Alveolar epithelial type 2 cells (AT2) respond to injury by proliferating and relining the alveolar surface as type 1 cells; they are also capable of developing basal and mesenchymal cell characteristics. Our aim was to determine whether AT2 cells could respond to cytokines by expressing pro-inflammatory or pro-fibrotic genes that could drive post-inflammatory pulmonary fibrosis.

We used human AT2 cells derived from induced pluripotent stem cells (iAT2) as a model. iAT2 cells were exposed for 48h to cytokines like those found in fibrotic lung lavage fluid (TGF-B [300 pg/mL], IL-1B [10], TNF-a [100], IL-8 [1500], MCP1 [700], IL-33 [40], TSLP [100], IL-13 [2500] and IL-4 [160]). RNA and protein were then isolated to assess expression of genes related to inflammation and fibrosis.

iAT2 cells expressed SP-C and SP-B proteins and contained lamellar bodies with tubular myelin. iAT2 cells expressed epithelial markers SFTPB, SFTPC and HOPX, in addition to KRT17, KRT19, CTGF and FN1, on single cell RNA-sequencing. After incubation with cytokines, cells expressed IL1-B, TNF-a, TGFB-1, a-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor-1, fibronectin, type I collagen and tenascin C mRNA. After 14d incubation of iAT2 cells with cytokines, cells lost expression of SP-B, an epithelial marker, and expressed on western blotting N-cadherin and vimentin, markers of mesenchymal differentiation. Cytokeratin 5 protein, found in basal cells, was also expressed after cytokines.

iAT2 cells respond to cytokines by expression of genes that produce inflammatory and fibrotic mediators, demonstrating the potential of AT2 cells to participate in lung inflammation and fibrosis.