Mitochondria/cytosol fractionation in vitro. Mitochondria and cytosol were fractionated using the Mitochondria/Cytosol Fractionation Kit according to the manufacturer's protocol (Enzo Life Sciences).Cytotoxicity assays. Cytotoxicity was assessed by measuring the release of LDH into the media (LDH-Cytotoxicity Colorimetric Assay Kit II; BioVision) according to the manufacturer's protocol.Flow cytometry. To discriminate live and dead cells, cells were simultaneously stained with green fluorescent calcein-AM to indicate intracellular esterase activity and red fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes). To assess the functional mitochondrial pool, cells were stained for 20 minutes at 37°C with 100 nM TMRE (Abcam), followed by CSE treatment. mtROS was measured in cells by MitoSOX (Invitrogen) staining (2.5 μM for 10 minutes at 37°C). Data were acquired with aIn vivo CS and chemical treatments. Age-matched mice (6-12 weeks old) were exposed to RA or CS in whole-body exposure chambers as described (5) Human lung bronchial epithelial Beas-2B cells were purchased from ATCC and maintained in DMEM containing 10% FBS and gentamicin (100 μg/ml). The primary alveolar epithelial cells of mouse lung were obtained as previously described and used for experiments before passage (70, 71). CSE was prepared and added to culture media as previously described (5, 6). , and Drp1 in human lung homogenate samples from control subjects and COPD patients. β-Actin served as the standard. PINK1, RIP3, and Drp1 expression was assessed by densitometry of immunoblots. Band intensities were normalized to β-actin. n = 9 samples/group. Representative immunohistochemical study (original magnification, ×200) for PINK1 (B) or RIP3 (C) in human lung sections from never-smokers (n = 3 patients, 5 images/patient) or COPD patients (n = 6 patients, 5 images/patient). Scale bar: 100 μm. Outlined areas are shown enlarged at right (scale bar: 20 μm). (D) Immunofluorescence staining (original magnification, ×40) for PINK1 (green), RIP3 (red), and nuclear (blue) in human lung tissue from never-smokers (n = 2 patients, 3 images/patient) and COPD patients (n = 2 patients, 3 images/patient). Scale bar: 50 μm. Yellow-outlined areas are shown enlarged in bottom panels (scale bar: 10 μm). Data represent the mean ± SEM (A). **P < 0.01 by unpaired, 2-tailed Student's t test (A). The Journal of Clinical Investigation R e s e a R c h a R t i c l e4 0 0 1

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1 +/-or Map1lc3B -/-) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6 -/Y ) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1 -/-, Map1lc3B -/-, and Hdac6 -/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.

Persistent inflammation within the respiratory tract underlies the pathogenesis of numerous chronic pulmonary diseases including chronic obstructive pulmonary disease, asthma and pulmonary fibrosis. Chronic inflammation in the lung may arise from a combination of genetic susceptibility and environmental influences, including exposure to microbes, particles from the atmosphere, irritants, pollutants, allergens, and toxic molecules. To this end, an immediate, strong, and highly regulated inflammatory defense mechanism is needed for the successful maintenance of homeostasis within the respiratory system. Macroautophagy/autophagy plays an essential role in the inflammatory response of the lung to infection and stress. At baseline, autophagy may be critical for inhibiting spontaneous pulmonary inflammation and fundamental for the response of pulmonary leukocytes to infection; however, when not regulated, persistent or inefficient autophagy may be detrimental to lung epithelial cells, promoting lung injury. This perspective will discuss the role of autophagy in driving and regulating inflammatory responses of the lung in chronic lung diseases with a focus on potential avenues for therapeutic targeting. Abbreviations AR allergic rhinitis AM alveolar macrophage ATG autophagy-related CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator COPD chronic obstructive pulmonary disease CS cigarette smoke CSE cigarette smoke extract DC dendritic cell IH intermittent hypoxia IPF idiopathic pulmonary fibrosis ILD interstitial lung disease MAP1LC3B microtubule associated protein 1 light chain 3 beta MTB Mycobacterium tuberculosis MTOR mechanistic target of rapamycin kinase NET neutrophil extracellular traps OSA obstructive sleep apnea PAH pulmonary arterial hypertension PH pulmonary hypertension ROS reactive oxygen species TGFB1 transforming growth factor beta 1 TNF tumor necrosis factor.

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element binding protein 2 (IRP2) as an important COPD susceptibility gene, with IRP2 protein increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RIP-Seq, RNA-Seq, gene expression and functional enrichment clustering analysis, we identified IRP2 as a regulator of mitochondrial function in the lung. IRP2 increased mitochondrial iron loading and cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice with higher mitochondrial iron loading had impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas synthesis of cytochrome c oxidase (Sco2)-deficient mice with reduced COX were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.