Novel netrin-1-Unc5b-Klf4 axis balances AT2 survival in experimental bronchopulmonary dysplasia

Alveolar growth and repair are central processes in development and chronic lung disease, such as bronchopulmonary dysplasia (BPD), a neonatal lung disease without curative therapy. Alveolar epithelial type 2 (AT2) cells are the endogenous progenitor pool giving rise to alveolar epithelial type 1 cells and promoting alveolar repair. Since netrin-1, a regulator of cell homeostasis and stemness, has been linked to lung diseases, we now investigated its signaling and function in AT2 cells in a hyperoxia-based model of BPD and in lungs of infants with BPD. First, we demonstrated that prolonged hyperoxia reduced both netrin-1 and its receptor Unc5b in neonatal mouse lungs and in primary AT2 cells. Second, ex vivo studies using precision-cut lung slices (PCLS) and primary murine AT2 cell culture showed that netrin-1 regulates AT2 cell survival and the expression of Krüppel-like factor 4 (Klf4) through Unc5b, a transcription factor regulating cell survival. Third, single-cell and bulk transcriptomic analysis, as well as proximity-dependent biotin identification assay, showed Klf4 to be upregulated in AT2 cells during alveolarization, downstream of netrin-1, and to regulate AT2 cell survival. In vivo, Klf4 gene expression and protein abundance was significantly reduced in total lung homogenates and in AT2 cells of neonatal mice exposed to hyperoxia. Finally, KLF4+ cells, KLF4+ epithelial, and specifically KLF4+ AT2 cells were reduced in clinical BPD. In summary, our data identify a novel netrin-1-Unc5b-Klf4 axis in AT2 cells that is disrupted in BPD and could offer a novel target for endogenous alveolar repair.