Single-cell transcriptomic atlas uncovers Immune/Structural lineage remodeling and fibrosis in Klebsiella pneumoniae- infected porcine lungs

Klebsiella pneumoniae (KP) is a predominant pathogen in respiratory infections, whose widespread antibiotic resistance poses severe public health risks and economic burdens. As the primary target of KP infection, the lung contains diverse immune cells, while pulmonary cellular responses to KP remain poorly understood. This study integrated single-cell RNA sequencing, flow cytometry, RT-qPCR, and immunofluorescence to investigate porcine lung cell response from healthy pigs and those infected with multidrug-resistant, highly-virulent KP. Post-infection, myeloid cells (neutrophils, monocytes, and interstitial macrophages) were rapidly activated with upregulated signatures related to neutrophil migration and interferon production. Alveolar macrophages (AM) exhibited marked heterogeneity: KP infection was associated with apoptosis in proliferating, monocyte-derived, and tissue-resident AM subsets, indicating a disruption of homeostasis, while infection-specific subsets (AM_MHCII, AM_CXCL2) displayed enhanced antigen presentation, inflammatory activity, and pro-fibrotic potential. Under homeostasis, γδ T and CD8αα+ T cells were the predominant T cell populations. After infection, CX3CR1−CD8αα+ T cells were activated, and CD8+ γδ T cells increased their interactions with myeloid cells, predominantly through ligand-receptor pairs such as TNF-TNFRSF1B and IFNG-IFNGR1/2. Furthermore, KP induced lung fibroblast proliferation and activation via the ROS-mediated TGF-β/SMAD pathway, WNT pathway, and aerobic glycolysis. Concurrently, infection reduced the proportion of ATII and transitional AT0 cells, which may impair their differentiation into ATI cells and compromise epithelial barrier function. Thus, this study is the first to characterize the dynamics of KP infection in porcine lungs at single-cell resolution. It advances our understanding of KP-host interactions, and provides a framework for anti-KP therapeutic development.