Search

Objectives: Metabolic changes are crucially involved in osteoclast development and may contribute to bone degradation in rheumatoid arthritis (RA). The enzyme aconitate decarboxylase 1 (Acod1) is known to link the cellular function of monocyte-derived macrophages to their metabolic status. As osteoclasts derive from the monocyte lineage, we hypothesised a role for Acod1 and its metabolite itaconate in osteoclast differentiation and arthritis-associated bone loss., Methods: Itaconate levels were measured in human peripheral blood mononuclear cells (PBMCs) of patients with RA and healthy controls by mass spectrometry. Human and murine osteoclasts were treated with the itaconate derivative 4-octyl-itaconate (4-OI) in vitro. We examined the impact of Acod1-deficiency and 4-OI treatment on bone erosion in mice using K/BxN serum-induced arthritis and human TNF transgenic (hTNFtg) mice. SCENITH and extracellular flux analyses were used to evaluate the metabolic activity of osteoclasts and osteoclast progenitors. Acod1-dependent and itaconate-dependent changes in the osteoclast transcriptome were identified by RNA sequencing. CRISPR/Cas9 gene editing was used to investigate the role of hypoxia-inducible factor (Hif)-1α in Acod1-mediated regulation of osteoclast development., Results: Itaconate levels in PBMCs from patients with RA were inversely correlated with disease activity. Acod1-deficient mice exhibited increased osteoclast numbers and bone erosion in experimental arthritis while 4-OI treatment alleviated inflammatory bone loss in vivo and inhibited human and murine osteoclast differentiation in vitro. Mechanistically, Acod1 suppressed osteoclast differentiation by inhibiting succinate dehydrogenase-dependent production of reactive oxygen species and Hif1α-mediated induction of aerobic glycolysis., Conclusion: Acod1 and itaconate are crucial regulators of osteoclast differentiation and bone loss in inflammatory arthritis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized., (© 2024. The Author(s).)

Eosinophils are involved in tissue homeostasis. Herein, we unveiled eosinophils as important regulators of bone homeostasis. Eosinophils are localized in proximity to bone-resorbing osteoclasts in the bone marrow. The absence of eosinophils in ΔdblGATA mice results in lower bone mass under steady-state conditions and amplified bone loss upon sex hormone deprivation and inflammatory arthritis. Conversely, increased numbers of eosinophils in IL-5 transgenic mice enhance bone mass under steady-state conditions and protect from hormone- and inflammation- mediated bone loss. Eosinophils strongly inhibit the differentiation and demineralization activity of osteoclasts and lead to profound changes in the transcriptional profile of osteoclasts. This osteoclast-suppressive effect of eosinophils is based on the release of eosinophil peroxidase causing impaired reactive oxygen species and mitogen-activated protein kinase induction in osteoclast precursors. In humans, the number and the activity of eosinophils correlates with bone mass in healthy participants and rheumatoid arthritis patients. Taken together, experimental and human data indicate a regulatory function of eosinophils on bone., (© 2024. The Author(s).)

Osteoclasts are polykaryons formed by cell-cell fusion of highly motile progenitors of the myeloid lineage. Osteoclast activity can preserve skeletal strength and bone homeostasis. However, osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA). Fc receptors activated by IgG immune complexes (IC) can boost osteoclast differentiation and bone loss in the course of RA. In contrast, interferon (IFN) γ secreted by immune cells blocks osteoclast activation. Despite their hypothetical importance in the regulation of osteoclast differentiation in RA, the interconnection between the two pathways has not been described so far. Here, we show by total internal reflection fluorescence (TIRF) microscopy that FcγR3 and IFNγ receptor (IFNγR) locate at close vicinity to each other on the human osteoclast surface. Moreover, the average distance increases during the differentiation process. Interestingly, FcγR and IFNγR activation shapes the position of both receptors to each other. Surprisingly, the inhibitory action of IFNγ on in-vitro human osteoclast differentiation depends on the osteoclast differentiation stage. Indeed, IFNγR activation in early osteoclast precursors completely inhibits the formation of polynucleated osteoclasts, while in premature osteoclasts, it further enhanced their fusion. In addition, gene expression analyses showed that IFNγR activation on early precursor cells but not on premature osteoclasts could induce FcγR expression, suggesting a co-regulation of both receptors on human osteoclast precursors. Phosphokinase array data of precursor cells demonstrate that the observed divergence of IFNγR signaling is dependent on the mitogen-activated protein kinase (MAPK) downstream signaling pathway. Overall, our data indicate that FcγR and IFNγR signaling pathways co-influence the differentiation and activity of osteoclasts dependent on the differentiation state, which might reflect the different stages in RA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Groetsch, Schachtschabel, Tripal, Schmid, Smith, Schett and Bozec.)

