Time-resolved single-cell RNA sequencing of mouse lungs during acute Pseudomonas aeruginosa pneumonia

Here, we performed time-resolved single-cell RNA sequencing (scRNA-seq) on murine lungs across five timepoints (0-96 h post-infection), capturing the dynamic continuum from acute injury to resolution and repair, profiling a total of 41,452 high-quality cells. To provide spatial context, we integrated these data with spatial transcriptomics (10x Visium) performed on matched tissue sections at key timepoints (0, 24, and 96 h).

We identified four neutrophil subsets, with the N3 population exhibiting potent antimicrobial activity. Furthermore, we traced the emergence of inflammatory fibroblasts during acute infection to Col13a1⁺ matrix fibroblasts, a process driven by a regulatory network centered on Stat1 and Nfkb1, Spatial transcriptomics with RCTD deconvolution revealed a 22.5% peak immune infiltration at 24 h, while COMMOT analysis uncovered 30-fold and 25-fold increases in Ccl2–Ccr2 and Cxcl2–Cxcr2 spatial signaling, respectively, pinpointing chemokine-driven recruitment foci.