Perinatal lung inflammation in a double-hit model of bronchopulmonary dysplasia results in short-term and long-term downregulation of pulmonary T cell receptor signaling

Purpose: Infants with bronchopulmonary dysplasia (BPD) have increased risk of viral respiratory infections throughout their lifetime. Lung resident immune cells, specifically, T lymphocytes are required for host defense against viral pathogens. Since a growing body of literature implicates that inflammatory environmental exposures during pregnancy and early postnatal period result in life-long alteration in immune cell health, we hypothesized that BPD induced by combined prenatal chorioamnionitis and postnatal hyperoxia results in short-term and long-term dysregulation of T cell receptor signaling in pulmonary immune cells. Methods: Pregnant Sprague-Dawley rats were injected with intra-amniotic LPS (1ug) or normal saline (NS) at 20 days of gestation. Upon delivery, pups were placed in hyperoxia chamber (85% O2) or room air (RA) for 14 days. Lungs were harvested at two weeks of age for short -term analysis or at two months of age for long-term analysis. Harvested lungs were processed into single-cell suspension. CD45+ immune cells were isolated from the single cell suspension using fluorescent activated cell sorting. Total RNA was extracted from immune cells and genome-wide microarray was performed using Affymetrix WT-plus Rat Clariom S Gene chip. Gene ontology terms of biological processes of differentially expressed genes with FDR < 0.01 were analyzed. Differentially expressed genes associated with T cell receptor signaling pathway were validated using RT-PCR. Results: Lungs from animals treated with prenatal LPS and postnatal O2 had significantly greater mean linear intercept indicating alveolar simplification (32±2 vs. 25±0.8; LPS+O2 vs. NS+RA, p < 0.001, n=5 per group). Purity of sorted immune cells was verified using qPCR. Percentage of CD45+ immune cells as a fraction of all pulmonary cells was similar between the two groups (27% vs. 21%, p = 0.25). Analysis of differentially expressed genes showed downregulation of several key T cell related biological processes (Fig.1). Specifically, genes involved in T cell receptor signaling were significantly downregulated in LPS+O2 group, compared to NS+ RA group. qPCR validation of select T-cell receptor genes showed significant downregulation of Cd3d, Cd3e, Cd5, Trat1, CD8a at two weeks (p <0.05, n = 5 per group) in LPS+O2 group compared to NS+RA group. Despite recovery in room air for six weeks, Cd8a gene expression remained significantly lower in LPS+O2 group compared to NS+RA group at 2 months (log2 fold change: -0.49±0.3 vs. 0±0.3; LPS+O2 vs. NS+RA respectively; p=0.01). Conclusion: Our study has highlighted that perinatal lung inflammation in a double-hit model of BPD results in short-term and long-term dysregulated pulmonary T lymphocyte gene expression, which may contribute to increased viral respiratory morbidities seen in children and adults with BPD.