Abstract
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.
Highlights
TNF is the major cytokine driving inflammation in rheumatoid arthritis (RA), a chronic inflammatory disease affecting about 1% of the world's population and characterized by synovial inflammation and joint destruction, leading to severe morbidity and premature mortality [1]
We found that in these CD11b+F4/80+ populations, Ly6C+Gr1- cells comprised 23.9%, 50.8% and 15.9% of M-Osteoclast precursors (OCPs), TNF-induced OCPs (T-OCPs) and RANKL-induced OCPs (R-OCPs), respectively, while Ly6C-Gr1- cells comprised 73%, 47% and 82.8% of these cells, respectively (Fig 1B middle panel), suggesting that TNF switched M-CSFinduced Ly6C-Gr1- M2 to Ly6C+Gr1- M1 macrophages, an effect similar to that induced by IFN-γ, which is a standard stimulator of M1 macrophage differentiation [22, 23]
M1 and M2 macrophages are linked to T helper 1 (TH1)- and TH2-type immune responses, respectively [33]
Summary
TNF is the major cytokine driving inflammation in rheumatoid arthritis (RA), a chronic inflammatory disease affecting about 1% of the world's population and characterized by synovial inflammation and joint destruction, leading to severe morbidity and premature mortality [1]. There is a need to better understand how TNF induces joint inflammation and destruction Inflammatory cells, such as lymphocytes, macrophages and mast cells, drive chronic inflammatory processes, including synovial inflammation, by producing cytokines and autoantibodies at involved sites. Receptor activator of nuclear factor-κB ligand (RANKL), a member of the TNF superfamily, mainly controls later phases of OC differentiation and activation [5], and its expression by synoviocytes and inflammatory cells in affected joints is promoted by TNF and other cytokines [6, 7]. Preclinical and clinical studies indicate that RANKL inhibitors do not significantly alter inflammatory processes in RA [11] These findings suggest that RANKL does not contribute significantly to TNF-induced inflammation in RA
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