Abstract
OBJECTIVES: Circulating myeloid precursors are responsible for post-natal osteoclast (OC) differentiation and skeletal health, although the exact human precursors have not been defined. Enhanced osteoclastogenesis contributes to joint destruction in rheumatoid arthritis (RA) and tumour necrosis factor (TNF) is a well-known pro-osteoclastogenic factor. Herein, we investigated the interplay between receptor activator of nuclear factor kappa-Β ligand (RANK-L), indispensable for fusion of myeloid precursors and the normal development of OCs, and TNF in directing the differentiation of diverse pre-OC populations derived from human peripheral blood. METHODS: Flow cytometric cell sorting and analysis was used to assess the potential of myeloid populations to differentiate into OCs. Transcriptomic, epigenetic analysis, receptor expression and inhibitor experiments were used to unravel RANK-L and TNF signalling hierarchy. RESULTS: TNF can act as a critical homoeostatic regulator of CD14(+) monocyte (MO) differentiation into OCs by inhibiting osteoclastogenesis to favour macrophage development. In contrast, a distinct previously unidentified CD14(-)CD16(-)CD11c(+) myeloid pre-OC population was exempt from this negative regulation. In healthy CD14(+) MOs, TNF drove epigenetic modification of the RANK promoter via a TNFR1-IKKβ-dependent pathway and halted osteoclastogenesis. In a subset of patients with RA, CD14(+) MOs exhibited an altered epigenetic state that resulted in dysregulated TNF-mediated OC homoeostasis. CONCLUSIONS: These findings fundamentally re-define the relationship between RANK-L and TNF. Moreover, they have identified a novel pool of human circulating non-MO OC precursors that unlike MOs are epigenetically preconditioned to ignore TNF-mediated signalling. In RA, this epigenetic preconditioning occurs in the MO compartment providing a pathological consequence of failure of this pathway.