Disclosure(s): No relevant financial relationship(s) to disclose.
Introduction: This study aims to investigate the heterogeneity and differentiation characteristics of neutrophil subsets during the acute phase of acute respiratory distress syndrome (ARDS), elucidating their role in ARDS fibrotic progression, thereby providing theoretical foundations and potential therapeutic targets for developing precision treatment strategies.
Methods: The ARDS mouse model was established through intratracheal administration of LPS (5 mg/kg; n=3), with control group receiving equal volume PBS (n=3). Twenty-four hours post-modeling, lung tissues were harvested for single-cell RNA sequencing (10× Genomics). Subsequent analyses included: neutrophil subset identification via Seurat v4.1 clustering, pseudotime trajectory analysis using SCUBA algorithm, functional pathway enrichment analysis (GO/KEGG), immunohistochemical validation of subset markers (S100a8/MT1) at protein level, and flow cytometric quantification of neutrophil subsets with CyclinD1/CD66b markers.
Results: Single-cell RNA sequencing demonstrated a significant expansion of neutrophils in LPS-treated mice (30% vs. 2% in controls, P < 0.001). Unsupervised clustering identified two distinct neutrophil subsets: a pro-inflammatory NEU-1 population characterized by elevated S100a8, S100a9, Il1b, and Ccl3 expression (log₂FC > 2.5, P < 0.001), and an antioxidative NEU-2 subset marked by Mt1, Mt2, and Retnlg upregulation (log₂FC > 2.0, P < 0.01). Immunohistochemistry confirmed these findings, showing increased S100a8 (P < 0.01) but decreased MT1 (P < 0.01) protein expression in LPS-exposed lungs. Pseudotime trajectory analysis revealed progressive differentiation from NEU-1 to NEU-2 (R² = 0.89), while flow cytometry demonstrated LPS dose-dependent accumulation of activated CyclinD1+CD66b+ neutrophils (68.2% vs. 1.63% in controls, P < 0.001). Pathway analysis further delineated their divergent roles, with NEU-1 enrichment in NF-κB/MAPK/TNF signaling versus NEU-2 predominance in ROS/PI3K-Akt pathways (FDR < 0.01).
Conclusions: In ARDS, neutrophils differentiate into functionally distinct subsets, with the pro-inflammatory NEU-1 subset driving fibrosis via the CCL3-IL-1β-TGF-β axis. Targeting these subsets or their signaling pathways represents a potential therapeutic strategy for ARDS-associated fibrosis.
