Abstract Rationale Lung regeneration is essential to maintaining/restoring respiratory function in the face of injury. During lung injury, lung epithelial progenitor cells must proliferate and differentiate to regenerate the progenitor pool and replace the lost epithelial cells, all while maintaining proximal-distal cell fate. Loss of normal proximal-distal patterning is a hallmark of several lung diseases including idiopathic pulmonary fibrosis (IPF). We previously identified increased LncRNA MEG3 expression in scRNA seq analysis of IPF epithelial cells. Herein we hypothesized that Meg3 regulates proximal-distal identity during alveolar repair following experimental models of fibrotic lung injury. Methods We found that Meg3 expression was increased in both the airway and alveolar epithelium following repetitive bleomycin injury. To determine the role of Meg3 during alveolar repair, we generated pan-epithelial (ShhCre), inducible lineage traced airway specific (Scgb1a1Creert2tdtomato), and inducible lineage traced AT2-cell specific (SftpcCreert2tdTomato) models of Meg3 deletion. Both Cre-negative litter mates and Cre specific WT mice were used as controls. For inducible models, a single 3mg dose of Tamoxifen was delivered via intraperitoneal injection. We then utilized the repetitive 8-dose bleomycin injury model (bleomycin injury or saline control delivered via intratracheal intubation) and assessed the lungs 2-weeks after the final bleomycin dose. Ashcroft scoring and fluorescent protein and RNA analysis was used to determine lung injury and cell fate of lineage traced cells. Results Deletion of pan-epithelial, airway specific, or AT2 cell specific Meg3 prior to repetitive bleomycin injury resulted in increased fibrotic remodeling. Analysis of cell-type specific markers found that Meg3 deleted secretory cells failed to migrate into the distal lung and maintained proximal cell fates rather than adopting alveolar cell fates associated with alveolar regeneration. AT2-cell specific deletion of Meg3 resulted in a population of lineage traced AT2 cells that lost alveolar cell-type markers. Single-cell RNA-sequencing and immunofluorescence analysis demonstrated these cells instead expressed markers associated with transitional cells (Krt19, Krt8) and proximal airway cell types including secretory (3.6% +/- 1.3 Scgb1a1+/Lin+), goblet (4.0% +/- 1.2 Muc5b+/Lin+), and ciliated (3.1% +/- 1.6 Foxj1+/Lin+) cells indicating a loss of proximal-distal identity. Conclusion These studies demonstrate that the LncRNA Meg3 is required to promote or maintain alveolar cell fate and proximal-distal identity during alveolar repair. This abstract is funded by: NHLBI
Gokey et al. (Fri,) studied this question.