Michael A. O’Reilly

The role of hyperoxia in the pathogenesis of experimental BPD

Supplemental oxygen is often used as a life-saving therapy in the treatment of preterm infants. However, its protracted use can lead to the development of bronchopulmonary dysplasia (BPD), and more recently, has been associated with adversely affecting the general health of children and adolescents who were born preterm. Efforts to understand how exposure to excess oxygen can disrupt lung development have historically focused on the interplay between oxidative stress and antioxidant defense mechanisms. However, there has been a growing appreciation for how changes in gene-environment interactions occurring during critically important periods of organ development can profoundly affect human health and disease later in life. Here, we review the concept that oxygen is an environmental stressor that may play an important role at birth to control normal lung development via its interactions with genes and cells. Understanding how changes in the oxygen environment have the potential to alter the developmental programing of the lung, such that it now proceeds along a different developmental trajectory, could lead to novel therapies in the prevention and treatment of respiratory diseases, such as BPD.

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12 h and basal respiration by 48 h. ROS were significantly increased at 24 h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity.

Bronchopulmonary dysplasia early changes leading to long-term consequences

Neonatal chronic lung disease, i.e., bronchopulmonary dysplasia, is characterized by impaired pulmonary development resulting from the impact of different risk factors including infections, hyperoxia, and mechanical ventilation on the immature lung. Remodeling of the extracellular matrix, apoptosis as well as altered growth factor signaling characterize the disease. The immediate consequences of these early insults have been studied in different animal models supported by results from in vitro approaches leading to the successful application of some findings to the clinical setting in the past. Nonetheless, existing information about long-term consequences of the identified early and most likely sustained changes to the developing lung is limited. Interesting results point towards a tremendous impact of these early injuries on the pulmonary repair capacity as well as aging related processes in the adult lung.

Neonatal Hyperoxia Increases Sensitivity of Adult Mice to Bleomycin-Induced Lung Fibrosis

Supplemental oxygen used to treat infants born prematurely constitutes a major risk factor for long-term deficits in lung function and host defense against respiratory infections. Likewise, neonatal oxygen exposure results in alveolar simplification in adult mice, and enhances leukocyte recruitment and fibrosis when adult mice are infected with a sublethal dose of influenza A virus. Because pulmonary fibrosis was not observed in infected adult mice exposed to room air as neonates, previous neonatal oxygen exposure may have reprogrammed how the adult lung responds to epithelial injury. By administering bleomycin to adult mice exposed to room air or hyperoxia as neonates, we tested the hypothesis that neonatal hyperoxia enhances fibrosis when the epithelium is injured by direct fibrotic stimulus. Increased sensitivity to bleomycin-induced lung fibrosis was observed in adult mice exposed to neonatal hyperoxia, and was associated with increased numbers of leukocytes and an accumulation of active transforming growth factor (TGF)-β1 in the lung. Fate mapping of the respiratory epithelium revealed that the epithelial-mesenchymal transition was not a significant source of fibroblasts in room air-exposed or oxygen-exposed mice treated with bleomycin. Instead, the treatment of mice with anti-Gr-1 antibody that depletes neutrophils and myeloid-derived suppressor cells reduced the early activation of TGF-β1 and attenuated hyperoxia-enhanced fibrosis. Because bleomycin and influenza A virus both cause epithelial injury, understanding how neonatal hyperoxia reprograms the epithelial response to these two different injurious agents could lead to new therapeutic opportunities for treating lung diseases attributed to prematurity.

Affect of Early Life Oxygen Exposure on Proper Lung Development and Response to Respiratory Viral Infections.

Children born preterm often exhibit reduced lung function and increased severity of response to respiratory viruses, suggesting that premature birth has compromised proper development of the respiratory epithelium and innate immune defenses. Increasing evidence suggests that premature birth promotes aberrant lung development likely due to the neonatal oxygen transition occurring before pulmonary development has matured. Given that preterm infants are born at a point of time where their immune system is also still developing, early life oxygen exposure may also be disrupting proper development of innate immunity. Here, we review current literature in hopes of stimulating research that enhances understanding of how the oxygen environment at birth influences lung development and host defense. This knowledge may help identify those children at risk for disease and ideally culminate in the development of novel therapies that improve their health.

DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.

Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.

