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.

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.

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.

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.