RESUMEN
Lung and brain development is often altered in infants born preterm and exposed to excess oxygen, and this can lead to impaired lung function and neurocognitive abilities later in life. Oxygen-derived reactive oxygen species and the ensuing inflammatory response are believed to be an underlying cause of disease because over-expression of some anti-oxidant enzymes is protective in animal models. For example, neurodevelopment is preserved in mice that ubiquitously express human extracellular superoxide dismutase (EC-SOD) under control of an actin promoter. Similarly, oxygen-dependent changes in lung development are attenuated in transgenic Sftpc EC-SOD mice that over-express EC-SOD in pulmonary alveolar epithelial type II cells. But whether anti-oxidants targeted to the lung provide protection to other organs, such as the brain is not known. Here, we use transgenic Sftpc EC-SOD mice to investigate whether lung-specific expression of EC-SOD also preserves neurodevelopment following exposure to neonatal hyperoxia. Wild type and Sftpc EC-SOD transgenic mice were exposed to room air or 100% oxygen between postnatal days 0-4. At 8 weeks of age, we investigated neurocognitive function as defined by novel object recognition, pathologic changes in hippocampal neurons, and microglial cell activation. Neonatal hyperoxia impaired novel object recognition memory in adult female but not male mice. Behavioral deficits were associated with microglial activation, CA1 neuron nuclear contraction, and fiber sprouting within the hilus of the dentate gyrus (DG). Over-expression of EC-SOD in the lung preserved novel object recognition and reduced the observed changes in neuronal nuclear size and myelin basic protein fiber density. It had no effect on the extent of microglial activation in the hippocampus. These findings demonstrate pulmonary expression of EC-SOD preserves short-term memory in adult female mice exposed to neonatal hyperoxia, thus suggesting anti-oxidants designed to alleviate oxygen-induced lung disease such as in preterm infants may also be neuroprotective.
RESUMEN
An acceptable level of oxygen exposure in preterm infants that maximizes efficacy and minimizes harm has yet to be determined. Quantifying oxygen exposure as an area-under-the curve (OAUC ) has been predictive of later respiratory symptoms among former low birth weight infants. Here, we test the hypothesis that quantifying OAUC in newborn mice can predict their risk for altered lung development and respiratory viral infections as adults. Newborn mice were exposed to room air or a FiO2 of 100% oxygen for 4 days, 60% oxygen for 8 days, or 40% oxygen for 16 days (same cumulative dose of excess oxygen). At 8 weeks of age, mice were infected intranasally with a non-lethal dose of influenza A virus. Adult mice exposed to 100% oxygen for 4 days or 60% oxygen for 8 days exhibited alveolar simplification and altered elastin deposition compared to siblings birthed into room air, as well as increased inflammation and fibrotic lung disease following viral infection. These changes were not observed in mice exposed to 40% oxygen for 16 days. Our findings in mice support the concept that quantifying OAUC over a currently unspecified threshold can predict human risk for respiratory morbidity later in life. Pediatr Pulmonol. 2015; 50:222-230. © 2014 Wiley Periodicals, Inc.
