Oxidative stress induces club cell proliferation and pulmonary fibrosis in Atp8b1 mutant mice.

Atp8b1 (ATPase, aminophospholipid transporter, class I, type 8B, member 1) is a cardiolipin transporter in the apical membrane of lung epithelial cells. While the role of Atp8b1 in pneumonia-induced acute lung injury (ALI) has been well studied, its potential role in oxidative stress-induced ALI is poorly understood. We herein show that Atp8b1G308V/G308V mice under hyperoxic conditions display exacerbated cell apoptosis at alveolar epithelium and aberrant proliferation of club cells at bronchiolar epithelium. This hyperoxia-induced ambivalent response in Atp8b1G308V/G308V lungs was followed by patchy distribution of non-uniform interstitial fibrosis at late recovery phase under normoxia. Since this club cell abnormality is commonly observed between Atp8b1G308V/G308V lungs under hyperoxic conditions and IPF lungs, we characterized this mouse fibrosis model focusing on club cells. Intriguingly, subcellular morphological analysis of IPF lungs, using transmission electron microscopy (TEM), revealed that metaplastic bronchiolar epithelial cells in fibrotic lesions and deformed type II alveolar epithelial cells (AECs) in alveoli with mild fibrosis, have common morphological features including cytoplasmic vacuolation and dysmorphic lamellar bodies. In conclusion, the combination of Atp8b1 mutation and hyperoxic insult serves as a novel platform to study unfocused role of club cells in IPF.

Resolvins Decrease Oxidative Stress Mediated Macrophage and Epithelial Cell Interaction through Decreased Cytokine Secretion.

BACKGROUND: Inflammation is a key hallmark of ALI and is mediated through ungoverned cytokine signaling. One such cytokine, interleukin-1beta (IL-1ß) has been demonstrated to be the most bioactive cytokine in ALI patients. Macrophages are the key players responsible for IL-1ß secretion into the alveolar space. Following the binding of IL-1ß to its receptor, "activated" alveolar epithelial cells show enhanced barrier dysfunction, adhesion molecule expression, cytokine secretion, and leukocyte attachment. More importantly, it is an important communication molecule between the macrophage and alveolar epithelium. While the molecular determinants of this inflammatory event have been well documented, endogenous resolution processes that decrease IL-1ß secretion and resolve alveolar epithelial cell activation and tissue inflammation have not been well characterized. Lipid mediator Aspirin-Triggered Resolvin D1 (AT-RvD1) has demonstrated potent pro-resolutionary effects in vivo models of lung injury; however, the contribution of the alveoli to the protective benefits of this molecule has not been well documented. In this study, we demonstrate that AT-RvD1 treatment lead to a significant decrease in oxidant induced macrophage IL-1ß secretion and production, IL-1ß-mediated cytokine secretion, adhesion molecule expression, leukocyte adhesion and inflammatory signaling. METHODS: THP-1 macrophages were treated with hydrogen peroxide and extracellular ATP in the presence or absence of AT-RvD1 (1000-0.1 nM). A549 alveolar-like epithelial cells were treated with IL-1ß (10 ng/mL) in the presence or absence of AT-RvD1 (0.1 µM). Following treatment, cell lysate and cell culture supernatants were collected for Western blot, qPCR and ELISA analysis of pro-inflammatory molecules. Functional consequences of IL-1ß induced alveolar epithelial cell and macrophage activation were also measured following treatment with IL-1ß ± AT-RvD1. RESULTS: Results demonstrate that macrophages exposed to H2O2 and ATP in the presence of resolvins show decreased IL-1ß production and activity. A549 cells treated with IL-1ß in the presence of AT-RvD1 show a reduced level of proinflammatory cytokines IL-6 and IL-8. Further, IL-1ß-mediated adhesion molecule expression was also reduced with AT-RvD1 treatment, which was correlated with decreased leukocyte adhesion. AT-RvD1 treatment demonstrated reduced MAP-Kinase signaling. Taken together, our results demonstrate AT-RvD1 treatment reduced IL-1ß-mediated alveolar epithelial cell activation. This is a key step in unraveling the protective effects of resolvins, especially AT-RvD1, during injury.