Single-cell sequencing of mouse lung epithelial cells after bleomycin injury and modulation of IRE1a

Epithelial cells that are committed to a differentiated state in homeostasis can change their identity in response to injury. In the lung, alveolar type 2 (AT2) cells are the resident secretory epithelial cells of the alveolus, and after injury can proliferate and differentiate into type 1 cells. This inducible plasticity is central to alveolar regeneration, fibrosis, and malignancy, but the regulatory pathways that govern the balance between fate maintenance and differentiation are incompletely understood. Epithelial cells that are committed to a differentiated state in homeostasis can change their identity in response to injury. In the lung, alveolar type 2 (AT2) cells are the resident secretory epithelial cells of the alveolus, and after injury can proliferate and differentiate into type 1 cells. This inducible plasticity is central to alveolar regeneration, fibrosis, and malignancy, but the regulatory pathways that govern the balance between fate maintenance and differentiation are incompletely understood. IRE1a is an ER transmembrane protein conserved throughout eukaryotes that senses misfolded protein in the ER lumen and initiates cellular efforts to regain protein folding homeostasis through its cytosolic kinase and RNase domains. We previously showed that IRE1a pharmacologic or genetic loss of function decreased the accumulation of damage-associated transient progenitors (DATPs), and also promoted their differentiation into new alveolar epithelial cells. To further detail the functions of IRE1a after AT2 injury, we performed single-cell transcriptome profiling of the AT2-to-AT1 transition after bleomycin injury, with pharmacologic or epithelial-specific genetic loss of IRE1a function. Overall design: This is single cell sequencing of mouse lung epithelial cells from a study with factorial design. The four groups are: wildtype mice exposed to saline; wildtype mice exposed to bleomycin; mice with conditional knockout of IRE1a in the lung epithelium -- Shh(Cre/+) Ern1(flox/flox) -- exposed to bleomycin; and wildtype mice exposed to bleomycin and treated daily with the IRE1a kinase inhibitor KIRA8. Lungs were harvested and dissociated on day 10 after bleomycin exposure, and sorted for live, CD45- CD31- Epcam+ single cells. Cells from multiple mice in each treatment group were pooled into a single GEM lane. Single-cell sequencing performed using 10X Single Cell 3' technology. The output of Cell Ranger software is provided in the processed data file.