RESUMEN
Premature infants requiring supplemental oxygen are at increased risk for developing bronchopulmonary dysplasia (BPD). Rodent models involving neonatal exposure to excessive oxygen concentrations (hyperoxia) have helped to identify mechanisms of BPD-associated pathology. Genome-wide assessments of the effects of hyperoxia in neonatal mouse lungs could identify novel BPD-related genes and pathways. Newborn C57BL/6 mice were exposed to 100% oxygen for 10 days, and whole lung tissue RNA was used for high-throughput, sequencing-based transcriptomic analysis (RNA-Seq). Significance Analysis of Microarrays and Ingenuity Pathway Analysis were used to identify genes and pathways affected. Expression patterns for selected genes were validated by qPCR. Mechanistic relationships between genes were further tested in cultured mouse lung epithelial cells. We identified 300 genes significantly and substantially affected following acute neonatal hyperoxia. Canonical pathways dysregulated in hyperoxia lungs included nuclear factor (erythryoid-derived-2)-like 2-mediated oxidative stress signaling, p53 signaling, eNOS signaling, and aryl hydrocarbon receptor (Ahr) pathways. Cluster analysis identified Ccnd1, Cdkn1a, and Ahr as critical regulatory nodes in the response to hyperoxia, with Ahr serving as the major effector node. A mechanistic role for Ahr was assessed in lung epithelial cells, and we confirmed its ability to regulate the expression of multiple hyperoxia markers, including Cdkn1a, Pdgfrb, and A2m. We conclude that a global assessment of gene regulation in the acute neonatal hyperoxia model of BPD-like pathology has identified Ahr as one driver of gene dysregulation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hiperoxia/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transcriptoma , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Línea Celular , Análisis por Conglomerados , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genoma , Humanos , Hiperoxia/genética , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de Hidrocarburo de Aril/genética , Transducción de SeñalRESUMEN
Supplemental oxygen used to treat infants born prematurely disrupts angiogenesis and is a risk factor for persistent pulmonary disease later in life. Although it is unclear how neonatal oxygen affects development of the respiratory epithelium, alveolar simplification and depletion of type II cells has been observed in adult mice exposed to hyperoxia between postnatal Days 0 and 4. Because hyperoxia inhibits cell proliferation, we hypothesized that it depleted the adult lung of type II cells by inhibiting their proliferation at birth. Newborn mice were exposed to room air (RA) or hyperoxia, and the oxygen-exposed mice were recovered in RA. Hyperoxia stimulated mRNA expressed by type II (Sftpc, Abca3) and type I (T1α, Aquaporin 5) cells and inhibited Pecam expressed by endothelial cells. 5-Bromo-2'-deoxyuridine labeling and fate mapping with enhanced green fluorescence protein controlled statically by the Sftpc promoter or conditionally by the Scgb1a1 promoter revealed increased Sftpc and Abca3 mRNA seen on Day 4 reflected an increase in expansion of type II cells shortly after birth. When mice were returned to RA, this expanded population of type II cells was slowly depleted until few were detected by 8 weeks. These findings reveal that hyperoxia stimulates alveolar epithelial cell expansion when it disrupts angiogenesis. The loss of type II cells during recovery in RA may contribute to persistent pulmonary diseases such as those reported in children born preterm who were exposed to supplemental oxygen.
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Células Epiteliales Alveolares/patología , Proliferación Celular , Hiperoxia/patología , Alveolos Pulmonares/patología , Células Epiteliales Alveolares/metabolismo , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Hiperoxia/genética , Hiperoxia/metabolismo , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Alveolos Pulmonares/metabolismo , Proteína C Asociada a Surfactante Pulmonar/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Uteroglobina/genética , Proteína Fluorescente RojaRESUMEN
Exposing preterm infants or newborn mice to high concentrations of oxygen disrupts lung development and alters the response to respiratory viral infections later in life. Superoxide dismutase (SOD) has been separately shown to mitigate hyperoxia-mediated changes in lung development and attenuate virus-mediated lung inflammation. However, its potential to protect adult mice exposed to hyperoxia as neonates against viral infection is not known. Here, transgenic mice overexpressing extracellular (EC)-SOD in alveolar type II epithelial cells are used to test whether SOD can alleviate the deviant pulmonary response to influenza virus infection in adult mice exposed to hyperoxia as neonates. Fibrotic lung disease, observed following infection in wild-type (WT) mice exposed to hyperoxia as neonates, was prevented by overexpression of EC-SOD. However, leukocyte recruitment remained excessive, and levels of monocyte chemoattractant protein (MCP)-1 remained modestly elevated following infection in EC-SOD Tg mice exposed to hyperoxia as neonates. Because MCP-1 is often associated with pulmonary inflammation and fibrosis, the host response to infection was concurrently evaluated in adult Mcp-1 WT and Mcp-1 knockout mice exposed to neonatal hyperoxia. In contrast to EC-SOD, excessive leukocyte recruitment, but not lung fibrosis, was dependent upon MCP-1. Our findings demonstrate that neonatal hyperoxia alters the inflammatory and fibrotic responses to influenza A virus infection through different pathways. Therefore, these data suggest that multiple therapeutic strategies may be needed to provide complete protection against diseases attributed to prematurity and early life exposure to oxygen.