Butyrophilins, which are members of the extended B7 family of immunoregulators structurally related to the B7 family, have diverse functions on immune cells as co-stimulatory and co-inhibitory molecules. Despite recent advances in the understanding on butyrophilins' role on adaptive immune cells during infectious or autoimmune diseases, nothing is known about their role in bone homeostasis. Here, we analyzed the role of one specific butyrophilin, namely Btn2a2, as we have recently shown that Btn2a2 is expressed on the monocyte/macrophage lineage that also gives rise to bone degrading osteoclasts. We found that expression of Btn2a2 on monocytes and pre-osteoclasts is upregulated by the receptor activator of nuclear factor κ-B ligand (RANKL), an essential protein required for osteoclast formation. Interestingly, in Btn2a2-deficient osteoclasts, typical osteoclast marker genes (Nfatc1, cathepsin K, TRAP, and RANK) were downregulated following RANKL stimulation. In vitro osteoclast assays resulted in decreased TRAP positive osteoclast numbers in Btn2a2-deficient cells. However, Btn2a2-deficient osteoclasts revealed abnormal fusion processes shown by their increased size. In vivo steady state µCT and histological analysis of bone architecture in complete Btn2a2-deficient mice showed differences in bone parameters further highlighting the fine-tuning effect of BTN2a2. Moreover, in rheumatoid arthritis patients and experimental arthritis, we detected significantly decreased serum levels of the secreted soluble Btn2a2 protein. Taken together, we identified the involvement of the immunomodulatory molecule Btn2a2 in osteoclast differentiation with potential future implications in basic and translational osteoimmunology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Frech, Schuster, Andes, Schett, Zaiss and Sarter.)

Regulation of osteoclast differentiation and function is a central element in bone homeostasis. While the role of soluble factors, such as cytokines, hormones and growth factors, in controlling osteoclast differentiation has been intensively characterized, the function of surface receptors is less well understood. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9 and its murine homolog Siglec-E are sialic acid-recognizing inhibitory receptors from the CD33-related Siglec-family and mainly expressed on myeloid cells. We found Siglec-9 and Siglec-E to be expressed at all stages of human and murine osteoclastogenesis, respectively. Siglec-E knockout mice displayed lower bone mass despite unchanged osteoclast numbers and an increased bone formation rate. Ex vivo osteoclast assays using Siglec-E knockout cells or a blocking antibody against human Siglec-9 confirmed the suppressive effect of Siglec-9/Siglec-E on osteoclast function. Although osteoclast numbers were unchanged or even slightly decreased, the blockade/absence of Siglec-9/Siglec-E resulted in an augmented resorption activity of mature osteoclasts. This increased resorption activity was associated with enlarged actin rings. Together, our results suggest Siglec-9/Siglec-E to inhibit osteoclast activation independently from osteoclast differentiation and thereby propose a new mechanism for the control of local bone resorption., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Osteoclasts are specialised bone resorbing cells that control both physiological and pathological bone turnover. Functional changes in the differentiation and activity of osteoclasts are accompanied by active metabolic reprogramming. However, the biological significance and the in vivo relevance of these events has remained unclear. Here we show that bone resorption of differentiated osteoclasts heavily relies on increased aerobic glycolysis and glycolysis-derived lactate production. While pharmacological inhibition of glycolysis did not affect osteoclast differentiation or viability, it efficiently blocked bone resorption in vitro and in vivo and consequently ameliorated ovariectomy-induced bone loss. Our experiments thus highlight the therapeutic potential of interfering with osteoclast-intrinsic metabolic pathways as possible strategy for the treatment of diseases characterized by accelerated bone loss.

Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.

While the role of T cells in the regulation of bone homeostasis is well defined, little is known about the role of innate lymphoid cells (ILCs) on bone. ILCs are innate immune cells that share cytokine expression patterns with T cells but lack the T cell receptor. In this study we show that type 2 ILCs (ILC2) potently inhibit the generation of bone resorbing osteoclasts in vitro as well as favorably influence bone homeostasis under steady state conditions in vivo using loss and gain of function models. Furthermore, adoptive transfer of ILC2 completely abrogated ovariectomy-induced bone loss by significantly down-regulating osteoclast numbers in vivo. The suppressive effects of ILC2s on osteoclasts in vitro and in vivo as well as the protection from ovariectomy-induced bone loss were linked to their expression of IL-4 and IL-13 as well as STAT6 activation on the myeloid target cell, since deletion of IL-4/IL-13 in ILC2s or STAT6 in osteoclast precursors abrogated the anti-osteoclastogenic effect of ILC2s. Taken together, these findings show that ILC2 have to be considered as potent regulators of bone homeostasis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by autoimmunity that triggers joint inflammation and tissue destruction. Traditional concepts of RA pathogenesis have strongly been focused on inflammation. However, more recent evidence suggests that autoimmunity per se modulates the disease and in particular bone destruction during the course of RA. RA-associated bone loss is caused by increased osteoclast differentiation and activity leading to rapid bone resorption. Autoimmunity in RA is based on autoantibodies such as rheumatoid factor (RF) and autoantibodies against citrullinated proteins (ACPA). These autoantibodies exert effector functions on immune cells and on bone resorbing osteoclasts, thereby facilitating bone loss. This review summarizes potential pathways involved in increased destruction of bone tissue in RA, particularly focusing on the direct and indirect actions of autoantibodies on osteoclast generation and function.

Osteoclasts are the only bone-resorbing cells in the body. Together with bone-forming osteoblasts, they are responsible for bone homeostasis and constant bone remodeling. Aberrant activation of osteoclasts leads to bone loss, as seen in postmenopausal osteoporosis or in autoimmune diseases like rheumatoid arthritis. Although much research has been performed to understand and prevent osteoclast-mediated bone loss, the mechanisms of osteoclast hyperactivation are not completely understood. This unit describes several protocols for ex vivo generation of murine and human osteoclasts, allowing study of the effects of specific cells, cytokines, or chemical substances on osteoclast formation and activity without the need for expensive and time-consuming animal experiments. In addition, we provide protocols for specific staining of osteoclasts and for analysis of resorption activity using calcium phosphate-coated surfaces or bone slices. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Local priming of osteoclast precursors (OCp) has long been considered the main and obvious pathway that takes place in the human body, where local bone lining cells and RANKL-expressing osteocytes may facilitate the differentiation of OCp. However, priming of OCp away from bone, such as in inflammatory tissues, as revealed in peripheral blood, may represent a second pathway, particularly relevant in individuals who suffer from systemic bone loss such as prevalent in inflammatory diseases. In this review, we used a systematic approach to review the literature on osteoclast formation in peripheral blood in patients with inflammatory diseases associated with bone loss. Only studies that compared inflammatory (bone) disease with healthy controls in the same study were included. Using this core collection, it becomes clear that experimental osteoclastogenesis using peripheral blood from patients with bone loss diseases in prevalent diseases such as rheumatoid arthritis, osteoporosis, periodontitis, and cancer-related osteopenia unequivocally point toward an intrinsically increased osteoclast formation and activation. In particular, such increased osteoclastogenesis already takes place without the addition of the classical osteoclastogenesis cytokines M-CSF and RANKL in vitro . We show that T-cells and monocytes as OCp are the minimal demands for such unstimulated osteoclast formation. In search for common and disease-specific denominators of the diseases with inflammation-driven bone loss, we demonstrate that altered T-cell activity and a different composition-such as the CD14+CD16+ vs. CD14+CD16- monocytes-and priming of OCp with increased M-CSF, RANKL, and TNF- α levels in peripheral blood play a role in increased osteoclast formation and activity. Future research will likely uncover the barcodes of the OCp in the various inflammatory diseases associated with bone loss.

