Virtual single-cell perturbation and genetic causal inference reveal CSF1R-dependent immunometabolic communication in...

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.