Related Experiment Video
Updated: Jan 9, 2026
ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils
Published on: January 3, 2025
Single-cell RNA Sequencing Analysis Reveals the Molecular Mechanisms of Neutrophil Dysfunction in Chronic Bone
Tiejian Li1, Shaokui Nan1, Hongbin Xie2
1Graduate School, Medical School of the Chinese PLA, Beijing, China.
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.
Insights
Chronic bone infection impairs neutrophil antimicrobial function, shifting them to an immunosuppressive state. This study reveals neutrophil dysfunction in a rat model, suggesting new therapeutic targets for persistent infections.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Neutrophils are crucial for host defense but their role in chronic bone infections is not fully understood.
- Neutrophils in chronic bone infections display heterogeneity and functional alterations.
- Methicillin-Resistant Staphylococcus Aureus (MRSA) is a common cause of chronic bone infections.
Purpose of the Study:
- To investigate neutrophil functional alterations in a rat model of MRSA-induced chronic bone infection.
- To identify specific neutrophil subsets and their molecular changes during chronic bone infection.
- To elucidate the impact of chronic infection on neutrophil antimicrobial and immune functions.
Main Methods:
- Established a rat model of MRSA-induced chronic bone infection.
- Analyzed bone marrow cells using single-cell RNA sequencing (scRNA-seq).
- Performed Gene Ontology (GO) and pathway enrichment analysis to identify differentially expressed genes (DEGs).
- Validated findings using immunofluorescence staining and reactive oxygen species (ROS) quantification.
Main Results:
- MRSA infection confirmed by bacterial colonization and inflammation in bone tissue.
- Neutrophils showed downregulated immune-related genes and upregulated immunosuppressive factors.
- Immune responses and neutrophil functions were significantly inhibited.
- Neutrophils exhibited polarization towards an N2 anti-inflammatory phenotype with reduced ROS production.
Conclusions:
- Identified 7 neutrophil subsets in chronic bone infection, with NeuP2ry10 showing significant changes.
- Neutrophils displayed suppressed chemotaxis, phagocytosis, and ROS production, indicating functional impairment.
- Chronic bone infection promotes a shift towards an immunosuppressive neutrophil phenotype, hindering antimicrobial capacity and promoting infection persistence.
- Targeting neutrophil function presents a potential therapeutic strategy for chronic bone infections.