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Interacciones Huésped-Patógeno/inmunología , Hiperoxia/complicaciones , Hiperoxia/virología , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/complicaciones , Infecciones por Orthomyxoviridae/virología , Transducción de Señal , Envejecimiento/inmunología , Envejecimiento/patología , Animales , Animales Recién Nacidos , Quimiocina CCL2/metabolismo , Progresión de la Enfermedad , Espacio Extracelular/enzimología , Femenino , Hiperoxia/enzimología , Hiperoxia/patología , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Infecciones por Orthomyxoviridae/enzimología , Infecciones por Orthomyxoviridae/patología , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/patología , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/enzimología , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/prevención & control , Superóxido Dismutasa/metabolismo , Análisis de SupervivenciaRESUMEN
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.
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Displasia Broncopulmonar/etiología , Hiperoxia/fisiopatología , Pulmón/crecimiento & desarrollo , Terapia por Inhalación de Oxígeno/efectos adversos , Respiración Artificial/efectos adversos , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Interacción Gen-Ambiente , Humanos , Hiperoxia/etiología , Recién Nacido , Recien Nacido Prematuro/crecimiento & desarrollo , Estrés OxidativoRESUMEN
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)-ß1 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-ß1 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.
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Animales Recién Nacidos , Hiperoxia/fisiopatología , Fibrosis Pulmonar/inducido químicamente , Animales , Secuencia de Bases , Cartilla de ADN , Inmunohistoquímica , Ratones , Fibrosis Pulmonar/fisiopatología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Oxygen exposure in premature infants is a major risk factor for bronchopulmonary dysplasia and can impair the host response to respiratory viral infections later in life. Similarly, adult mice exposed to hyperoxia as neonates display alveolar simplification associated with a reduced number of alveolar epithelial type II cells and exhibit persistent inflammation, fibrosis, and mortality when infected with influenza A virus. Because type II cells participate in innate immunity and alveolar repair, their loss may contribute to oxygen-mediated sensitivity to viral infection. A genomewide screening of type II cells identified eosinophil-associated RNase 1 (Ear1). Ear1 was also detected in airway epithelium and was reduced in lungs of mice exposed to neonatal hyperoxia. Electroporation-mediated gene delivery of Ear1 to the lung before infection successfully reduced viral replication and leukocyte recruitment during infection. It also diminished the enhanced morbidity and mortality attributed to neonatal hyperoxia. These findings demonstrate that novel epithelial expression of Ear1 functions to limit influenza A virus infection, and its loss contributes to oxygen-associated epithelial injury and fibrosis after infection. People born prematurely may have defects in epithelial innate immunity that increase their risk for respiratory viral infections.
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Neurotoxina Derivada del Eosinófilo/metabolismo , Epitelio/metabolismo , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Oxígeno/farmacología , Ribonucleasas/metabolismo , Envejecimiento/patología , Aire , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Animales Recién Nacidos , Electroporación , Epitelio/efectos de los fármacos , Epitelio/patología , Epitelio/virología , Femenino , Técnicas de Transferencia de Gen , Hiperoxia/complicaciones , Hiperoxia/patología , Hiperoxia/virología , Virus de la Influenza A/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
Oxygen exposure in preterm infants has been associated with altered lung development and increased risk for respiratory viral infections later in life. Although the dose of oxygen sufficient to exert these changes in humans remains unknown, adult mice exposed to 100% oxygen between postnatal days 1-4 exhibit alveolar simplification and increased sensitivity to influenza virus infection. Additionally, two nonlinear thresholds of neonatal oxygen exposures were previously identified that promote modest (between 40% and 60% oxygen) and severe (between 80% and 100% oxygen) changes in lung development. Here, we investigate whether these two thresholds correlate with the severity of lung disease following respiratory viral infection. Adult mice exposed to 100% oxygen at birth, and to a lesser extent 80% oxygen, demonstrated enhanced body weight loss, persistent inflammation, and fibrosis following infection compared with infected siblings exposed to room air at birth. In contrast, the host response to infection was indistinguishable between mice exposed to room air and 40% or 60% oxygen. Interestingly, levels of monocyte chemoattractant protein (MCP)-1 were equivalently elevated in infected mice that had been exposed to 80% or 100% oxygen as neonates. However, reducing levels of MCP-1 using heterozygous Mcp-1 mice did not affect oxygen-dependent changes in the response to infection. Thus lung development and the host response to respiratory viral infection are disrupted by different doses of oxygen. Our findings suggest that measuring lung function alone may not be sufficient to identify individuals born prematurely who have increased risk for respiratory viral infection.