Background/Purpose:

TNF-α is well known to be involved in inflammatory-induced bone destruction. This is widely supported by clinical studies showing reduced bone pathology after anti-TNF-α therapies. Numerous in vivo and in vitro studies in rodents have demonstrated that TNFα directly contributes to osteoclast(OC)-mediated joint destruction. Moreover, specific murine OC precursors are expand during inflammatory arthritis. Despite this, it is still unclear which population within the human myeloid compartment primarily contributes to OC-mediated bone erosion, and whether or not TNF-α has a direct impact on all pre-cursors equally. Here we have investigated the direct effect of TNF-α on different circulating myeloid populations and their subsequent differentiation into osteoclasts.

Methods: Total CD14⁺ monocytes, monocyte subsets (CD14⁺CD16^–, CD14⁺CD16⁺, CD14^dimCD16⁺), and myeloid dendritic cell precursors (pre-DCs; CD16^–CD14^–CD11c⁺) were isolated from peripheral blood of healthy donors using either magnetic separation or FACS. Selected populations were differentiated into OC with M-CSF and RANK-L in the presence or absence of TNF-α. Mature OCs were stained with tartrate-resistant acid phosphatase and quantified by light microscopy. Mineral-coated surfaces were used to assess the resorption activity of mature OCs. Flow cytometry analysis was used to assess surface protein expression during OC differentiation. Antibody blocking studies and selective inhibition of IKK-β were performed to determine the role of TNFR1 and TNFR2 in OC development. Gene expression was assessed by RT-qPCR.

Results: Simultaneous stimulation of CD14⁺ monocytes with RANK-L and TNF-α resulted in complete abolishment of OC differentiation and resorption. Notably, these cells acquired a macrophage phenotype. In contrast, delayed addition of TNF-α (72h pre-stimulation with RANKL) enhanced osteoclast numbers. Importantly, CD14⁺ monocytes expressed both TNFR1 and TNFR2 but only the level of TNFR1 decreased during OC differentiation. Blockade of TNFR1 negated the inhibitory capacity of simultaneous TNF-α whilst blockade of TNFR2 reverted the enhancing capacity of TNF-α. Inhibition of IKK-β revealed that the TNFR1-driven inhibitory signal was dependent on the canonical NF-κB pathway. At a molecular level, TNF-α signalling in CD14⁺ monocytes blocked the M-CSF-driven up-regulation of RANK transcript.

In comparison to CD14⁺ monocytes, pre-DCs differentiated quicker and more efficiently into OCs under M-CSF and RANK-L stimulation. Interestingly, pre-DCs were found to express less TNFR1 than CD14⁺ monocytes and TNF-α failed to inhibit RANK expression and thus their differentiation into functional OCs.

Conclusion: We have shown that the hierarchy between TNF-α and RANK-L is crucial for driving the differentiation of CD14⁺ monocytes into either macrophage or osteoclasts. In comparison, this axis does not influence the ability of pre-DCs to differentiate down the OC lineage. This study provides new insight in the contribution of different circulating myeloid precursors to bone destruction in a TNF-α rich environment.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tnf-%ce%b1-overrides-the-ability-of-rank-ligand-to-induce-osteoclast-differentiation-of-classical-circulating-precursors/