NR4A1 (Nur77 or NGFI-B), an orphan member of the nuclear receptor superfamily, has been identified as a key regulator of the differentiation and function of myeloid, lymphoid, and mesenchymal cells. The detailed role of NR4A1 in bone biology is incompletely understood. Here, we report a role for NR4A1 as novel factor controlling the migration and recruitment of osteoclast precursors during bone remodeling. Myeloid-specific but not osteoblast-specific deletion of NR4A1 resulted in osteopenia due to an increase in the number of bone-lining osteoclasts. Although NR4A1-deficient osteoclast precursors displayed a regular differentiation into mature osteoclasts, they showed a hyper-motile phenotype that was largely dependent on increased osteopontin expression, suggesting that expression of NR4A1 negatively controlled osteopontin-mediated recruitment of osteoclast precursors to the trabecular bone. Pharmacological activation of NR4A1, in turn, inhibited osteopontin expression and osteopontin-dependent migration of osteoclast precursors resulted in reduced abundance of bone-resorbing osteoclasts in vivo as well as in an ameliorated bone loss after ovariectomy in mice. This study identifies NR4A1 as a crucial player in the regulation of osteoclast biology and bone remodeling and highlights this nuclear receptor as a promising target for therapeutic intervention during the treatment of osteoporosis. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc., (© 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.)

Microbial metabolites are known to modulate immune responses of the host. The main metabolites derived from microbial fermentation of dietary fibers in the intestine, short-chain fatty acids (SCFA), affect local and systemic immune functions. Here we show that SCFA are regulators of osteoclast metabolism and bone mass in vivo. Treatment of mice with SCFA as well as feeding with a high-fiber diet significantly increases bone mass and prevents postmenopausal and inflammation-induced bone loss. The protective effects of SCFA on bone mass are associated with inhibition of osteoclast differentiation and bone resorption in vitro and in vivo, while bone formation is not affected. Mechanistically, propionate (C3) and butyrate (C4) induce metabolic reprogramming of osteoclasts resulting in enhanced glycolysis at the expense of oxidative phosphorylation, thereby downregulating essential osteoclast genes such as TRAF6 and NFATc1. In summary, these data identify SCFA as potent regulators of osteoclast metabolism and bone homeostasis.

