P2C379: TGFβ Regulates Type 1 Pulmonary Epithelial Cell Plasticity and Extracellular Matrix Gene Transcription During Neonatal Lung Development

Background: The TGFβ superfamily is a suspected contributor to bronchopulmonary dysplasia (BPD) pathogenesis although the mechanism is unclear. Prior work indicates that both TGFβ overexpression and knockout models in pulmonary epithelial cells result in arrested alveolarization in mice, a hallmark of BPD. In type 1 pulmonary alveolar (AT1) cells, TGFβ is enriched and involved in in vitro cellular spreading. Furthermore, recent evidence points to a role for AT1 cells as regulators of the extracellular (ECM) matrix, which, in other cell types, is controlled by TGFβ and influenced by mechanical force.

Methods: Mice lacking the main TGFβ receptor, Tgfbr2, in AT1s under a tamoxifen-inducible reporter were generated (HopxCreERT2:Tgfbr2:R26REYFP). AT1-AT2 cell reprogramming, histology, and other analyses were evaluated embryonically (E15.5 trace to E18.5) or postnatally (P0 trace to P5 or P42). A model of oligohydramnios to evaluate limited embryonic alveolar stretch on AT1 cell identity was performed with surgical aspiration of amniotic fluid at E15.5 with analysis at E18.5. RNA sequencing was performed on fluorescence-sorted AT1 cells at P5 with qRT-PCR at P5 and P42. In vitro experiments of fluorescence-sorted primary AT1 cells were performed on fibronectin-coated plates with addition of a TGFβ inhibitor or ligand. All experiments contained at least 3 replicates with equal sex distribution.

Results: Loss of Tgfbr2 prenatally resulted in increased AT1 to AT2 cell reprogramming with no difference in proliferation, apoptosis, or total AT2 cell numbers. On histology, loss of Tgfbr2 yielded a BPD-like phenotype with increase mean linear intercept and mean septal thickness (MST). Removal of amniotic fluid to induce oligohydramnios resulted in attenuated lung development with elevated MST but also increased AT1 cell reprogramming as well as an altered AT1:AT2 cell ratio. RNA sequencing and qRT-PCR of AT1 cells from the Tgfbr2 knockout model showed a significant decrease in several ECM-related genes including glycoproteins, AT1-specific collagen IV subtypes, and ECM regulators. In vitro, addition of TGFβ ligand or inhibitor altered AT1 cell spreading ability, which was mediated by changes in integrin expression. However, in both instances, ECM gene expression was reduced.

Conclusion: Together, this data suggests that alterations in biophysical force through reduced integrin-mediated cell spreading with loss of Tgfbr2 or in a model of oligohydramnios are important in AT1 cell fate determinations. During development, AT1 cells serve an integral function as hubs for intercellular communication and ECM remodeling that, when affected, perturb the normal developmental programming and contribute to neonatal lung pathology such as BPD.