Abstract / Summary
Abstract Background Sepsis-associated acute lung injury (ALI) contributes substantially to sepsis mortality, but the cellular heterogeneity and molecular regulators driving its progression remain incompletely understood. This study integrated multiple public single-cell transcriptomic datasets to characterize pulmonary cellular remodeling and identify key regulators during sepsis-associated ALI. Methods Mouse lung scRNA-seq datasets (GSE207651, GSE249835, GSE278767; sham and CLP at 12, 24, and 48 h) were integrated with a human PBMC dataset (GSE279452, healthy controls vs. sepsis) and a mouse lung bulk RNA-seq dataset (GSE279484) used for validation. Data were processed in Seurat, with ambient RNA removed via scCDC/DecontX and cell types annotated using CellMarker 2.0. Compositional dynamics were assessed by RO/E ratios, and pathway/M1-M2 activity was scored with decoupleR. Monocyte/macrophage subclusters were characterized using AUCell, GSEApy, and Diffusion Pseudotime analysis. hdWGCNA, pySCENIC, and LIANA were applied to construct co-expression networks, TF regulatory networks, and cell communication maps, respectively; scTenifoldKnk validated candidate TFs in AT2 cells. A CLP mouse model with immunofluorescence staining validated KLF6/CD86 colocalization in vivo. Results Integrated analysis identified major pulmonary populations—including fibroblasts, endothelial cells, epithelial subtypes, and immune lineages that underwent time-dependent remodeling during CLP, particularly within innate immunity. Monocyte/macrophage populations showed pronounced M1/M2 heterogeneity, with M1 scores peaking early and M2 scores rising progressively toward 48 h. Pseudotime analysis revealed a continuous monocyte-to-M1 transition, and hdWGCNA identified associated co-expression modules (M1-M5, M1-M7, M1-M8), nominating Bhlhe40, Glul, and Klf6 as core candidate regulators. Klf6 showed highly specific regulatory activity in M1 macrophages, which communicated with AT2 and endothelial cells via multiple ligand-receptor axes. In PBMCs, a KLF6-high inflammatory monocyte subset was significantly expanded in sepsis and correlated with inflammation score. In AT2 cells, Fli1 and other TFs showed increased activity at CLP24h, with targets linked to vascular and tissue remodeling. Immunofluorescence confirmed increased KLF6⁺CD86⁺ cells in CLP-treated lungs. Conclusions This multi-omic integration delineates the dynamic remodeling of the pulmonary cellular landscape and monocyte/macrophage polarization in sepsis-associated ALI, identifying KLF6 as a key regulator linking macrophage M1 polarization to peripheral inflammatory monocyte activation offering new mechanistic insight and potential therapeutic targets.