Single-cell transcriptomics of mouse alveolar type 2 cells after bleomycin treatment

Aging-related impairment in alveolar regeneration has been identified in a variety of acute and chronic lung diseases. As the aging population increases, understanding the mechanisms involved in age-dependent deterioration in alveolar stem cell function can support the development of new therapies for lung diseases. In this study, we investigated the cellular and molecular mechanisms underlying AT2 cell differentiation and how aging affected post-injury alveolar regeneration. We discovered that the process of AT2 cells differentiating into AT1 cells is an energetically costly process. During the differentiation of AT2 cells, activated AMPK-PFKFB2 signaling up-regulates glycolysis, which is essential to enhance cellular ATP production and to support the intracellular energy expenditure that is required for cell shape change and cytoskeletal remodeling during AT2 cells differentiating into AT1 cells. AT2 cells in aging lungs show reduced AMPK-PFKFB2 signaling and decreased ATP production, resulting in impaired AT2 cell differentiation. Activating AMPK-PFKFB2 signaling in aged AT2 cells can rescue defects in alveolar regeneration. Thus, beyond demonstrating that the activation of AMPK-PFKFB2 signaling facilitates the great energy demands of AT2 cells during alveolar regeneration, our study suggests that modulating these pathways could promote the alveolar repair in aged lungs. Overall design: Lineage labeled alveolar epithelial type 2 (GFP+ AT2) cells of 3-month old and 12-month old mice were harvested by FACS. AT2 cells isolated from three replicates of each group were identically mixed and then processed to sc-RNA seq following the 10X Genomics protocol. Four single-cell RNA sequencing datasets were generated named as "3-mo day0","3-mo day30","12-mo day0", and "12-mo day30". The "3-mo day0" and "12-mo day0" datasets were sequenced for AT2s of PBS-treated 3-month old or 12-month old Sftpc-creER;Rosa26-mTmG mice. The "3-mo day30" and "12-mo day30" datasets were sequenced for AT2s of 3-month old or 12-month old Sftpc-creER;Rosa26-mTmG mice on post-bleomycin day30.