial dysfunction and mesenchymal transition in chronic obstructive pulmonary disease: Role of Oct-4.

The airway epithelium is a dynamic tissue that undergoes slow but constant renewal. Dysregulation of airway epithelial function related to cigarette smoke exposure plays an important role in the pathophysiology of COPD. Oct4 is a transcription factor responsible for maintaining cellular self-renewal and regeneration, and CD146 and CD105/Endoglin are adhesion molecules involved in cell proliferation, differentiation, epithelial-mesenchymal-transition and tissue remodeling. Bronchial biopsy specimens (BBs) were obtained from 7 healthy controls (HC) and 10 COPD and subjected to paraffin embedding; BBs from HC were also used for epithelial cell expansion and pHBEC/ALI (air-liquid interface) culture. pHBEC/ALI were exposed to cigarette smoke extract (CSE) for 7, 14 and 21 days. In BBs, Oct4, CD146 and CD105 were evaluated by immunohistochemistry. In pHBEC/ALI, the expression of Oct4, CD146, CD105 and acetyl-αtubulin was evaluated by Western Blot, MUC5AC and IL-8 measurements by ELISA. The Oct4 epithelial immunoreactivity was lower in COPD than in HC, whilst CD146 and CD105 expression was higher in COPD than in HC. In pHBEC/ALI, Transepithelial Electrical Resistance values, measured over 7 to 21 days of differentiation, decreased by 18% (2.5% CSE) and 29% (5% CSE) compared to untreated samples. Oct4 and acetyl-αtubulin were induced after one-week differentiation and downregulated by CSE in reconstituted epithelium; CD146, CD105, MUC5AC and IL-8 were increased by CSE. Oct4 de-regulation and CD146 and CD105 overexpression, induced by cigarette smoke exposure, might play a role in airway epithelial dysfunction by causing changes in self-renewal and mesenchymal transition mechanisms, leading to alteration of epithelium homeostasis and abnormal tissue remodeling involved in progression of COPD.

Non-Coding RNAs in Airway Diseases: A Brief Overview of Recent Data.

Inflammation of the human lung is mediated in response to different stimuli (e.g., physical, radioactive, infective, pro-allergenic, or toxic) such as cigarette smoke and environmental pollutants. These stimuli often promote an increase in different inflammatory activities in the airways, manifesting themselves as chronic diseases (e.g., allergic airway diseases, asthma chronic bronchitis/chronic obstructive pulmonary disease, or even lung cancer). Non-coding RNA (ncRNAs) are single-stranded RNA molecules of few nucleotides that regulate the gene expression involved in many cellular processes. ncRNA are molecules typically involved in the reduction of translation and stability of the genes of mRNAs s. They regulate many biological aspects such as cellular growth, proliferation, differentiation, regulation of cell cycle, aging, apoptosis, metabolism, and neuronal patterning, and influence a wide range of biologic processes essential for the maintenance of cellular homeostasis. The relevance of ncRNAs in the pathogenetic mechanisms of respiratory diseases has been widely established and in the last decade many papers were published. However, once their importance is established in pathogenetic mechanisms, it becomes important to further deepen the research in this direction. In this review we describe several of most recent knowledge concerning ncRNA (overall miRNAs) expression and activities in the lung.

PBDEs affect inflammatory and oncosuppressive mechanisms via the EZH2 methyltransferase in airway epithelial cells.

AIMS: We aimed to investigate the effect of PBDEs (47, 99, 209) on cellular events involved in epigenetic modification, inflammation, and epithelial mesenchymal transition (EMT). MATERIALS AND METHODS: We studied: 1) ERK1/2 phosphorylation; 2) Enhancer of Zester Homolog 2 (EZH2); 3) Histone H3 tri-methylated in lysine 27 (H3K27me3); 4) K-RAS; 5) silencing disabled homolog 2-interacting protein gene (DAB2IP), 6) let-7a; 7) Muc5AC/Muc5B, and 8) IL-8 in a 3D in vitro model of epithelium obtained with primary Normal Human Bronchial Epithelial cells (pNHBEs) or A549 cell line, chronically exposed to PBDEs (47, 99, 209). KEY FINDINGS: PBDEs (10 nM, 100 nM and 1 µM) increased ERK1/2 phosphorylation, and EZH2, H3K27me3, and K-RAS protein expression, while decreased DAB2IP and Let-7a transcripts in pNHBEs ALI culture. Furthermore PBDEs (47, 99) (100 nM) increased Muc5AC and Muc5B mRNA, and PBDE 47 (100 nM) IL-8 mRNA via EZH2 in pNHBEs. Finally, PBDEs (100 nM) affected EZH2, H3K27me3, K-RAS protein expression, and DAB2IP, Let-7a transcripts and cell invasion in A549 cells. Gsk343 (methyltransferase EZH2 inhibitor) (1 mM) and U0126 (inhibitor of MEK1/2) (10 µM) were used to show the specific effect of PBDEs. SIGNIFICANCE: PBDE inhalation might promote inflammation/cancer via EZH2 methyltransferase activity and H3K27me3, k-RAS and ERk1/2 involvement, generating adverse health outcomes of the human lung.

Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers.

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

Endotyping Seasonal Allergic Rhinitis in Children: A Cluster Analysis.

BACKGROUND: Seasonal Allergic Rhinitis (SAR) is a heterogeneous inflammatory disease. We hypothesized that a cluster analysis based on the evaluation of cytokines in nasal lavage (NL) could characterize distinctive SAR endotypes in children. METHODS: This cross-sectional study enrolled 88 children with SAR. Detailed medical history was obtained by well-trained physicians. Quality of life and sleep quality were assessed through standardized questionnaires [Pediatric Rhinoconjunctivitis Quality of Life Questionnaire (PRQLQ) and Pittsburgh Sleep Quality Index (PSQI) respectively]. Children were grouped through K-means clustering using Interleukin (IL)-5, IL-17, IL-23, and Interferon (INF)-γ in NL. RESULTS: Out of the 88 patients enrolled, 80 were included in the cluster analysis, which revealed three SAR endotypes. Cluster 1 showed lower levels of IL-5 and IL-17 and intermediate levels of IL-23 and IFN-γ; Cluster 2 had higher levels of IL-5 and intermediate levels of IL-17, IL-23, and IFN-γ; Cluster 3 showed higher levels of IL-17, IL-23, and IFN-γ and intermediate levels of IL-5. Cluster 1 showed intermediate values of nasal pH and nasal nitric oxide (nNO), and a lower percentage of neutrophils at nasal cytology than Clusters 2 and 3. Cluster 2 had a lower level of nasal pH, a higher nNO, higher scores in the ocular domain of PRQLQ, and worse sleep quality than Clusters 1 and 3. Cluster 3 showed a higher percentage of neutrophils at nasal cytology than Clusters 1 and 2. CONCLUSIONS: Our study identified three endotypes based on the evaluation of cytokines in NL, highlighting that childhood SAR is characterized by heterogeneous inflammatory cytokines.

Can PBDEs affect the pathophysiologic complex of epithelium in lung diseases?

Brominated flame-retardant (BFRs) exposure promotes multiple adverse health outcomes involved in oxidative stress, inflammation, and tissues damage. We investigated BFR effects, known as polybrominated diphenyl ethers (PBDEs) (47, 99 and 209) in an air-liquid-interface (ALI) airway tissue derived from A549 cell line, and compared with ALI culture of primary human bronchial epithelial cells (pHBEC). The cells, exposed to PBDEs (47, 99 and 209) (0.01-1 µM) for 24 h, were studied for IL-8, Muc5AC and Muc5B (mRNAs and proteins) production, as well as NOX-4 (mRNA) expression. Furthermore, we evaluated tight junction (TJ) integrity by Trans-Epithelial Electrical Resistance (TEER) measurements, and zonula occludens-1 (ZO-1) expression in the cells, and pH variations and rheological properties (elastic G', and viscous G″, moduli) in apical washes of ALI cultures. N-acetylcysteine (NAC) (10 mM) effects were tested in our experimental model of A549 cells. PBDEs (47, 99 and 209) exposure decreased TEER, ZO-1 and pH values, and increased IL-8, Muc5AC, Muc5B (mRNAs and proteins), NOX-4 (mRNA), and rheological parameters (G', G″) in ALI cultures of A549 cell line and pHBEC. NAC inhibited PBDE effects in A549 cells. PBDE inhalation might impairs human health of the lungs inducing oxidative stress, inflammatory response, loss of barrier integrity, unchecked mucus production, as well as altered physicochemical and biological properties of the fluids in airway epithelium. The treatment with anti-oxidants restored the negative effects of PBDEs in epithelial cells.

