Increased osteoclast (OC) differentiation and activity is the critical event that results in bone loss and joint destruction in common pathological bone conditions, such as osteoporosis and rheumatoid arthritis (RA). RANKL and its decoy receptor, osteoprotegerin (OPG), control OC differentiation and activity. However, there is a specific concern of a rebound effect of denosumab discontinuation in treating osteoporosis. TNFα can induce OC differentiation that is independent of the RANKL/RANK system. In this review, we discuss the factors that negatively and positively regulate TNFα induction of OC formation, and the mechanisms involved to inform the design of new anti-resorptive agents for the treatment of bone conditions with enhanced OC formation. Similar to, and being independent of, RANKL, TNFα recruits TNF receptor-associated factors (TRAFs) to sequentially activate transcriptional factors NF-κB p50 and p52, followed by c-Fos, and then NFATc1 to induce OC differentiation. However, induction of OC formation by TNFα alone is very limited, since it also induces many inhibitory proteins, such as TRAF3, p100, IRF8, and RBP-j. TNFα induction of OC differentiation is, however, versatile, and Interleukin-1 or TGFβ1 can enhance TNFα-induced OC formation through a mechanism which is independent of RANKL, TRAF6, and/or NF-κB. However, TNFα polarized macrophages also produce anabolic factors, including insulin such as 6 peptide and Jagged1, to slow down bone loss in the pathological conditions. Thus, the development of novel approaches targeting TNFα signaling should focus on its downstream molecules that do not affect its anabolic effect.

Angiogenesis is involved in the pathogenesis of inflammatory arthritis, but little is known about the role of lymphangiogenesis in this setting. Here, we examined whether tumor necrosis factor (TNF) stimulates osteoclast precursors (OCPs) to produce the lymphatic growth factor, vascular endothelial growth factor-C (VEGF-C), and induce lymphangiogenesis. We used TNF-transgenic (Tg) mice and mice with serum-induced arthritis. OCPs were purified by fluorescence-activated cell sorting of CD11b⁺/Gr-1^-/lo blood or bone marrow cells and subjected to microarray analysis or were generated from spleen or joint cells and treated with TNF. Expression of VEGFs was analyzed and examined by real-time reverse transcription-polymerase chain reaction and Western blotting. Immunostaining and magnetic resonance imaging were used to quantify lymphatic vessels and volumes of synovium and draining lymph nodes. TNF stimulated VEGF-C expression by OCPs and increased nuclear factor-kappa B (NF-κB) binding to an NF-κB sequence in the VEGF-C promoter. OCPs from joints of TNF-Tg mice express high levels of VEGF-C. Lymphatic vessel numbers and size were markedly increased in joint sections of TNF-Tg mice and mice with serum-induced arthritis. The severity of synovitis correlated with draining lymph node size. In summary, TNF induces OCPs to produce VEGF-C through NF-κB, leading to significantly increased lymphangiogenesis in joints of arthritic mice. The lymphatic system may play an important role in the pathogenesis of inflammatory arthritis.

Inflammaging induces osteoporosis by promoting bone destruction and inhibiting bone formation. TRAF3 limits bone destruction by inhibiting RANKL-induced NF-κB signaling in osteoclast precursors. However, the role of TRAF3 in mesenchymal progenitor cells (MPCs) is unknown. Mice with TRAF3 deleted in MPCs develop early onset osteoporosis due to reduced bone formation and enhanced bone destruction. In young mice TRAF3 prevents β-catenin degradation in MPCs and maintains osteoblast formation. However, TRAF3 protein levels decrease in murine and human bone samples during aging when TGFβ1 is released from resorbing bone. TGFβ1 induces degradation of TRAF3 in murine MPCs and inhibits osteoblast formation through GSK-3β-mediated degradation of β-catenin. Thus, TRAF3 positively regulates MPC differentiation into osteoblasts. TRAF3 deletion in MPCs activated NF-κB RelA and RelB to promote RANKL expression and enhance bone destruction. We conclude that pharmacologic stabilization of TRAF3 during aging could treat/prevent age-related osteoporosis by inhibiting bone destruction and promoting bone formation.