Bone tissue undergoes permanent and lifelong remodeling with a concerted action of bone-building osteoblasts and bone-resorbing osteoclasts. A precise cooperation between those two cell types is critical in the complex process of bone renewal. Galectin-3 is a member of the β-galactoside-binding lectin family playing multiple roles in cell growth, differentiation and aggregation. As it has been described to be expressed in bone, galectin-3 might influence bone homeostasis by regulating the function and/or interplay of osteoblasts and osteoclasts. Here, we investigated the role of galectin-3 in osteoclastogenesis and osteoblast-osteoclast interactions. Bone histomorphometric analysis and μCT measurements revealed a decreased trabecular bone volume and an increased osteoclast number in 12weeks old male galectin-3 knockout mice compared to wildtype littermates. Galectin-3 deficient bone marrow cells displayed a higher osteoclastogenic capacity in ex vivo differentiation assays, associated with elevated TRAF6 mRNA levels, suggesting an intrinsic inhibition of osteoclastogenesis by galectin-3 interfering with RANKL-mediated signaling. Furthermore, the addition of extracellular galectin-3 to murine or human osteoclastogenesis assays inhibited osteoclast formation and osteoclast numbers were higher in co-culture assays with galectin-3 deficient osteoblasts. In conclusion, our data suggest the secretion of galectin-3 as a novel mechanism for osteoblasts to control osteoclastogenesis and to maintain trabecular bone homeostasis independently of the RANKL/OPG-axis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator differentially expressed in FDCP 6 homolog (Def6) as a novel inhibitor of osteoclastogenesis in physiological and inflammatory conditions. Def6 deficiency in Def6 -/- mice enhanced the sensitivity of osteoclast precursors to the physiological osteoclastogenic inducer receptor activator for NF-κB ligand, and Def6 -/- osteoclasts formed actin rings. Furthermore, Def6 deficiency markedly increased TNF-α-induced osteoclastogenesis in vitro and in vivo and enhanced bone resorption in an inflammatory osteolysis mouse model. TNF-α serum levels correlated negatively with Def6 expression levels in osteoclast precursors obtained from RA patients, and the osteoclastogenic capacity of the osteoclast precursors was significantly inversely correlated with their Def6 expression levels, indicating that Def6 functions as an inhibitor of excessive osteoclast formation and bone destruction in RA. Mechanistically, Def6 suppressed osteoclastogenesis and the expression of key osteoclastogenic factors NFATc1, B lymphocyte-induced maturation protein-1, and c-Fos by regulating an endogenous IFN-β-mediated autocrine feedback loop. The Def6-dependent pathway may represent a novel therapeutic target to prevent pathological bone destruction., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objectives: Adiponectin is an effector molecule in the pathophysiology of rheumatoid arthritis, e.g. by inducing cytokines and matrix degrading enzymes in synovial fibroblasts. There is growing evidence that adiponectin affects osteoblasts and osteoclasts although the contribution to the aberrant bone metabolism in rheumatoid arthritis is unclear. Therefore, the adiponectin effects on rheumatoid arthritis-derived osteoblasts and osteoclasts were evaluated., Methods: Adiponectin and its receptors were examined in bone tissue. Primary human osteoblasts and osteoclasts were stimulated with adiponectin and analysed using realtime polymerase chain-reaction and immunoassays. Effects on matrix-production by osteoblasts and differentiation and resorptive activity of osteoclasts were examined., Results: Immunohistochemistry of rheumatoid arthritis bone tissue showed adiponectin expression in key cells of bone remodelling. Adiponectin altered gene expression and cytokine release in osteoblasts and increased IL-8 secretion by osteoclasts. Adiponectin inhibited osterix and induced osteoprotegerin mRNA in osteoblasts. In osteoclasts, MMP-9 and tartrate resistant acid phosphatase expression was increased. Accordingly, mineralisation capacity of osteoblasts decreased whereas resorptive activity of osteoclasts increased., Conclusions: The results confirm the proinflammatory potential of adiponectin and support the idea that adiponectin influences rheumatoid arthritis bone remodelling through alterations in osteoblast and osteoclast.

Background: Pasteurella multocida toxin (PMT) is a potent inducer of osteoclast formation. Pigs suffering from an infection with toxigenic Pasteurella multocida strains develop atrophic rhinitis characterised by a loss of turbinate bones and conchae. However, on the molecular level the process of bone loss remains largely uncharacterised., Results: Recently it was found that PMT activates the serine/threonine kinase mammalian target of rapamycin (mTOR) in fibroblasts. Using RAW264.7 macrophages, we investigated the role of the mTOR complex 1 (mTORC1) in PMT-mediated osteoclast formation. PMT induces the differentiation of RAW264.7 macrophages into multinucleated, tartrate resistant acid phosphatase (TRAP) positive osteoclasts that are capable to resorb bone. In the presence of the mTORC1 inhibitor rapamycin, PMT was significantly less able to induce the formation of TRAP-positive osteoclasts. Accordingly, the resulting resorption of bone was strongly reduced. A major target of mTOR is the 70 kDa ribosomal protein S6 kinase 1 (p70 S6K1). Activated p70 S6K1 decreases the expression of programmed cell death protein 4 (PDCD4), a negative transcriptional regulator of osteoclastogenesis, at the protein and gene level. Ultimately this results in the activation of c-Jun, a component of the activator protein 1 (AP-1) complex, which is a major transcription factor for the induction of osteoclast-specific genes. We now demonstrate that c-Jun and its downstream target, the osteoclast-specific bone degrading protease cathepsin K, are upregulated upon PMT treatment in an mTOR-dependent manner., Conclusions: Activation of mTOR signalling plays a central role in the formation of osteoclasts through the bacterial toxin PMT. On the molecular level, PMT-induced activation of mTOR leads to down regulation of PDCD4, a known repressor of AP-1 complex, culminating in the activation of c-Jun, an essential transcription factor for triggering osteoclastogenesis.

