Single-cell RNA Sequencing Analysis Reveals the Molecular Mechanisms of Neutrophil Dysfunction in Chronic Bone Infection

INTRODUCTION: Neutrophils play a key role in host immune defense. At present, neutrophils in chronic bone infections exhibit significant heterogeneity but functional alterations that remain poorly understood. MATERIALS AND METHODS: A rat model of chronic bone infection induced by Methicillin- Resistant Staphylococcus Aureus (MRSA) was established. Bone marrow cells were analyzed using scRNA-seq with Gene Ontology (GO) and pathway enrichment analysis. Differentially Expressed Genes (DEGs) were identified to assess neutrophil dysfunction, validated by immunofluorescence staining and ROS quantification. RESULTS: MRSA-induced chronic bone infection was confirmed by Gram and H&E staining, which showed bacterial colonization and inflammation. Neutrophils from infected rats showed downregulated immune-related genes (e.g., Clec7a, Ccr5) and upregulated immunosuppressive factors (e.g., Nfkbia, IL10ra). Enrichment analysis showed that immune responses and neutrophil functions were inhibited. Immunofluorescence showed neutrophil polarization towards N2 phenotype and reduced Reactive Oxygen Species (ROS) production in the infection group. DISCUSSION: This study established a rat model of MRSA-induced chronic bone infection and identified 7 neutrophil subsets via scRNA-seq analysis, with the NeuP2ry10 subset showing the most significant changes. Neutrophils displayed decreased chemotaxis, phagocytosis, and ROS production, along with elevated anti-inflammatory gene expression, suggesting functional suppression and a shift toward an immunosuppressive state. CONCLUSION: Chronic bone infection drives neutrophil polarization toward an N2 anti-inflammatory phenotype, reducing antimicrobial capacity and promoting infection persistence. Targeting neutrophil function may offer new therapeutic strategies for chronic bone infection.