464 - Loss of TGFβ Impacts Type 1 Alveolar Epithelial Cell-mediated Extracellular Matrix Production and Cell Plasticity

Poster Number: 464
Publication Number: 464.432

Danielle Callaway, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Ian Penkala, University of Pennsylvania, Philadelphia, PA, United States; Su Zhou, Perelman School of Medicine at the University of Pennsylvania, Mickleton, NJ, United States; Michael Morley, Perelman School of Medicine at the University of Pennsylvania, philadelphia, PA, United States; Edward Morrisey, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States

Background: Bronchopulmonary dysplasia (BPD) is the most frequent sequelae of extreme prematurity and characterized as arrest of alveolarization with enlarged alveoli and thickened septa. TGFβ, a superfamily involved in cellular processes such as proliferation, extracellular matrix (ECM) synthesis, and cell fate, has been implicated in BPD development but the mechanism remains unclear as models of both gain and loss of function yield a BPD-like phenotype. RNA sequencing from our lab revealed that type 1 alveolar cells (AT1s) are enriched for TGFβ. We have also shown that in a neonatal hyperoxia model of BPD, AT1s transdifferentiate into alveolar type 2 cells (AT2s), which is not readily apparent in adult animals after hyperoxic injury, demonstrating age-specific plasticity of AT1s that may be regulated by TGFβ.

Objective: To elucidate how loss of TGFβ in AT1s influences cell fate and contributes to BPD pathogenesis.

Design/Methods: Mice lacking the main TGFβ receptor, Tgfbr2, in AT1s under a tamoxifen-inducible reporter were generated (HopxCreERT2:Tgfbr2:R26REYFP). In embryonic experiments, pregnant dams were injected with tamoxifen at E15.5 and lungs were collected at E18.5. Postnatally, pups were injected at P0 and harvested at P5 or P42. Lungs were fixed for immunohistochemistry or underwent tissue digestion and flow cytometry sorting of AT1s for RNA sequencing or qRT-PCR.

Results: Loss of Tgfbr2 at all time points resulted in increased numbers of lineage traced AT2 cells. Mean septal thickness was higher in knockout animals at E18.5 compared to controls. RNA sequencing of AT1 cells in knockouts compared to controls at P5 identified downregulation of P3h2, an enzyme responsible for collagen IV hydroxylation. Examination of single-cell RNA sequencing data from mice and human neonates revealed that most components of the ECM are expressed in AT1s during late development. Intriguingly, genes for collagen IV (Col4a3 and Col4a4) and laminin-332 were specific to AT1s. Validation with qRT-PCR indicated loss of Tgfbr2 in AT1s at P5 and P42 yielded a decrease in multiple ECM genes including multiple collagen IV chains and the enzymes P3h2 and Plod2.Conclusion(s): Loss of Tgfbr2 led to an increase in AT1 cellular plasticity and increased septal wall thickness indicative of an abnormal pulmonary basement membrane. RNA sequencing and qRT-PCR of AT1 cells lacking Tgfbr2 revealed downregulation of genes for collagen IV and associated enzymes. Combined with findings from single-cell RNA sequencing, this data points to a novel function for AT1s as key contributors to the pulmonary ECM that appears to be regulated by TGFβ.