We tested the hypothesis that innate immune signaling in utero could disrupt the structural development of the fetal lung, contributing to the pathogenesis of bronchopulmonary dysplasia. Injection of Escherichia coli lipopolysaccharide (LPS) into the amniotic fluid of E15 BALB/cJ mice increased the luminal volume density of fetal mouse lungs at embryonic day (E) 17 and E18. LPS also increased luminal volume and decreased distal lung branching in fetal mouse lung explants. This effect required NF-B activation and functional Toll-Like Receptor 4. Airway branching may require fibronectin-dependent epithelial-mesenchymal interactions, representing a potential target for innate immune signaling. Anti-fibronectin antibodies and LPS both blocked distal lung branching. By immunofluorescence, fibronectin localized to the clefts between newly formed airways but was restricted to peripheral mesenchymal cells in LPS-exposed explants. These data suggest that LPS may alter the expression pattern of mesenchymal fibronectin, potentially disrupting epithelial-mesenchymal interactions and inhibiting distal airway branching and alveolarization. This mechanism may link innate immune signaling with defects in structural development of the fetal lung. Developmental Dynamics 233:553-561, 2005.

Hyperoxia contributes to the development of bronchopulmonary dysplasia in former premature infants. Injurious environmental factors such as hyperoxia may disrupt distal airway branching and alveolar septation, as these critical stages in lung development occur following birth in extremely premature infants. To test if hyperoxia directly inhibited distal airway branching, we cultured E16 fetal mouse lung explants in either 20% (control) or 95% oxygen (hyperoxia). Hyperoxia reduced the number of distal airways to less than 50% of controls. Explants cultured in 95% oxygen also had fewer complex distal airways compared with controls. Mesenchymal cells adjacent to distal airways in hyperoxic explants appeared apoptotic by phase microscopy. Consistent with increased apoptosis, explants cultured in hyperoxia had increased caspase 3/7 activity compared with controls. Hyperoxia also increased mesenchymal caspase 3 expression and annexin V binding within cultured explants as visualized by fluorescence microscopy. We measured increased annexin V binding in isolated primary fetal lung mesenchymal cells cultured in 95% oxygen suggesting a direct effect on cells within the mesenchyme. Hyperoxia can lead to NF-B activation, which mediates inflammatory cascades and may protect cells from apoptosis. We detected NF-B activation and nuclear p65 localization in explants exposed to 48 h of hyperoxia. Inhibition of NF-B prevented the hyperoxia-induced activation of caspase 3. NF-B activation may therefore contribute to apoptosis in the developing fetal mouse lung following hyperoxia exposure. Our data suggest hyperoxia inhibits distal airway branching and directly induces apoptosis of the fetal mouse lung mesenchyme. (Pediatr Res 59: [185][186][187][188][189][190] 2006) B ranching of distal airway saccules into immature alveoli begins in the canalicular phase of human fetal lung development at 20 -22 wk gestation, equivalent to embryonal day 16 (E16) in mice (1-4). As the fetal lung progresses from the canalicular to alveolar phase of development (30 wk gestation in humans, E18 in mice), mesenchymal cells adjacent airways undergo apoptosis, allowing formation of thin interstitial septae (5). Alveoli then continue to divide and septate following birth. Extremely premature infants delivered between 23-27 wk are therefore born before distal lung branching is complete and before alveolarization begins. Abnormal distal airway branching and dysregulated mesenchymal apoptosis in these premature infants may contribute to the pathogenesis of bronchopulmonary dysplasia(6).The lungs of infants with bronchopulmonary dysplasia contain large, simplified alveolar structures (6). With this pathology, the surface area for gas exchange is reduced and the lungs are prone to heterogeneous patterns of atelectasis and hyperinflation. Both arrested lung development and lung injury contribute to the pathogenesis of bronchopulmonary dysplasia (7). In addition to contributing to lung injury, hyperoxia inhibits normal alveolar development in roden...