Dynamic orchestration of structural and immune cells in acute Pseudomonas aeruginosa pneumonia revealed by time-resolved single-cell and spatial transcriptomics.

BACKGROUND: Pseudomonas aeruginosa pneumonia triggers a complex, dynamic host response, yet the cellular coordination of inflammation and repair remains poorly defined. METHODS: We performed time-resolved single-cell RNA sequencing (scRNA-seq) on murine lungs across five timepoints (0-96 h post-infection) and integrated these data with spatial transcriptomics (10x Visium) performed on matched tissue sections at key timepoints (0, 24, and 96 h), profiling a total of 41,452 high-quality cells. RESULTS: We identified four neutrophil subsets, with the N3 population exhibiting potent antimicrobial activity. 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. Notably, spatial mapping distinguished tissue remodeling activity from cell-type abundance, identifying acute matrix remodeling zones dominated by Timp1 rather than canonical fibroblast markers. Alveolar type 2 epithelial cells transitioned through three distinct states-homeostatic, inflammatory, and reparative. Notably, the circadian gene Dbp was specifically upregulated at 96 h, suggesting a role for circadian programs in resolution of pathology. Crucially, we uncovered a pivotal role for group 2 innate lymphoid cells (ILC2s): epithelial-derived IL-33 activated ILC2s via the ST2 receptor (Il1rl1) during acute infection (24 hpi), promoting a type 2 immune response that limits immunopathology. CONCLUSION: Our integrative time-resolved spatial transcriptomic atlas elucidates how structural and immune cells orchestrate the balance between bacterial clearance and tissue repair, highlighting novel targets for host-directed therapies in P. aeruginosa pneumonia.