# Combination of single-cell and bulk RNA-seq reveals changes in the immune landscape in osteomyelitis

## TL;DR

This study uses RNA sequencing to explore immune changes in osteomyelitis, identifying key genes and macrophage subtypes involved in disease progression.

## Contribution

The study combines single-cell and bulk RNA-seq to uncover immune cell dynamics and signaling pathways in osteomyelitis.

## Key findings

- Six gene clusters with distinct expression patterns were identified, linked to immune activation and bone formation.
- Macrophage subtypes like Arg1+Sdc4+ and Cxcl1+Ccl4+ showed increased infiltration in osteomyelitis.
- Ccl3–Ccr1 and Cxcl2–Cxcr2 signaling pathways were highlighted as key in immune regulation during the disease.

## Abstract

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.

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.

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.

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.