Virtual single‐cell perturbation and genetic causal inference reveal CSF1R ‐dependent immunometabolic communication in iron metabolism‐associated osteoarthritis

Abstract Iron dysregulation has emerged as a contributor to osteoarthritis (OA), yet the cell‐communication mechanisms connecting iron‐related genetic signals to joint degeneration remain insufficiently defined. Here, we established an integrative computational framework combining transcriptomic screening, machine learning, Mendelian randomization, immune and metabolite mediation analysis, single‐cell transcriptomics, virtual gene perturbation, molecular docking, molecular dynamics simulation, and experimental validation to identify signaling regulators involved in iron metabolism‐associated OA. By intersecting iron metabolism‐related genes, OA differentially expressed genes, and eQTL‐supported genes, we identified 27 shared candidates. Machine learning‐based prioritization and genetic causal inference further highlighted CSF1R as a central regulatory gene. Single‐cell analysis localized CSF1R expression predominantly to macrophages, indicating a macrophage‐centered role in the osteoarthritic microenvironment. Mediation analysis integrating 731 immune‐cell traits and 1400 circulating metabolites identified CD14 + CD16 + monocytes as a significant cellular mediator linking CSF1R activity to OA susceptibility, suggesting that CSF1R may promote disease progression mainly through monocyte–macrophage remodeling rather than isolated metabolic alteration. Genetic colocalization further supported a shared regulatory signal between CSF1R expression and OA risk. Virtual single‐cell perturbation revealed distinct downstream consequences of CSF1R modulation. Simulated CSF1R depletion enhanced antigen processing, major histocompatibility complex class II presentation, and phagosome‐related programs, whereas simulated CSF1R overexpression preferentially activated extracellular matrix organization, integrin signaling, and cartilage development‐associated pathways. Structural analyses identified stable interactions between CSF1R and candidate inhibitory compounds, and inflammatory stimulation of macrophages confirmed increased CSF1R protein expression. Collectively, this study identifies CSF1R as a macrophage‐associated immunometabolic signaling hub linking iron dysregulation to OA. These findings provide a mechanistic basis for targeting CSF1R ‐mediated monocyte–macrophage communication and offer a computational strategy for prioritizing therapeutic targets in degenerative joint disease.