Preclinical studies on susceptibility to viral infection driving lung injury and disruptions to lung development in neonates with bronchopulmonary dysplasia

Abstract

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth, with significant morbidity and mortality, and is experimentally modelled by exposure of newborn mice to hyperoxia to mimic oxygen supplementation of preterm infants. Infants with BPD have a higher risk of acquiring a respiratory virus infection and have a worse disease course. The pathophysiological mechanisms underlying this increased risk of infection and more severe pathology have not been clarified, but include oxygen creating a more permissive environment for virus infection, or oxygen modulating the lung inflammatory response to virus infection. When neonatal mice were exposed to hyperoxia (85% O2) from birth, and then challenged with pneumonia virus of mice (PVM) on postnatal day (P)4, all hyperoxia-exposed pups infected at a 1:1,000 dilution of viral stock exhibited 100% mortality by P12. In contrast, mice maintained in room air survive PVM infection over a range of virus doses (1,1000, 1:2,500, or 1:5,000), highlighting a stark, dose-dependent synergy between oxygen toxicity and viral challenge. By P11, lungs from hyperoxia-exposed mice, with or without PVM infection, exhibit pronounced alveolar simplification, with enlarged airspaces and diminished septation. The PVM infection under conditions of hyperoxia provokes marked inflammatory infiltration, whereas lungs infected under conditions of normoxia (21% O2) retain normal architecture. Whole-body plethysmography revealed a dose-dependent decline in tidal volume, expiratory volume, minute volume, and breathing frequency in hyperoxia-infected mice. Despite identical early viral replication across both oxygen conditions, the addition of hyperoxia before and concomitant with virus infection reshapes inflammatory signaling and tissue remodeling. Gene expression in lung homogenates indicated early increases of Ccl2, Cxcl10, and Ccl3 mRNA transcript abundance under hyperoxia. Antibody microarrays revealed a shift from homeostatic chemokines (CCL2, IL-25) to pro-inflammatory and fibrotic mediators (IL-6, IL-8, TGF-β1-3) upon infection, with prior and concomitant hyperoxia magnifying CCL17, IL-15, and lung fibrosis pathways. Immune cell profiling using single-cell RNA sequencing revealed that hyperoxia depleted tissue-resident alveolar macrophages (TR-AM) and expanded exudate macrophages, dendritic cells, and neutrophils. Under normoxia, PVM infection enriches a virus-responsive TR-AM subset (AM_PVM), while hyperoxia completely eliminates these protective responses. Single-cell transcriptomics confirmed the loss of AM_HYX (a stress-adapted macrophage subset) and AM_PVM cells in hyperoxia-infected mice, with extensive dendritic cell expansion. Intranasal clodronate liposome–mediated TR-AM depletion in normoxia-treated, virus-infected animals mimicked the lethal phenotype noted in hyperoxia-infected animals, with 100% mortality by P15. These mice exhibited excessive lung inflammation, impaired respiratory function, and a hyperoxia-like proteomic signature, even as viral loads reduced. Therefore, TR-AM dysfunction and skewed inflammatory chemokine signaling, and not viral load, were associated with fatal outcome. Together, these findings reveal that hyperoxia disrupts macrophage-mediated immune regulation in the newborn mouse lung, converting a balanced antiviral defense into maladaptive inflammation. Strategies to preserve or restore TR-AM function may therefore protect preterm infants from severe respiratory viral disease. These novel findings might explain the predisposition of BPD patients to a high risk of respiratory virus infection and worse clinical outcomes.