Cytokines, including receptor activator of nuclear factor κB ligand (RANKL) and TNF, induce increased osteoclast (OC) formation and bone loss in postmenopausal osteoporosis and inflammatory arthritides. RANKL and TNF can independently induce OC formation in vitro from WT OC precursors via TNF receptor-associated factor (TRAF) adaptor proteins, which bind to their receptors. Of these, only TRAF6 is required for RANKL-induced osteoclastogenesis in vitro. However, the molecular mechanisms involved remain incompletely understood. Here we report that RANKL induced the formation of bone-resorbing OCs from TRAF6^−/− OC precursors when cultured on bone slices but not on plastic. The mechanisms involved increased TNF production by TRAF6^−/− OC precursors resulting from their interaction with bone matrix and release of active TGFβ from the resorbed bone, coupled with RANKL-induced autophagolysosomal degradation of TRAF3, a known inhibitor of OC formation. Consistent with these findings, RANKL enhanced TNF-induced OC formation from TRAF6^−/− OC precursors. Moreover, TNF induced significantly more OCs from mice with TRAF3 conditionally deleted in myeloid lineage cells, and it did not inhibit RANKL-induced OC formation from these cells. TRAF6^−/− OC precursors that overexpressed TRAF3 or were treated with the autophagolysosome inhibitor chloroquine formed significantly fewer OCs in response to TNF alone or in combination with RANKL. We conclude that RANKL can enhance TNF-induced OC formation independently of TRAF6 by degrading TRAF3. These findings suggest that preventing TRAF3 degradation with drugs like chloroquine could reduce excessive OC formation in diseases in which bone resorption is increased in response to elevated production of these cytokines.

TNF induces bone loss in common bone diseases by promoting osteoclast formation directly and indirectly, but it also limits osteoclast formation by inducing expression of NF-κB p100. Osteoclast precursors (OCPs) are derived from M1 (inflammatory) and M2 (resident) macrophages. However, it is not known if TNF stimulates or limits osteoclast formation through regulation of M1 or M2 differentiation or if RelB, a partner of p100, is involved. To investigate these questions, we treated bone marrow cells (BMCs) with M-CSF alone or in combination with TNF to enrich for OCPs, which we called M-OCPs and T-OCPs, respectively. We found that TNF switched CD11b⁺F4/80⁺ M-OCPs from Ly6C^-Gr1^- M2 to Ly6C⁺Gr1^-CD11c⁺ and Ly6C^-Gr1^-CD11c⁺ M1 cells. RANKL induced osteoclast formation from both Ly6C⁺Gr1^- and Ly6C^-Gr1^- T-OCPs, but only from Ly6C⁺Gr1^- M-OCPs, which formed significantly fewer osteoclasts than T-OCPs. Importantly, Ly6C⁺Gr1^- cells from both M- and T-OCPs have increased expression of the M1 marker genes, iNOS, TNF, IL-1β and TGFβ1, compared to Ly6C^-Gr1^- cells, and Ly6C^-Gr1^- cells from T-OCPs also have increased expression of iNOS and TGFβ1 compared to cells from M-OCPs. Both RANKL and TNF increased RelB mRNA expression. TNF significantly increased RelB protein levels, but RANKL did not because it also induced RelB proteasomal degradation. TNF inhibited RANKL-induced NFATc1 mRNA expression and osteoclast formation from M-OCPs, but not from T-OCPs, and it did not induce Ly6C⁺Gr1^-CD11c⁺ or Ly6C^-Gr1^-CD11c⁺ M1 macrophages from RelB-/- BMCs. Furthermore, overexpression of RelB in M-OCPs reduced RANKL-induced osteoclast formation and NFATc1 mRNA expression, but it increased TNF-induced OC formation without affecting NFATc1 levels. Thus, TNF induction of RelB directly mediates terminal osteoclast differentiation independent of NFATc1 and limits RANKL-induced osteoclastogenesis by inhibiting NFATc1 activation. However, the dominant role of TNF is to expand the OCP pool by switching the differentiation of M-CSF-induced M2 to M1 macrophages with enhanced osteoclast forming potential. Strategies to degrade RelB could prevent TNF-induced M2/M1 switching and reduce osteoclast formation.

TGFβ1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFβ1⁺CCR5⁺) is the major source of TGFβ1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFβ1⁺CCR5⁺ neutrophil numbers in BM of young mice. During aging, TGFβ1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFβ1⁺CCR5⁺ neutrophil numbers in BM where they induce bone loss. TGFβ1⁺CCR5⁺ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFβ1⁺CCR5⁺ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFβRII specifically deleted in MPCs had lower numbers of TGFβ1⁺CCR5⁺ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFβ1⁺CCR5⁺ neutrophil numbers in BM could treat or prevent age-related osteoporosis.