APOE and CCR2: Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

Dongyi Wang,1,2 Le Lu,1 Yuping Zhang,1 Wei Shang1 1Department of Integrated Traditional and Western Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu, People’s Republic of China; 2Postdoctoral Research Station, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu, People’s Republic of ChinaCorrespondence: Wei Shang, Email [email protected]: Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder in which macrophages play crucial roles. Given macrophage heterogeneity, novel biomarkers are needed for timely diagnosis and severity assessment. This study aimed to identify macrophage-specific hub genes in RA and investigate their biological functions.Methods: Bulk and single-cell RNA-seq datasets were downloaded from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) in RA synovial macrophages were identified from the GSE97779 dataset using the Limma R package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine the biological processes and pathways associated with the DEGs, followed by Gene Set Enrichment Analysis (GSEA) for further validation. Hub genes were identified using the STRING database and Cytoscape. Based on the single‑cell dataset GSE192504, cell clusters were annotated with Seurat to determine macrophage‑specific hub genes, whose associated biological processes were explored via gene set variation analysis (GSVA). Further sub‑clustering revealed distinct macrophage subtypes. Finally, immunofluorescence staining was performed to identify molecular markers of macrophage subtypes, while RT-qPCR and ELISA were used to validate the mRNA and protein expression of macrophage-specific hub genes in in vitro experiments.Results: We identified 334 DEGs enriched in immune-related pathways. Ten hub genes (FN1, CXCL10, FOS, CCR2, GZMB, CD69, CXCL9, FCGR1A, APOE, IGF1) were identified, with APOE and CCR2 specifically expressed in macrophages and strongly associated with inflammatory response. As expected, IL-4 stimulation upregulated APOE in RAW264.7 cells, while LPS/IFN-γ increased CCR2 expression. Additionally, six macrophage subsets were found, with an expanded APOE+ subset following IL-4 exposure.Conclusion: APOE and CCR2 are specifically highly expressed in synovial macrophages and correlate with inflammatory responses, highlighting their potential as biomarkers for disease progression and promising therapeutic targets in RA. Further investigation is required to elucidate the underlying mechanisms.Keywords: rheumatoid arthritis, macrophages, immune infiltration, APOE, CCR2, biomarkers