Multimodal peripheral blood immunophenotyping and single cell epigenetic analysis of knee osteoarthritis reveals expansion and epigenetic dysregulation of regulatory T-cell and nonclassical monocyte populations.

Abstract

Osteoarthritis (OA) involves systemic inflammation, yet peripheral blood immunophenotypes and their underlying epigenetic landscapes remain poorly defined. We characterized cellular and chromatin accessibility profiles in knee OA and radiographic progressors (RP). We performed multimodal immunophenotyping using mass cytometry (CyTOF) on peripheral blood from 21 knee OA patients and 11 healthy controls (HC). To confirm findings, we developed DNA methylation-based imputation models and applied them to two independent validation cohorts (n=723). Finally single-cell ATAC-seq (scATAC-seq) was performed to interrogate chromatin accessibility landscapes in a patient subset. CyTOF identified significant expansions of CD8+ central memory T-cells, CD4+ regulatory T-cells (T-regs), and nonclassical monocytes in OA vs. HC (all P≤0.05). These expansions were robustly confirmed in the large-scale validation cohorts (all P<0.0001). Conversely, RPs displayed validated reductions in circulating CD4+ and CD8+ central memory pools. scATAC-seq revealed extensive epigenetic remodeling, particularly within monocytes and Tregs. Paradoxically, unsupervised clustering indicated that epigenetically defined pro-inflammatory clusters were depleted in OA blood. Pathway analysis revealed that these depleted clusters possess activated, migratory phenotypes, whereas the expanded circulating cells among OA patients display quiescent, non-migratory epigenetic signatures. Knee OA is characterized by validated systemic expansions of specific T-reg and monocyte subsets. Single-cell epigenetic profiling suggests epigenetically distinct subpopulations driven by age, OA, comorbid conditions, or migration from circulation to the periphery.