Alveolar type 2 (AT2) to type 1 (AT1) transition is a key process in lung repair after injury. It involves transitional cell states such as activated AT2 and Krt8+ alveolar differentiation intermediate cells (ADI). The dysfunction of this transition impairs the repair process and is described in lung fibrosis. However, how AT2 to AT1 transition is affected in emphysema and the role of this dysfunction in this disease remains to be determined. We aimed to characterize the AT2 to AT1 transition in elastase-induced emphysema in mice and emphysematous patients, and to determine the impact of alveolar differentiation impairment on emphysema development.

Murine emphysema is established in the elastase model 21days after elastase instillation (established emphysema) and persists at late time points (150days). We performed single cell RNAseq of EpCAM+ cells in established and late emphysema and clustered the different alveolar epithelial cells populations. We next assessed the functionality of the emphysematous epithelial cells compared to controls by analyzing their ability to form organoids obtained by co-culturing epithelial cells (EpCAM+ from elastase or control lung) with control lung fibroblasts. Single cell RNAseq was next performed on elastase and control organoids. In human lung tissue section, we quantified the number of SpC+ Krt8+ and Krt8+/Krt17+ cells by immunofluorescence in emphysematous or non-emphysematous lung.

In the elastase model, we profiled 91.094 cells and identified 6 alveolar cell states: AT2 (Lyz2+/Etv5+), inflammatory AT2 (Lcn2+/IL33+/Lrg1+), Sprr1a-pre-ADI and Sppr1a+ Krt8-ADI (Cldn4+, Itgb6+, Lgals3+), proliferating AT2 (Top2a+/Mki67+) and AT1 (Hopx+/Ager+). Among these populations, we identified only the subset of inflammatory AT2 to be increased in frequency in established and late emphysema. Cell cycle was reduced in each emphysema epithelial cells compared to controls (phase G2M). The gene set SenMayo to evaluate senescent cells was slightly increase in the alveolum of elastase mice vs. control (pooled time points). Transcirptomic analysis in epithelial cells reveal an upregulation of P53 and TNF signaling and a downregultation of E2F and mitotic spindle pathways. Pseudotime trajectory analysis of alveolar lineages revealed that inflammatory AT2 cells located at the branches from the middle of AT2 cells. Regarding organoids formation, they were fewer and smaller when they were obtained from elastase-treated WT epithelial cells than control mice. In organoids, we identified the same clusters than in the lung but did not find any increase in frequency of one of thess clusters in elastase mice derived organoids compared to controls. In human, no difference was observed in frequency of ADI cells (SpC-Krt8+ and Krt8+ Krt17+ cells) compared to non-emphysematous lungs.

Emphysema is associated with an increased of activated AT2 that may be in competition with the AT2 to AT1 transition and limit the ability of AT2 to repair the alveolar destruction.