Alternative Progenitor Lineages Regenerate the Adult Lung Depleted of Alveolar Epithelial Type 2 Cells

&NA; An aberrant oxygen environment at birth increases the severity of respiratory viral infections later in life through poorly understood mechanisms. Here, we show that alveolar epithelial cell (AEC) 2 cells (AEC2s), progenitors for AEC1 cells, are depleted in adult mice exposed to neonatal hypoxia or hyperoxia. Airway cells expressing surfactant protein (SP)‐C and ATP binding cassette subfamily A member 3, alveolar pod cells expressing keratin (KRT) 5, and pulmonary fibrosis were observed when these mice were infected with a sublethal dose of HKx31, H3N2 influenza A virus. This was not seen in infected siblings birthed into room air. Genetic lineage tracing studies in mice exposed to neonatal hypoxia or hyperoxia revealed pre‐existing secretoglobin 1a1+ cells produced airway cells expressing SP‐C and ATP binding cassette subfamily A member 3. Pre‐existing Kr5+ progenitor cells produced squamous alveolar cells expressing receptor for advanced glycation endproducts, aquaporin 5, and T1&agr; in alveoli devoid of AEC2s. They were not the source of KRT5+ alveolar pod cells. These oxygen‐dependent changes in epithelial cell regeneration and fibrosis could be recapitulated by conditionally depleting AEC2s in mice using diphtheria A toxin and then infecting with influenza A virus. Likewise, airway cells expressing SP‐C and alveolar cells expressing KRT5 were observed in human idiopathic pulmonary fibrosis. These findings suggest that alternative progenitor lineages are mobilized to regenerate the alveolar epithelium when AEC2s are severely injured or depleted by previous insults, such as an adverse oxygen environment at birth. Because these lineages regenerate AECs in spatially distinct compartments of a lung undergoing fibrosis, they may not be sufficient to prevent disease.

Oxygen-dependent changes in lung development do not affect epithelial infection with influenza A virus

Infants born prematurely often require supplemental oxygen, which contributes to aberrant lung development and increased pulmonary morbidity following a respiratory viral infection. We have been using a mouse model to understand how early-life hyperoxia affects the adult lung response to influenza A virus (IAV) infection. Prior studies showed how neonatal hyperoxia (100% oxygen) increased sensitivity of adult mice to infection with IAV [IAV (A/Hong Kong/X31) H3N2] as defined by persistent inflammation, pulmonary fibrosis, and mortality. Since neonatal hyperoxia alters lung structure, we used a novel fluorescence-expressing reporter strain of H1N1 IAV [A/Puerto Rico/8/34 mCherry (PR8-mCherry)] to evaluate whether it also altered early infection of the respiratory epithelium. Like Hong Kong/X31, neonatal hyperoxia increased morbidity and mortality of adult mice infected with PR8-mCherry. Whole lung imaging and histology suggested a modest increase in mCherry expression in adult mice exposed to neonatal hyperoxia compared with room air-exposed animals. However, this did not reflect an increase in airway or alveolar epithelial infection when mCherry-positive cells were identified and quantified by flow cytometry. Instead, a modest increase in the number of CD45-positive macrophages expressing mCherry was detected. While neonatal hyperoxia does not alter early epithelial infection with IAV, it may increase the activity of macrophages toward infected cells, thereby enhancing early epithelial injury.

The Impact of DNA Damage on Epithelial Cell Maintenance of the Lung

The primary function of the lung is to facilitate the exchange of oxygen and carbon dioxide between air and blood and to exclude or defend against infectious agents and other airborne pollutants. As such, the respiratory epithelium is under constant attack by reactive oxygen species derived from metabolic respiration and the inflammatory response to pathogens in the airway. While reactive oxygen species can damage all macromolecules, oxidative damage to DNA is of great importance because it can affect how cells and hence organs function. DNA lesions activate a family of phosphatidylinositol-3 kinase-related kinases (PIKKs) that phosphorylate numerous substrates, including the tumor suppressor p53, which is critically important for maintaining genome integrity. While oxidized DNA is historically thought to be detrimental to cell function, emerging evidence suggest that it may also be an important post-replication modification that controls gene expression. Understanding how oxidation of nuclear and mitochondrial DNA affects cell function could provide new opportunities for treating lung diseases attributed to oxidant injury to the respiratory epithelium.