Background/Purpose: Although TNF inhibitors suppress bone destruction in patients with rheumatoid arthritis (RA), TNFα stimulation alone does not directly induce osteoclasts, bone-resorbing cells. With the assumption that the combination of TNFα and IL-6 induces differentiation of osteoclast-like cells, we examined the effect of the combination on bone marrow-derived monocytes/macrophages (BMMs) and on intracellular signaling pathways.

Methods: BMMs were cultured with macrophage-colony stimulating factor (M-CSF), TNFα, IL-6, and receptor activator of nuclear factor κB ligand (RANKL). The activities of nuclear factor-κB (NF-κB), c-Fos, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) were analyzed using an ELISA-based system, western blot analysis and immunostaining. In vivo administration of the cytokines into the supracalvariae of mice was performed. The effects of osteoprotegerin (OPG), the decoy receptor for RANKL, the NFAT inhibitor tacrolimus, anti-IL-1β antibody, ERK inhibitors and a novel JAK inhibitor tofacitinib were examined. The effects of RNAi for c-Fos and the genetic ablation of Stat3 were also evaluated.

Results: The combination of TNFα and IL-6 induced the differentiation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast-like cells in a RANKL-independent manner. The osteoclast-like cells resorbed dentin slices in a manner similar to osteoclasts induced by RANKL. Activity of c-Fos and NFATc1 was significantly upregulated by TNFα and IL-6 used in combination compared to TNFα or IL-6 alone. The differentiation of osteoclast-like cells was completely inhibited by tacrolimus but not by OPG or anti-IL-1β antibody. In addition, silencing of c-Fos significantly decreased the expression level of NFATc1 mRNA and the number of osteoclast-like cells. Most interestingly, we observed no significant difference in the induction of osteoclast-like cells derived from conditional Stat3-knockout mice and control mice. In contrast, ERK inhibitors clearly exerted an inhibitory effect on the differentiation of osteoclast-like cells compared to that of osteoclasts induced by RANKL. Thus, the JAK-ERK signaling pathway is likely to regulate the differentiation of osteoclast-like cells. On the other hand, the number of TRAP-positive cells on the calvariae was significantly higher in mice administered with TNFα plus IL-6 than in mice administered with PBS, IL-6 or TNFα alone. Furthermore, tofacitinib suppressed the differentiation of osteoclast-like cells in vitro, and decreased the number of TRAP-positive cells and inhibited bone resorption induced by the combination of TNFα and IL-6 in vivo.

Conclusion: The combination of TNFα and IL-6 is a potent mediator of bone resorptive osteoclast-like cells even in the absence of RANKL. These results suggest that TNF inhibitors might suppress bone destruction by inhibiting the differentiation of osteoclast-like cells induced by the combination of TNFα and IL-6.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/combination-of-tnf-and-il-6-induces-osteoclast-like-cells-with-bone-resorption-activity-both-in-vitro-and-in-vivo/