Abstract Rationale In premature infants, bacterial inflammation is associated with development of bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by impaired lung development and airway hyperresponsiveness. We have shown that in immature mice, chronic airway exposure to lipopolysaccharide (LPS), a bacterial endotoxin, induces hypoalveolarization and inflammation, reminiscent of human BPD. We have also shown that IL-17a-producing gamma-delta (γδ) T cells are required for LPS-induced neonatal lung immunopathology (Am J Respir Crit Care Med 2025;211:A7325). We hypothesized that neonatal lung γδ T cells display unique transcriptional profiles after acute and chronic LPS exposure. Methods Immature C57BL/6J mice were inoculated with 3µg/10ul LPS or 10ul PBS intranasally on day of life (DOL) 3, 5, 7, and 10. Lung γδ T cells were analyzed by flow cytometry and sorted after acute (DOL4) and chronic (DOL11) LPS exposure. RNA was extracted and processed with a low-input RNA-Seq Kit. Sequences were aligned and annotated. Differentially expressed genes for acute LPS versus PBS and chronic LPS versus acute LPS were determined using limma-voom. Significance was based on absolute log2 fold change 1 and adjusted P 0.05. Gene set enrichment analysis determined enriched pathways. Results The number of lung γδ T cells was doubled after acute and quadrupled after chronic LPS exposure. Principal component analysis visualized acute and chronic LPS samples distinct from their PBS controls and from each other. All samples expressed high levels of Cd3e, Cd3g, Cd3d, consistent with γδ T cells. All samples showed selective high expression levels of Il17ra, Il17a, and the toll-like receptors Tlr2, Tlr4, indicating capacity for a pro-inflammatory feedback loop and for direct response to bacterial immunogens. The most significant differences were between acute LPS and PBS and between chronic and acute LPS with over 680 and 2580 differentially expressed genes respectively. Pathways enriched in acute LPS versus PBS included Th17 polarization and T cell activation. Pathways enriched in chronic versus acute LPS included effector and memory T cell development. Chronic LPS induced the expression of tissue-resident memory markers, including Cd69, Itgae, Cd44, and others. This phenotype, specific for lung γδ T cells, was confirmed by flow cytometry. Conclusions We demonstrate that γδ T cells acquire unique pro-inflammatory and tissue-resident phenotypes after acute and chronic LPS exposure in early life. Future studies will identify links between phenotypic and functional characteristics of neonatal lung γδ T cells, and the mechanisms by which these cells contribute to long-term respiratory morbidity. This abstract is funded by: NIH grant R01 HL167716
Cui et al. (Fri,) studied this question.