Aging is a major risk factor for lung fibrosis. While bleomycin injury in young mice leads to resolving fibrosis, aged mice develop non-resolving fibrosis, resembling human disease.

Here, we conducted a longitudinal single-cell RNA profiling of young (3 mo) and aged (18 mo) mice after bleomycin-induced lung injury (days 0/3/10/20/30/37 p.i., n=55 mice), coupled to multiplexed immunofluorescence to uncover spatially-resolved cellular programs responsible for this impaired regeneration. 

Single-cell analyses of over 160,000 cells across 69 cell states combined with imaging revealed key differences in cell type abundances, gene expression and cell-cell communication in aged vs young mice at baseline and during injury and repair. Aged mice showed a significant change in the lymphocyte repertoire, including the emergence of pro-inflammatory B and T-cell populations, and exhibited a persistent alveolar-mesenchymal crosstalk driven mainly by Krt8+ alveolar differentiation intermediates, leading to sustained fibroblast activation. This was accompanied by increased and prolonged cellular senescence and DNA damage. Digital sorting of senescent alveolar cells based on the canonical senescence genes showed an age-specific gene expression and SASP profile, including Macrophage migration inhibitory factor (MIF). MIF was predicted to cause fibroblast activation in aged mice, which was confirmed by in vitro MIF stimulation of mouse fibroblasts. 

Our study provides a comprehensive single-cell map of the molecular changes of age-related lung fibrosis and identifies inflammaging and persistent senescence as crucial pathogenic mechanisms.