Cytotoxic and genotoxic effects of the flame retardants (PBDE-47, PBDE-99 and PBDE-209) in human bronchial epithelial cells.

Polybrominated diphenyl ethers (PBDEs) are widespread as flame-retardants in different types of consumer products. PBDEs present in the air or dust and their inhalation can damage human health by influencing the respiratory system. We evaluated the effects of environment relevant concentrations (0.01-1  µM) of PBDE-47, PBDE-99 and PBDE-209 on the mechanism of oxidative stress, dysregulation of cell proliferation, apoptosis, and DNA damage and repair (in term of H2AX phosphorylation ser139) in an in-vitro/ex-vivo model of bronchial epithelial cells. PBDEs (-47, -99 and -209) at the environment relevant concentrations (0.01 and 1  µM) induce oxidative stress (in term of NOX-4 expression as well as ROS and JC-1 production), activate the mechanism of DNA-damage and repair affecting Olive Tail length (comet assay) production and H2AX phosphorylation (ser139) in normal human bronchial epithelial cells. Furthermore PBDEs, although do not affect cell viability, induce cell apoptosis and single cell capacity to grow into a colony (like a cancer phenotype) in bronchial epithelial cells. Finally, PBDE-47 had a greater effect than -99 and -209. PBDE-47, -99 and -209 congeners exert cytotoxic and genotoxic effects, and play a critical role in the dysregulation of oxidative stress, damaging DNA and the related gene expression in bronchial epithelial cells. Our findings might suggest that PBDEs inhalation might have adverse effect on human health regarding pulmonary diseases in the areas of environmental pollution.

A 3D "In Vitro" Model to Study Hyaluronan Effect in Nasal Epithelial Cell Line Exposed to Double-Stranded RNA Poly(I:C).

Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an "in vitro" model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G' and viscous G"), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D "in vitro" model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.

Corrigendum to "Effect of High, Medium, and Low Molecular Weight Hyaluronan on Inflammation and Oxidative Stress in an In Vitro Model of Human Nasal Epithelial Cells".

[This corrects the article DOI: 10.1155/2016/8727289.].

Cigarette smoke affects the onco-suppressor DAB2IP expression in bronchial epithelial cells of COPD patients.

Cigarette smoke is a risk factor for COPD and lung cancer. In cancer, epigenetic modifications affect the expression of Enhancer of Zester Homolog 2 (EZH2), and silenced disabled homolog 2 interacting protein gene (DAB2IP) (onco-suppressor gene) by Histone H3 tri-methylation in lysine 27 (H3K27me3). In"ex vivo"studies, we assessed EZH2, H3K27me3 and DAB2IP immunoreactivity in bronchial epithelial cells from COPD patients (smokers, ex-smokers), Smoker and control subjects. In"in vitro" experiments we studied the effect of cigarette smoke extract (CSE) on EZH2/H3K27me3/DAB2IP expression, apoptosis, invasiveness, and vimentin expression in 16HBE, primary cells, and lung cancer cell lines (A549) long-term exposed to CSE. Finally, in "in vitro"studies, we tested the effect of GSK343 (selective inhibitor of EZH2). EZH2 and H3K27me3 expression was higher, while DAB2IP was lower levels, in bronchial epithelium from COPD and Smokers than in Controls. CSE increased EZH2, H3K27me3 expression and decreased DAB2IP, cell apoptosis and invasiveness in epithelial cells. GSK343 restored the effects of CSE. Cigarette smoke affects EZH2 expression, and reduced DAB2IP via H3K27me3 in COPD patients. The molecular mechanisms associated with EZH2 expression, generate a dysregulation of cell apoptosis, mesenchymal transition, and cell invasiveness in bronchial epithelial cells, encouraging the progression of airway inflammation toward lung cancer in COPD patients.

Targeting quality of life in asthmatic children: The MyTEP pilot randomized trial.

