Long‐Living Alveolar Macrophages Modulate Severity of Lung Injury and Postnatal Lung Development in Hyperoxia‐Induced Lung Injury Mouse Model

Abstract

RationaleDespite advances in neonatology, bronchopulmonary dysplasia (BPD) continues to be a serious medical problem with long‐term consequences. Alveolar macrophages are long‐living cells which populate lung soon after birth and maintain their population via proliferation, without contribution from circulating monocytes. Normally alveolar macrophages play an essential role in maintaining lung homeostasis and limiting inflammatory response to the pathogens. We hypothesize that neonatal exposure to hyperoxia, via epigenetic mechanisms, may shape these long‐living alveolar macrophages into pro‐inflammatory cells, which contribute to the development of BPD. We sought to investigate the kinetic and phenotype of alveolar macrophages in a mouse model of BPD and to test whether depletion of hyperoxia‐primed alveolar macrophages and recruitment of the monocyte‐derived alveolar macrophages under the normoxic conditions may improve outcomes in mouse model of hyperoxia‐induced lung injury/BPD.MethodsNewborn mice were exposed to room air (normoxia) or 85% O2 (hyperoxia) for 14 days (P0–P14). Alveolar macrophages were depleted via intranasal instillation of clodronate‐loaded liposomes at P14, control group received PBS. Lungs were analyzed using histology, immunohistochemistry, and flow cytometry at P14 and P28. In addition, we performed gene expression profiling of the FACSorted alveolar macrophages, alveolar type 2 epithelial cells and endothelial cells at P14 and 28 via RNA‐seq.ResultsHyperoxia exposure affected lung development (increased mean alveolar area, decreased alveolar count, decreased number of small vessels at P14 and P28). At P14, the number of alveolar macrophages was decreased in hyperoxia‐exposed mice, and alveolar macrophages had an inflammatory phenotype. By P28, the number of alveolar macrophages was expanded in hyperoxia‐exposed mice. Intranasal administration of clodronate‐loaded liposomes at P14 depleted the preexisting pool of alveolar macrophags and induced recruitment of the circulating monocytes to repopulate the niche. This depletion of hyperoxia‐primed inflammatory alveolar macrophages resulted in partial reversal of hyperoxia‐induced lung injury/BPD phenotype (decrease in mean alveolar area, increase in mean alveolar count and number of small vessels). To unravel the contribution of alveolar macrophages to the development and amelioration of hyperoxia lung injury phenotype, we performed transcriptional profiling via RNA‐seq of the whole lung tissue, and FACSorted alveolar macrophages, alveolar type 2 cells and lung endothelial cells during the course of hyperoxia‐induced lung injury with and without treatment with clodronate‐loaded liposomes. We found that at P28 alveolar macrophages from mice exposed to hyperoxia from P0 to P14 had dramatically different gene expression profiles, with a prevalent pro‐inflammatory signature, which was attenuated after treatment with clodronate‐loaded liposomes.ConclusionNeonatal hyperoxia exposure can induce a long‐lasting pro‐inflammatory alveolar macrophage phenotype, most likely via epigenetic mechanisms, which can contribute to the development of hyperoxia‐induced lung injury. Depletion of hyperoxia‐primed alveolar macrophages and recruitment of the new alveolar macrophages under normoxia conditions resulted in a partial reversal of the lung injury/BPD phenotype in mouse.Support or Funding InformationThis work was supported by HL109478 for Dr. Farrow and ATS/Scleroderma Foundation Research Grant for Dr. Misharin.