Transcriptional Profiling of Lung Macrophages from Preterm Infants Identifies Disease Related Programs

To understand the molecular mechanisms of human lung macrophage development, function, and role in BPD pathogenesis, we conducted a clinical study using isolated tracheal aspirate macrophages from intubated preterm infants born before 30 wk gestation. One hundred twenty-eight patients intubated for respiratory distress syndrome and surfactant administration were consented for the study. Tracheal aspirate cells were collected from intubated preterm infants as detailed below. After instillation of 0.5 ml of normal saline into the endotracheal tube, a suction catheter was introduced into the endotracheal tube and tracheal secretions were aspirated into a sterile mucus trap. The suction catheter was subsequently rinsed with an additional 1.5 ml of sterile saline to clear any remaining aspirate from the catheter into the mucus trap. Cells were collected by centrifugation and resuspended in DMEM with 10% fetal bovine serum, penicillin, and streptomycin. Equal aliquots of resuspended cells were divided between two separate tissue culture treated plates and incubated at 37 ˚C in a humidified environment of 95% air and 5% CO2 to allow macrophage attachment. After 30 min, nonadherent cells were removed by gentle washing and fresh cell culture media was applied. Lipopolysaccharide from E. coli (strain 055:B5, gel purified, Sigma-Aldrich L2637; 250 ng/ml) was added to one of the plates and both were incubated for 4 h at 37 ˚C. After incubation, the media was removed and cells were lysed in TRIzol monophasic solution containing phenol and guanidine isothiocyanate (Thermo Fisher). RNA was isolated using standard protocols for TRIzol extraction. RNA yield and quality were measured using High Sensitivity RNA ScreenTape Analysis (Agilent Tapestation).