BACKGROUND: Quality of life (QoL) is an important outcome in the management of children with asthma. Mobile Health (m-Health) and Therapeutic Education Programs (TEPs) are increasingly recognized as essential components of pediatric asthma management to improve disease outcomes. OBJECTIVE: To evaluate the effect of an education program (MyTherapeutic Education Program, MyTEP) that couples multidisciplinary TEP intervention with an m-Health Program (mHP) in improving QoL in asthmatic children. METHODS: This single-center study employed a nonblinded randomized clinical trial design. Italian-speaking children (6-11 years) with mild-moderate asthma were eligible for participation. Participants were randomly paired 1:1 with a control group that received mHP (smartphone app) or an intervention group that received MyTEP (TEP plus a smartphone app). Patients were followed up for 3 months. Descriptive statistics, Least Square (LS) mean change and Generalized Linear Mixed model were used for analysis. RESULTS: Fifty patients were enrolled. The Pediatric Asthma Quality of Life Questionnaire (PAQLQ) score improved in both MyTEP (p = 0.014) and mHP (p = 0.046) with the minimally clinically significant difference of ⩾0.5 points reached in 23% of MyTEP and in 16% of mHP. Changes in PAQLQ scores were significantly greater in MyTEP than in mHP (LS mean difference: 0.269 p = 0.05). PAQLQ score was: positively associated with MyTEP (p = 0.023) and study time (p = 0.002); and inversely associated with current passive smoke exposure (p = 0.003). CONCLUSION: Despite the small sample size and short observation period, this study demonstrated that implementing a multidisciplinary TEP with an m-Health program results in gains in QoL of children with asthma.

IL-17A-associated IKK-α signaling induced TSLP production in epithelial cells of COPD patients.

Thymic stromal lymphopoietin (TSLP) is a cytokine expressed in the epithelium, involved in the pathogenesis of chronic disease. IL-17A regulates airway inflammation, oxidative stress, and reduction of steroid sensitivity in chronic obstructive pulmonary disease (COPD). TSLP and IL-17A were measured in induced sputum supernatants (ISs) from healthy controls (HC), healthy smokers (HS), and COPD patients by enzyme-linked immunosorbent assay. Human bronchial epithelial cell line (16HBE) and normal bronchial epithelial cells were stimulated with rhIL-17A or ISs from COPD patients to evaluate TSLP protein and mRNA expression. The effects of the depletion of IL-17A in ISs, an anticholinergic drug, and the silencing of inhibitor kappa kinase alpha (IKKα) on TSLP production were evaluated in 16HBE cells. Coimmunoprecipitation of acetyl-histone H3(Lys14)/IKKα was evaluated in 16HBE cells treated with rhIL-17A and in the presence of the drug. TSLP and IL-17A levels were higher in ISs from COPD patients and HS compared with HC. TSLP protein and mRNA increased in 16HBE cells and in normal bronchial epithelial cells stimulated with ISs from COPD patients compared with ISs from HC and untreated cells. IKKα silencing reduced TSLP production in 16HBE cells stimulated with rhIL-17A and ISs from COPD patients. RhIL-17A increased the IKKα/acetyl-histone H3 immunoprecipitation in 16HBE cells. The anticholinergic drug affects TSLP protein and mRNA levels in bronchial epithelial cells treated with rhIL-17A or with ISs from COPD patients, and IKKα mediated acetyl-histone H3(Lys14). IL-17A/IKKα signaling induced the mechanism of chromatin remodeling associated with acetyl-histone H3(Lys14) and TSLP production in bronchial epithelial cells. Anticholinergic drugs might target TSLP derived from epithelial cells during the treatment of COPD.

Cigarette smoke and non-neuronal cholinergic system in the airway epithelium of COPD patients.

Acetylcholine (ACh), synthesized by Choline Acetyl-Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: "ex vivo" in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and "in vitro" in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co-localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co-localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non-neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.

Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells.

IL-17A is overexpressed in the lung during acute neutrophilic inflammation. Acetylcholine (ACh) increases IL-8 and Muc5AC production in airway epithelial cells. We aimed to characterize the involvement of nonneuronal components of cholinergic system on IL-8 and Muc5AC production in bronchial epithelial cells stimulated with IL-17A. Bronchial epithelial cells were stimulated with recombinant human IL-17A (rhIL-17A) to evaluate the ChAT expression, the ACh binding and production, the IL-8 release, and the Muc5AC production. Furthermore, the effectiveness of PD098,059 (inhibitor of MAPKK activation), Bay11-7082 (inhibitor of IkBα phosphorylation), Hemicholinium-3 (HCh-3) (choline uptake blocker), and Tiotropium bromide (Spiriva®) (anticholinergic drug) was tested in our in vitro model. We showed that rhIL-17A increased the expression of ChAT, the levels of ACh binding and production, and the IL-8 and Muc5AC production in stimulated bronchial epithelial cells compared with untreated cells. The pretreatment of the cells with PD098,059 and Bay11-7082 decreased the ChAT expression and the ACh production/binding, while HCh-3 and Tiotropium decreased the IL-8 and Muc5AC synthesis in bronchial epithelial cells stimulated with rhIL-17A. IL-17A is involved in the IL-8 and Muc5AC production promoting, via NFκB and ERK1/2 pathway activation, the synthesis of ChAT, and the related activity of autocrine ACh in bronchial epithelial cells.

