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Although the pathogenesis of pulmonary fibrosis remains unclear, it is known to involve epithelial injury and epithelial-mesenchymal transformation (EMT) as a consequence of cigarette smoke (CS) exposure. Moreover, smoking deposits iron in the mitochondria of alveolar epithelial cells. Iron overload in mitochondria causes the Fenton reaction, leading to reactive oxygen species (ROS) production, and ROS leakage from the mitochondria induces cell injury and inflammation in the lungs. Nevertheless, the mechanisms underlying iron metabolism and pulmonary fibrosis are yet to be elucidated. In this study, we aimed to determine whether iron metabolism and mitochondrial dysfunction are involved in the pathogenesis of pulmonary fibrosis. We demonstrated that administration of the iron chelator deferoxamine (DFO) reduced CS-induced pulmonary epithelial cell death, mitochondrial ROS production, and mitochondrial DNA release. Notably, CS-induced cell death was reduced by the administration of an inhibitor targeting ferroptosis, a unique iron-dependent form of non-apoptotic cell death. Transforming growth factor-β-induced EMT of pulmonary epithelial cells was also reduced by DFO. The preservation of mitochondrial function reduced Transforming growth factor-β-induced EMT. Furthermore, transbronchial iron chelation ameliorated bleomycin-induced pulmonary fibrosis and leukocyte migration in a murine model. Our findings indicate that iron metabolism and mitochondrial dysfunction are involved in the pathogenesis of pulmonary fibrosis. Thus, they may be leveraged as new therapeutic targets for pulmonary fibrosis.

Background: Inhaled corticosteroids enhance airway epithelial barrier integrity. However, the mechanism by which they accomplish this is unclear. Therefore, we investigated steroid-inducible genes and signaling pathways that were involved in enhancing airway epithelial barrier integrity. Methods: A human bronchial epithelial cell line (16HBE cells) was cultured with 10−6 M dexamethasone (DEX) for 3 days to enhance epithelial barrier integrity. After measuring transepithelial electrical resistance (TER) and paracellular permeability, we extracted total RNA from 16HBE cells and performed microarray and pathway analysis. After we identified candidate genes and a canonical pathway, we measured TER and immunostained for tight junction (TJ) and adherent junction (AJ) proteins in cells that had been transfected with specific small interfering RNAs (siRNAs) for these genes. Results: We identified a nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response pathway which was primarily involved in the steroid-induced enhancement of airway epithelial barrier integrity. Transfecting cells with Nrf2 specific siRNA reduced the steroid-induced enhancement of airway epithelial barrier integrity and the accumulation of TJ and AJ proteins at sites of cell–cell contact. Moreover, based on pathway analysis, aldehyde oxidase 1 (AOX1) was identified as a downstream enzyme of Nrf2. Transfecting cells with AOX1-specific siRNA also reduced the steroid-induced enhancement of airway epithelial barrier integrity. Conclusions: Our results indicated that the Nrf2/AOX1 pathway was important for enhancing airway epithelial barrier integrity. Because the airway epithelium of asthmatics is susceptible to reduced barrier integrity, this pathway might be a new therapeutic target for asthma.

Background: Allergic sensitization is a key step in the pathogenesis of asthma. However, little is known about the molecules that are critical regulators for establishing allergic sensitization of the airway. Thus, we conducted global gene expression profiling to identify candidate genes and signaling pathways involved in house dust mite (HDM)-induced allergic sensitization in the murine airway. Methods: We sensitized and challenged mice with HDM or saline as a control through the airway on days 1 and 8. We evaluated eosinophilia in bronchoalveolar lavage fluid (BALF), airway inflammation, and mucus production on days 7 and 14. We extracted total RNA from lung tissues of HDM- and saline-sensitized mice on days 7 and 14. Microarray analyses were performed to identify up-regulated genes in the lungs of HDM-sensitized mice compared to the control mice. Data analyses were performed using GeneSpring software and gene networks were generated using Ingenuity Pathways Analysis (IPA). Results: We identified 50 HDM-mediated, stepwise up-regulated genes in response to allergic sensitization and amplification of allergic airway inflammation. The highest expressed gene was myeloid differentiation-2 (MD-2), a lipopolysaccharide (LPS)-binding component of Toll-like receptor (TLR) 4 signaling complex. MD-2 protein was expressed in lung vascular endothelial cells and was increased in the serum of HDM-sensitized mice, but not in the control mice. Conclusions: Our data suggest MD-2 is a critical regulator of the establishment of allergic airway sensitization to HDM in mice. Serum MD-2 may represent a potential biomarker for the amplification of allergic sensitization and allergic inflammation.