Combination of single-cell and bulk RNA-seq reveals changes in the immune landscape in osteomyelitis
Objective This study presented a comprehensive characterization of the osteomyelitis immune microenvironment, identified driver genes and pathogenic cell populations underlying disease progression, and uncovered potential therapeutic targets through single-cell and bulk transcriptomic analysis. Methods We analyzed time-series transcriptomic sequencing data from mouse osteomyelitis samples in the dataset GSE168896. Fuzzy c-means clustering was applied to reveal gene sets linked to disease progression. Immune cell infiltration analysis was conducted through the online tool ImmuCellAI-mouse. Furthermore, by leveraging single-cell sequencing data, we characterized immune cell subpopulations and pinpointed the key cell subtypes that were present in the osteomyelitis mice. Results We identified six gene clusters exhibiting distinct temporal expression patterns and functional roles in osteomyelitis, such as leukocyte and lymphocyte activation and ossification. Single-cell sequencing analysis further showed seven distinct cellular subpopulations. Among these, macrophages demonstrated a significant increase following osteomyelitis, and the infiltration of Mif + Cd63 + , Arg1 + Sdc4 + , and Cxcl1 + Ccl4 + macrophages significantly increased. Moreover, Ccl3–Ccr1 and Cxcl2–Cxcr2 ligand–receptors contributed mostly in immune cells. Conclusion Our findings tracked the transcriptional dynamics and evolving immune landscape of osteomyelitis, highlighting macrophages as central regulators of disease progression. We identified that significant infiltration of Arg1 + Sdc4 + , Cxcl1 + Ccl4 + , and Mif + Cd63 + macrophages may affect osteomyelitis through the Ccl3–Ccr1 and Cxcl2–Cxcr2 signaling pathways. These findings offer a new perspective on immune regulation in osteomyelitis.