Effect of High, Medium, and Low Molecular Weight Hyaluronan on Inflammation and Oxidative Stress in an In Vitro Model of Human Nasal Epithelial Cells.

IL-17A is involved in the activation of oxidative stress and inflammation in nasal epithelial cells. Hyaluronan (HA) in its high molecular weight form (HMW-HA) shows anti-inflammatory responses in contrast to low and medium molecular weight HA (LMW-HA and MMW-HA). The aim of this study was to investigate the pro- or anti-inflammatory biologic function of HA at different molecular weight in an in vitro model of nasal inflammation IL-17A mediated. We evaluated the ERK1/2 and IκBα phosphorylation, NF-κB signal pathway activation, ROS production, IL-8 and NOX-4 protein, and mRNA levels, in nasal epithelial cells RPMI 2650 stimulated with recombinant human (rh) IL-17A. Furthermore, the cells were treated with HMW-HA, MMW-HA, LMW-HA, and U0126. Our results showed that rhIL-17A increased the ERK1/2, IκBα phosphorylation and NF-κB signal pathway activation, ROS production, IL-8 and NOX-4 proteins, and mRNA levels. The addiction of HMW-HA or U0126 showed a significant downregulatory effect on inflammation due to the rhIL-17A stimulation in nasal epithelial cells. IL-17A is able to generate oxidative stress and inflammation via the activation of ERK1/2/NF-κB pathway in nasal epithelial cells. The HMW-HA might represent a coadjuvant of the classic anti-inflammatory/antioxidative treatment of nasal epithelial cells during IL-17A nasal inflammation.

IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: Effect of Tiotropium.

AIMS: IL-17A plays a key role in the persistence of airway inflammation, oxidative stress, and reduction of steroid-sensitivity in COPD. We studied the effect of IL-17A on chromatin remodeling and IL-8 production. MAIN METHODS: We measured the levels of IL-8 and IL-17A in induced sputum supernatants (ISS) from healthy controls (HCs), healthy smokers (HSs), and COPD patients by enzyme-linked immunosorbent assay (ELISA). A human bronchial epithelial cell line (16HBE) was stimulated with ISS from HCs, HSs, or COPD subjects. IL-8 was evaluated in 16HBE by Western blot and real-time polymerase chain reaction (PCR). Histone deacetylase 2 (HDAC2), acetyl histone H3 (Ac-His H3) (k9) and inhibitor kappa kinase alpha (IKKα) levels were evaluated in the nuclear extract by Western blot. Finally, we evaluated the effect of IL-17A depletion in ISS, the silencing of IKKα, and the anti-inflammatory effects of Tiotropium Spiriva® (100nM) on 16HBE. KEY FINDINGS: IL-8 and IL-17A levels were higher in ISS from COPD patients and HSs than from HCs. IL-8 protein and messenger RNA (mRNA) levels were increased in 16HBE stimulated with ISS from COPD patients compared with untreated cells. Furthermore, ISS from COPD patients reduced the nuclear levels of HDAC2 while increasing the activity of both Ac-His H3 (k9) and IKKα in stimulated 16HBE. IL-17A depletion in ISS and the IKKα silencing in 16HBE significantly increased the nuclear levels of HDAC2, reduced Ac-His H3 (k9), and promoted IL-8 synthesis in stimulated 16HBE. Tiotropium controls the proinflammatory activity generated by ISS from COPD patients in 16HBE. SIGNIFICANCE: IL-17A present in the airway of COPD patients, which induces chromatin remodeling, promotes the release of IL-8 in the bronchial epithelium. Tiotropium is able to control this proinflammatory activity.