Loss of circadian protection against influenza infection in adult mice exposed to hyperoxia as neonates.

Adverse early-life exposures have a lasting negative impact on health. Neonatal hyperoxia that is a risk factor for bronchopulmonary dysplasia confers susceptibility to influenza A virus (IAV) infection later in life. Given our previous findings that the circadian clock protects against IAV, we asked if the long-term impact of neonatal hyperoxia vis-à-vis IAV infection includes circadian disruption. Here, we show that neonatal hyperoxia abolishes the clock-mediated time of day protection from IAV in mice, independent of viral burden through host tolerance pathways. We discovered that the lung intrinsic clock (and not the central or immune clocks) mediated this dysregulation. Loss of circadian protein, Bmal1, in alveolar type 2 (AT2) cells recapitulates the increased mortality, loss of temporal gating, and other key features of hyperoxia-exposed animals. Our data suggest a novel role for the circadian clock in AT2 cells in mediating long-term effects of early-life exposures to the lungs.

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.

Neonatal hyperoxia alters the host response to influenza A virus infection in adult mice through multiple pathways.

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.

Neonatal oxygen increases sensitivity to influenza A virus infection in adult mice by suppressing epithelial expression of Ear1.

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.

Lung development and the host response to influenza A virus are altered by different doses of neonatal oxygen in mice.

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.