Impaired autophagy in the lower airways and lung parenchyma in stable COPD.

Background: There is increasing evidence of autophagy activation in COPD, but its role is complex and probably regulated through cell type-specific mechanisms. This study aims to investigate the autophagic process at multiple levels within the respiratory system, using different methods to clarify conflicting results reported so far. Methods: This cross-sectional study was performed on bronchial biopsies and peripheral lung samples obtained from COPD patients (30 and 12 per sample type, respectively) and healthy controls (25 and 22 per sample type, respectively), divided by smoking history. Subjects were matched for age and smoking history. We analysed some of the most important proteins involved in autophagosome formation, such as LC3 and p62, as well as some molecules essential for lysosome function, such as lysosome-associated membrane protein 1 (LAMP1). Immunohistochemistry was used to assess the autophagic process in both sample types. ELISA and transcriptomic analysis were performed on lung samples. Results: We found increased autophagic stimulus in smoking subjects, regardless of respiratory function. This was revealed by immunohistochemistry through a significant increase in LC3 (p<0.01) and LAMP1 (p<0.01) in small airway bronchiolar epithelium, alveolar septa and alveolar macrophages. Similar results were obtained in bronchial biopsy epithelium by evaluating LC3B (p<0.05), also increased in homogenate lung tissue using ELISA (p<0.05). Patients with COPD, unlike the others, showed an increase in p62 by ELISA (p<0.05). No differences were found in transcriptomics analysis. Conclusions: Different techniques, applied at post-transcriptional level, confirm that cigarette smoke stimulates autophagy at multiple levels inside the respiratory system, and that autophagy failure may characterise COPD.

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.

Extracellular vesicles from PBDE-47 treated M(LPS) THP-1 macrophages modulate the expression of markers of epithelial integrity, EMT, inflammation and muco-secretion in ALI culture of airway epithelium.

AIMS: The lung epithelial cells form a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli. These cells interact with several other cellular components, of which macrophages are some of the most relevant. We analysed the effects of the PBDE-47 on the microRNA cargo of THP-1 macrophage like derived small Extracellular Vesicles (sEVs) and the effects on A549 lung epithelial cells. MAIN METHODS: sEVs from M(LPS) THP-1 macrophage-like cells after PBDE-47 treatment (sEVsPBDE+LPS) were characterized by nanoparticle tracking analysis and their microRNA cargo studied by qPCR. Confocal microscopy was applied to study sEVs cellular uptake by A549 cells. The expression of tight junctions (TJs), adhesion molecules, inflammation markers and mucus production in A549 cultured in air liquid interface (ALI) conditions were studied by Real Time PCR and confocal microscopy. KEY FINDINGS: sEVsPBDE+LPS microRNA cargo analysis showed that the PBDE-47 modulated the expression of the miR-15a-5p, miR29a-3p, miR-143-3p and miR-122-5p. Furthermore, ALI cultured A549 cells incubated with sEVsPBDE+LPS showed that zonula occludens-1 (p ≤ 0.04), claudin (p ≤ 0.02), E-cadherin (p ≤ 0.006) and Vimentin (p ≤ 0.0008) mRNAs were increased in A549 cells after sEVsPBDE+LPS treatment. Indeed, Interleukin (IL)-8 (p ≤ 0.008) and mucin (MUC5AC and MUC5B) (p ≤ 0.03 and p ≤ 0.0001) mRNA expression were up- and down-regulated, respectively. SIGNIFICANCE: PBDE-47 treated macrophages secrete sEVs with altered microRNA cargo that affect the mRNA expression of TJs, adhesion molecules, cytokines and EMT markers damaging the normal function of the lung epithelium, potentially contributing to the development of lung diseases.

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.

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.

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.

Expression/Activation of PAR-1 in Airway Epithelial Cells of COPD Patients: Ex Vivo/In Vitro Study.

The role of PAR-1 expression and activation was described in epithelial cells from the central and distal airways of COPD patients using an ex vivo/in vitro model. PAR-1 immunoreactivity was studied in epithelial cells from surgical specimens of the central and distal airways of COPD patients and healthy control (HC). Furthermore, PAR-1 expression and activation were measured in both the human bronchial epithelial cell line (16HBE) and normal human bronchial epithelial cells (NHBEs) exposed to cigarette smoke extract (CSE) (10%) or thrombin. Finally, cell proliferation, apoptosis, and IL-8 release were detected in stimulated NHBEs. We identified higher levels of PAR-1 expression/activation in epithelial cells from the central airways of COPD patients than in HC. Active PAR-1 increased in epithelial cells from central and distal airways of COPD, with higher levels in COPD smokers (correlated with pack-years) than in COPD ex-smokers. 16HBE and NHBEs exposed to CSE or thrombin showed increased levels of active PAR-1 (localized in the cytoplasm) than baseline conditions, while NHBEs treated with thrombin or CSE showed increased levels of IL-8 proteins, with an additional effect when used in combination. Smoking habits generate the upregulation of PAR-1 expression/activation in airway epithelial cells, and promoting IL-8 release might affect the recruitment of infiltrating cells in the airways of COPD patients.

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.

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.

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.

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.

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.

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.

Crosstalk between mAChRM3 and ß2AR, via acetylcholine PI3/PKC/PBEP1/Raf-1 MEK1/2/ERK1/2 pathway activation, in human bronchial epithelial cells after long-term cigarette smoke exposure.

BACKGROUND: Cigarette smoke extract (CSE) affects the expression of non-neuronal components of cholinergic system in bronchial epithelial cells and, as PEBP1/Raf-mediated MAPK1/2 and ERK1/2 pathway, promotes inflammation and oxidative stress. AIMS: We studied whether Acetylcholine (ACh) is involved in the mechanism of crosstalk between mAChRM3 and ß2Adrenergic receptors (ß2AR) promoting, via PI3/PKC/PBEP1/Raf/MEK1/2/ERK1/2 activation, ß2AR desensitization, inflammation and, oxidative stress in a bronchial epithelial cell line (16HBE) after long-term exposure to cigarette smoke extract (LECSE). METHODS: We evaluated mAChRM3 and Choline Acetyltransferase (ChAT) expression, ACh production, PEBP1, ERk1/2, and ß2AR phosphorylation, as well as NOX-4, ROS production and IL-8 release in 16HBE after LECSE. The inhibitory activity of Hemicholinium (HCh-3) (a potent choline uptake blocker), LY294002 (a highly selective inhibitor of PI3 kinase), Tiotropium (Spiriva®) (anticholinergic drug) and Olodaterol (ß2AR agonist), were tested in 16HBE after LECSE. RESULTS: mAChRM3, ChAT, ACh activity, pPEBP1, pß2AR, pERK1/2, ROS, NOX-4 and IL-8 increased after LECSE in 16HBE LECSE compared to untreated cells. HCh-3 and LY294002 (alone or in combination) as well as Tiotropium (Spiriva®) or Olodaterol (alone or in combination) all reduced the levels of pPEBP1, pß2AR, pERK1/2, ROS, NOX-4, and IL-8 in 16HBE LECSE compared to untreated cells. CONCLUSIONS: LECSE promotes ACh production which enhances PI3/PKC/PEBP1/Raf-ERK1/2 pathway activation, heterologous ß2AR desensitization, as well as release of inflammatory and oxidative mediators in bronchial epithelial cells. The use of anticholinergic drugs and long-acting ß2-agonists, alone or in combination may be dampen these inflammatory mechanisms when used in combination in some epithelial cell types.