TNF Induction of NF-κB RelB Enhances RANKL-Induced Osteoclastogenesis by Promoting Inflammatory Macrophage Differentiation but also Limits It through Suppression of NFATc1 Expression

RESEARCH ARTICLE TNF Induction of NF-κB RelB Enhances RANKL-Induced Osteoclastogenesis by Promoting Inflammatory Macrophage Differentiation but also Limits It through Suppression of NFATc1 Expression a11111 Zhijun Zhao1☯, Xiaodong Hou1,2☯, Xiaoxiang Yin1,2, Yanyun Li2, Rong Duan2, Brendan F. Boyce2, Zhenqiang Yao2* 1 Department of Medical Imaging, Henan University First Affiliated Hospital, 357 Ximen Street, Kaifeng, Henan 475001, P.R. China, 2 University of Rochester Medical Center, Department of Pathology and Laboratory Medicine and Center for Musculoskeletal Research, Box 626, Room 1–2105, 601 Elmwood Ave, Rochester, NY 14642, United States of America ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Zhao Z, Hou X, Yin X, Li Y, Duan R, Boyce BF, et al. (2015) TNF Induction of NF-κB RelB Enhances RANKL-Induced Osteoclastogenesis by Promoting Inflammatory Macrophage Differentiation but also Limits It through Suppression of NFATc1 Expression. PLoS ONE 10(8): e0135728. doi:10.1371/journal.pone.0135728 Editor: Juha Tuukkanen, University of Oulu, FINLAND Received: December 29, 2014 Accepted: July 26, 2015 Published: August 19, 2015 Copyright: © 2015 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by National Natural Science Foundation of China 81373191 to ZY, National Institute for Arthritis, Musculoskeletal and Skin Diseases AR43510 to BFB, and P30AR061307 pilot grant (from Edward M. Schwarz) to ZY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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 PLOS ONE | DOI:10.1371/journal.pone.0135728 August 19, 2015 1 / 20 TNF Induced Osteoclast Formation Competing Interests: The authors have declared that no competing interests exist. 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. Introduction 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]. Transgenic mice over-expressing TNF (TNF-Tg mice) develop a form of arthritis that is very similar to human RA [2]. Although anti-TNF therapies have significantly reduced the morbidity and joint destruction in RA, they are expensive, and only about 60% of patients have a good response to these agents [3, 4]. In non-responding patients, TNF inhibitors typically are administered for several months before a decision is made to switch to an alternative treatment, which is often another TNF inhibitor that also may be ineffective. Thus, 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. Joint destruction in RA is mediated by ectopic differentiation of osteoclasts (OCs) from their monocyte-macrophage lineage precursors in affected joints. 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]. RANKL expression is also required for normal B cell development and lymph node formation [8], suggesting that it might have a role to promote joint inflammation in RA. However, TNF-Tg mice generated to have deficiency of RANKL also develop synovial inflammation, but not joint destruction because OCs do not form in these mice [9, 10]. 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. TNF can induce osteoclastogenesis directly from Rank–/–OC precursors (OCPs) in vitro when the cells are co-cultured with [12] or without [13] TGF-β1, which is released from bone matrix during bone resorption and activated by the acidic microenvironment in resorption lacunae as a result of acid release from OCs [14, 15]. However, the numbers of OCs induced by TNF from WT OCPs are much lower than those induced by RANKL [16]. Despite these findings, it was puzzling that TNF did not induce OC formation when administered in vivo to Rank–/–mice [17]. We have reported tha (...truncated)