Figure 4 – BDNF identified as important for alveolar epithelial regeneration.

(A) AT2 cells were isolated from an uninjured C57BL/6 mouse and from a C57BL/6 mouse that underwent acid-induced lung injury 24 hours prior. Single-cell transcriptomic analysis was performed and cells were grouped into populations using t-SNE. There was little overlap between AT2 cells from uninjured and injured mice. (B) K-means analysis was used to sub-divide AT2 cells into groups based on their individual gene expression. (C) Genes whose expression was unique to each cluster are shown. Cluster 3 has gene expression from both AT2 and AT1 cells (Sftpc and Pdpn) and AEP marker Tm4sf1. (D) Bdnf is the only bona fide STAT3 target gene with newly accessible chromatin following acid-induced lung injury that is differentially expressed in cluster 3. (E) Data from ATAC-seq (shown in Fig. 1) demonstrate increased accessibility of the Bdnf promotor in cells isolated from mice 24 hours after acid-induced lung injury. (F) AT2 cells from tamoxifen-exposed Sftpc^CreERT2:Stat3^LoxP/LoxP mice express less Bdnf than vehicle exposed mice (n=4 mice per group) (G) BAL fluid from Sftpc^CreERT2:Stat3^LoxP/LoxP mice treated with either tamoxifen or corn oil (vehicle) prior to undergoing acid-induced lung injury shows that mice lacking AT2-specific Stat3 have significantly less BDNF in their BAL fluid (n=3 mice per group per time point). (H) AT2 cells from Sftpc^{CreERT2-Rosa26TdTomato}:Hopx^3FlagGFP mice were co-cultured with PDGFRα+ mesenchymal cells from C57BL/6 mice in the presence of BDNF which increased alveolar organoid forming efficiency of murine AT2 cells. Right-hand panels show quantification from n=3 distinct cultures per condition. (I) Primary human AT2 cells were isolated and co-cultured with MRC5 fibroblasts for three weeks. We observed increased organoid size and forming efficiency when BDNF was added to the culture media. Right-hand panels show the average organoid forming efficiency and size from n=3 different donors. Individual points are shown with the mean and standard error of the mean depicted. For panels (F), (G), (H), and (I), data is shown as the mean +/− SEM. Statistical significance was determined with a two-tailed Student’s t-test (F), (H), and (I), and with a two-way ANOVA (G). Scale bars, 1mm.