Osteoporosis can arise in systemic lupus erythematosus (SLE) patients secondary to medication and/or chronic inflammation. To analyze if patients with SLE have phenotypically-impaired osteoclastogenesis, we differentiated ex vivo monocytes from 72 SLE patients and 15 healthy individuals into osteoclasts followed by TRAP staining and counting. We identified a subgroup of SLE patients (45%) with a significantly impaired osteoclast differentiation, relative to the other SLE patients or healthy individuals (OR 11.2; 95% CI 1.4-89.9). A review of medication indicated that patients with osteoclast counts equal to healthy donors were significantly more likely to be treated with mycophenolate mofetil (MMF) compared to patients with impaired osteoclastogenesis. We analyzed expression of RANKL and the MMF target genes IMPDH1 and IMPDH2 in osteoclasts by qPCR, but detected no difference. Since MMF might influence interferon-α (IFNα) and -γ (IFNγ) we measured serum IFNα and IFNγ levels. Patients with very low osteoclast counts also had comparably higher IFNα serum levels than patients with normal osteoclast counts. We conclude that in vitro osteoclastogenesis is impaired in a subgroup of SLE patients. This correlates inversely with MMF treatment and high IFNα serum levels. Further observational study will be required to determine whether this translates into a clinically meaningful effect.

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

Bone resorption is seminal for the physiological remodeling of bone during life. However, this process needs to be strictly controlled; excessive bone resorption results in pathologic bone loss, osteoporosis, and fracture. We describe a control mechanism of bone resorption by the adaptive immune system. CD80/86, a pair of molecules expressed by antigen-presenting cells and involved in T cell costimulation, act as negative regulator for the generation of bone-resorbing osteoclasts. CD80/86-deficient mice were osteopenic because of increased osteoclast differentiation. CD80/86-deficient osteoclasts escaped physiological inhibition by CTLA-4 or regulatory T cells. Mechanistically, engagement of CD80/86 by CTLA-4 induced activation of the enzyme indoleamine 2,3-dioxygenase (IDO) in osteoclast precursors, which degraded tryptophan and promoted apoptosis. Concordantly, IDO-deficient mice also showed an osteopenic bone phenotype with higher numbers of osteoclast precursors and osteoclasts. Also, IDO-deficient mononuclear cells escaped the anti-osteoclastogenic effect of CTLA-4. This molecular mechanism was also present in humans because targeting CD80/86 by abatacept, a CTLA-4-immunoglobulin fusion protein, reduced, whereas blockade of CTLA-4 by ipilimumab antibody enhanced, the frequency of peripheral osteoclast precursors and osteoclastogenesis. In summary, these data show an important role of the adaptive immune system, in particular T cell CD80/86 costimulation molecules, in the physiological regulation of bone resorption and preservation of bone mass, as well as affect the understanding of the function of current and future drugs fostering or blocking the effects of CTLA-4 in humans.

In rheumatoid arthritis (RA), the presence of autoantibodies such as the rheumatoid factor and antibodies against citrullinated proteins is highly correlated with the severity of disease and bone loss. For many years, the involvement of autoantibodies in bone resorption has merely been attributed to enhanced tissue infiltration and the production of inflammatory cytokines that promote osteoclastogenesis. However, recent research provides evidence for a direct activation of osteoclasts and their precursors by autoantibodies, which is independent of inflammation. The depletion of B-cells with rituximab that substantially reduces autoantibody levels seems to be as effective as the well-established treatment with tumor necrosis factor-antagonists in RA patients that do not respond to methotrexate, highlighting the significance of autoantibodies for RA and bone loss.

Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38α is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3-/- cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3-/- and MKK6-/- mice by micro-CT analysis. Bone loss was partially inhibited in MKK3-/- as well as MKK6-/- mice, despite normal osteoclastogenesis in MKK6-/- cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation.

