Single-Cell and Spatial Transcriptomics Identify the Mono-S100A4 Subset Associated with Sepsis-Induced Lung Injury...

In sepsis-induced immune dysregulation, monocytes contribute critically through their dual pro-inflammatory and immunosuppressive functions. Leading to multi-organ injury, the lungs are the most vulnerable initial target, dictating clinical outcomes. Thus, studying monocyte dynamics is essential to understand sepsis-induced acute lung injury (ALI) pathophysiology.

We analyzed publicly available mouse-derived single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic (ST) datasets for sepsis-induced ALI from the GEO database. Computational characterization of monocyte heterogeneity included cell-cell communication, single-cell trajectory analysis, and functional enrichment analyses to identify subtype-specific signaling and injury-related signatures. Spatial mapping further confirmed monocyte subtype localization. Experimental validation was performed in a mouse model of sepsis-induced ALI established by intraperitoneal LPS injection. Histological evaluation, immunohistochemistry, immunofluorescence, Quantitative real-time PCR, and Western blot were integrated to verify the spatial distribution of the Mono-S100A4 subset and its associated pathway molecules.

Clustering analysis revealed a restructured immune landscape in septic ALI, characterized by both cellular heterogeneity and prominent monocyte accumulation. Spatial transcriptomics revealed that inflammatory infiltration mediated by monocytes and other immune cells predominantly localized to bronchial and bronchiole regions. Monocyte heterogeneity analysis identified a distinct Mono-S100A4 subset, which exhibited enhanced intercellular communication capacity, influencing other subclusters through the (MIF)-(CD74+CD44) axis. KEGG and GSEA analyses of Mono-S100A4 revealed significant enrichment in the IL-17 signaling pathway and Th17 cell differentiation, suggesting a potential role in exacerbating inflammatory responses. In vivo experiments revealed a significant upregulation in both the mRNA and protein expression levels of S100A4, IL-17A, MIF, and CD74 in the lung tissue of ALI mice.

The Mono-S100A4 subset is strongly associated with the activation of the Th17 signaling pathway via the MIF-(CD74+CD44) axis, and this correlative relationship supports a potential pathogenic role of Mono-S100A4 in sepsis-induced ALI, which forms a key hypothesis to be validated by subsequent functional experiments.