Glycopyrronium bromide inhibits lung inflammation and small airway remodeling induced by subchronic cigarette smoke exposure in mice.

The effects of long-acting muscarinic receptor antagonists (LAMAs) have not been evaluated in a model with simultaneous lung inflammation and small airway remodeling induced by cigarette smoke (CS). We exposed the mice to CS for four weeks with daily treatment with a LAMA (glycopyrronium bromide, NVA237) or its vehicle. Human bronchial epithelial cells (PBECs) and lung fibroblasts were exposed to CS extract (CSE) or acetylcholine with or without NVA237 treatment. We found that NVA237, but not its vehicle, suppressed elevations in inflammatory score, epithelial thickness, and peribronchial collagen deposition in CS-exposed mice. NVA237 alleviated CS-induced increased levels of chemokines, inflammatory cells, and total protein in the bronchoalveolar lavage fluid. NVA237 suppressed acetylcholine- or CSE-induced elevations in IL-8 production in PBECs and elevations in proliferation and collagen production in lung fibroblasts. These phenomena were also prevented by a p44/42 MAPK inhibitor. In conclusion, NVA237 exerted a potent suppressive effect on lung inflammation and small airway remodeling induced by subchronic CS exposure.

miRNA-146a expression positively regulates tumor necrosis factor-α-induced interleukin-8 production in mesenchymal stem cells and differentiated lung epithelial-like cells.

Bone marrow-derived mesenchymal stem cells (BM-MSC) can be differentiated into lung epithelial-like cells (MSC-EC) in vitro. The response of BM-MSC and MSC-EC to stimuli may vary because of their character and differentiation. We aimed to investigate the factors that may influence in vitro differentiation of BM-MSC to MSC-EC. We determined the response of BM-MSC, MSC-EC, bronchial epithelial cells, and alveolar epithelial cells to tumor necrosis factor (TNF)-α stimulation. We also investigated the changes in micro(mi)RNA-146a, miRNA-155, and TNF receptor 1 (TNFR1) expression after stimulation. Our results demonstrate that the addition of transforming growth factor-ß(1) and extracellular matrix collagen are required to facilitate such differentiation. After 3 weeks of culture, the morphological appearance and expression of airway epithelial markers, cytokeratin and Clara cell secretory protein, in MSC-EC were characteristics of lung epithelial cells. In response to TNF-α stimulation, the maximal interleukin (IL)-8 production by BM-MSC at the 24-h time point was 4.8 times greater compared with MSC-EC. TNF-α induced a significant increase in the expression of miRNA-146a in BM-MSC as compared with MSC-EC. miRNA-155 expression remained unchanged after stimulation. TNFR1 mRNA also significantly increased in BM-MSC after TNF-α stimulation. This was not observed in MSC-EC. Transfection with miRNA-146a mimics resulted in a significant increase of miRNA-146a expression and IL-8 production in both types of cells. In contrast, miRNA-146a inhibitors reduced miRNA-146a expression and IL-8 production. Overexpression of miRNA-146a, which positively regulates TNF-α-induced IL-8 release, may enhance the inflammatory response in both BM-MSC and MSC-EC. The expression of miRNA-146a and the response to stimuli may be modulated through mature differentiation of BM-MSC.

Exposure of airway epithelium to bile acids associated with gastroesophageal reflux symptoms: a relation to transforming growth factor-beta1 production and fibroblast proliferation.

RATIONALE: Gastroesophageal reflux (GER) is common in patients with various airway diseases. Airway epithelial cells can release growth factors that promote fibroblast proliferation. Exposure of airway epithelium to bile acids may induce a fibrotic response. OBJECTIVES: To determine how bile acids interact with airway epithelium; particularly, whether transforming growth factor-beta1 secretion and fibroblast proliferation are affected. METHODS: Induced sputum from patients with asthma, GER, or asthma associated with GER symptoms, or from healthy control subjects was collected. Total bile acids were measured by a spectrophotometric enzymatic assay. The major components of bile acids, chenodeoxycholic acid (CD) and glycochenodeoxycholic acid (GCD), were used to stimulate primary airway epithelial cells. Quantitative polymerase chain reaction and Western blotting were applied for messenger RNA expression and signal pathway analysis, respectively. Conditioned medium following CD stimulation was coincubated with fibroblasts for proliferation study. RESULTS: The amount of total bile acids in induced sputum was significantly higher in patients with GER and asthma-associated GER symptoms compared to that of healthy control subjects (p<0.005). CD, but not GCD, significantly induced TGF-beta1 production. TGF-beta1 messenger RNA expression was 2.5-fold increased compared to unstimulated cells. This occurred via p38 mitogen-activated protein (MAP) kinase and activating transcription factor-2 activation. Pretreatment with dexamethasone inhibited TGF-beta1 production at both messenger RNA and protein levels by inhibiting p38 MAP kinase phosphorylation. Conditioned medium from CD-treated epithelial cells enhanced fibroblast proliferation. CONCLUSIONS: Aspiration of bile acids may induce airway fibrosis through the production of TGF-beta1 and fibroblast proliferation. Early intervention to attenuate these processes may reduce fibrogenesis in various airway diseases associated with GER.

Leukotriene C4 induces TGF-beta1 production in airway epithelium via p38 kinase pathway.

Cysteinyl leukotrienes (CysLTs) play an important role in the pathogenesis of airway remodeling. We investigated the interaction between epithelium and CysLTC4, and the contribution of this interaction to airway fibrosis. Human airway epithelial cells were grown on air-liquid interface culture inserts. CysLTC4 was employed to stimulate the cells. Conditioned medium following CysLTC4 stimulation was coincubated with human lung fibroblasts. Our results have demonstrated that CysLTC4 stimulates airway epithelial cells, through a p38 mitogen-activated protein kinase (MAPK) activation mechanism, to produce transforming growth factor beta1 (TGF-beta1), which results in fibroblast proliferation. The selective p38 MAPK inhibitor S203580 successfully inhibits p38 MAPK phosphorylation and subsequent TGF-beta1 production. CysLT1 receptor antagonist montelukast and corticosteroid inhibit TGF-beta1 production at the mRNA and protein levels. When treated with LTC4, the conditioned medium from epithelial cells enhances fibroblast proliferation, this mitogenic effect being attributed to TGF-beta1 and LTC4 remaining in the culture medium. In addition, LTC4 itself acts as a potential growth factor for lung fibroblasts. These data indicate that interactions between LTC4 and airway epithelial cells may contribute to the pathogenesis of airway remodeling. Early intervention to stop these processes may be useful in preventing airway fibrosis in chronic allergic inflammation.