Destruction of bone tissue by osteoclasts represents a severe pathological phenotype during inflammatory arthritis and results in joint pain and bone malformations. Previous studies have established the essential role of cytokines including TNFα and receptor-ligand interactions, such as the receptor activator of nuclear factor-kappa B-receptor activator of nuclear factor-kappa B ligand interaction for osteoclast formation during joint inflammation. Moreover, autoantibodies contribute to joint inflammation in inflammatory arthritis by triggering cellular fragment crystallizable (Fc)γ receptors (FcγR), resulting in the release of proinflammatory cytokines and chemokines essential for recruitment and activation of innate immune effector cells. In contrast, little is known about the expression pattern and function of different FcγRs during osteoclast differentiation. This would allow osteoclasts to directly interact with autoantibody immune complexes, rather than being influenced indirectly via proinflammatory cytokines released upon immune complex binding to other FcγR-expressing innate immune cells. To address this question, we studied FcγR expression and function on osteoclasts during the steady state and during acute joint inflammation in a model of inflammatory arthritis. Our results suggest that osteoclastogenesis is directly influenced by IgG autoantibody binding to select activating FcγRs on immature osteoclasts, resulting in enhanced osteoclast generation and, ultimately, bone destruction.

Rapid clearance of apoptotic cells, frequently referred to as efferocytosis, is crucial for the maintenance of tissue homeostasis and the prevention of autoimmunity. The common model of apoptotic cell clearance involves a system of released "Find me" and exposed "Eat me" signals on apoptotic cells, detected and recognized by matching receptors on macrophages or dendritic cells (DC), referred to as the phagocytic synapse. Osteoclasts share the monocyte lineage with these professional mononuclear phagocytes, thus raising the question if, in addition to bone resorption, osteoclasts can act as scavengers for apoptotic cells. Our qPCR data clearly show that osteoclasts express most of the genes required for dying cell clearance at mRNA levels similar to or even higher than those observed in M1-macrophages, M2-macrophages or DC. Our microscopical analyses reveal that osteoclasts in fact can bind and/or engulf apoptotic cells in an essentially serum-independent fashion. Together with our data on the abundance of the respective mRNAs, these results identify the vitronectin receptor (integrin α(ν)β(3))/milk fat globule-EGF factor 8 protein (MFG-E8) axis, the scavenger receptors (CD36, CD68 and class A macrophage scavenger receptor (SR-A)), the complement/complement receptor axis, the Mer/Tyro3/Protein S axis, and the phosphatidylserine (PS) receptor brain-specific angiogenesis inhibitor 1 (BAI1) as the most promising candidates to be involved in osteoclast-mediated efferocytosis.

Osteoporosis is characterized by enhanced differentiation of bone-resorbing osteoclasts, resulting in a rapid loss of functional trabecular bone. Bone-forming osteoblasts and osteoblast-derived osteocytes perform a key role in the regulation of osteoclast development by providing both the pro-osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) and its natural decoy receptor osteoprotegerin (OPG). By regulating the RANKL/OPG ratio, osteoblasts hence determine the rate of both osteoclast differentiation and bone turnover. Here, we describe a novel role for liver X receptors (LXRs) during the crosstalk of bone-forming osteoblasts and bone-resorbing osteoclasts. By using a system of osteoblast/osteoclast cocultures, we identify LXRs as regulator of RANKL expression and the RANKL/OPG ratio in osteoblasts. Activation of LXRs drastically reduced the RANKL/OPG ratio and interfered with osteoblast-mediated osteoclast differentiation in vitro. During an ovariectomy (OVX)-induced model of postmenopausal osteoporosis, the application of an LXR agonist shifted the RANKL/OPG ratio in vivo, ameliorated the enhanced osteoclast differentiation, and provided complete protection from OVX-induced bone loss. These results reveal an unexpected involvement of LXRs in the regulation of bone turnover and highlight a potential role for LXRs as novel targets in the treatment of osteoporosis and related diseases., (Copyright © 2012 American Society for Bone and Mineral Research.)

Bone erosion is a central feature of rheumatoid arthritis and is associated with disease severity and poor functional outcome. Erosion of periarticular cortical bone, the typical feature observed on plain radiographs in patients with rheumatoid arthritis, results from excessive local bone resorption and inadequate bone formation. The main triggers of articular bone erosion are synovitis, including the production of proinflammatory cytokines and receptor activator of nuclear factor κB ligand (RANKL), as well as antibodies directed against citrullinated proteins. Indeed, both cytokines and autoantibodies stimulate the differentiation of bone-resorbing osteoclasts, thereby stimulating local bone resorption. Although current antirheumatic therapy inhibits both bone erosion and inflammation, repair of existing bone lesions, albeit physiologically feasible, occurs rarely. Lack of repair is due, at least in part, to active suppression of bone formation by proinflammatory cytokines. This Review summarizes the substantial progress that has been made in understanding the pathophysiology of bone erosions and discusses the improvements in the diagnosis, monitoring and treatment of such lesions.

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Background: Osteoclasts play a critical role in bone resorption under basal conditions, but they also contribute to pathological bone loss during diseases including postmenopausal osteoporosis. Phospholipase Cγ2 (PLCγ2) is an important signalling molecule in diverse haematopoietic lineages. Here, we tested the role of PLCγ2 in basal and ovariectomy-induced bone resorption, as well as in in vitro osteoclast cultures using PLCγ2-deficient (PLCγ2(-/-) ) mice., Materials and Methods: The trabecular architecture of long bone metaphyses was tested by micro-CT and histomorphometric analyses. Postmenopausal osteoporosis was modelled by surgical ovariectomy. Osteoclast development and function, gene expression and PLCγ2 phosphorylation were tested on in vitro osteoclast and macrophage cultures., Results:   PLCγ2(-/-) mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. PLCγ2 was required for in vitro development and resorptive function of osteoclasts, but not for upregulation of osteoclast-specific gene expression. PLCγ2 was phosphorylated in a Src-family-dependent manner upon macrophage adhesion but not upon stimulation by M-CSF or RANKL. Surprisingly, ovariectomy-induced bone resorption in PLCγ2(-/-) mice was similar to, or even more robust than, that in wild-type animals., Conclusions: Our results indicate that PLCγ2 participates in bone resorption under basal conditions, likely because of its role in adhesion receptor signalling during osteoclast development. In contrast, PLCγ2 does not appear to play a major role in ovariectomy-induced bone loss. These results suggest that basal and oestrogen deficiency-induced bone resorption utilizes different signalling pathways and that PLCγ2 may not be a suitable therapeutic target in postmenopausal osteoporosis., (© 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.)

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2(-/-) bone marrow led to more bone loss compared with the chimeras with ST2(+/+) bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206(+) AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL-1) and IL-18, two cytokines of the IL-1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL-33, another IL-1 family member, has not been addressed yet. Since we observed that the expression of IL-33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL-33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1-deficient mice, which lack the IL-33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL-33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL-33 completely abolished the generation of TRACP(+) multinucleated osteoclasts, even in the presence of RANKL and macrophage colony-stimulating factor (M-CSF). Although our molecular studies revealed that IL-33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL-33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL-33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL-33 as an inhibitor of bone resorption., (Copyright © 2011 American Society for Bone and Mineral Research.)

Objective: Activation of the mammalian target of rapamycin (mTOR) pathway is important for immune cell activation and bone metabolism. To date, the contribution of mTOR signaling to joint inflammation and structural bone and cartilage damage is unknown. The aim of this study was to investigate the potential of inhibiting mTOR as a treatment of inflammatory arthritis., Methods: Human tumor necrosis factor-transgenic mice in which inflammatory arthritis was developing were treated with 2 different mTOR inhibitors, sirolimus or everolimus. The effects of treatment on clinical disease activity, inflammation, and localized joint and cartilage destruction were studied. In addition, the effects of mTOR inhibition on osteoclast survival and expression of key molecules of osteoclast function were analyzed in vitro. Moreover, synovial tissue from patients with rheumatoid arthritis (RA) was assessed for activation of the mTOR pathway., Results: Inhibition of mTOR by sirolimus or everolimus reduced synovial osteoclast formation and protected against local bone erosions and cartilage loss. Clinical signs of arthritis improved after mTOR inhibition, and histologic evaluation showed a decrease in synovitis. In vitro, mTOR inhibition down-regulated the expression of digestive enzymes and led to osteoclast apoptosis. Moreover, mTOR signaling was shown to be active in the synovial membrane of patients with RA, particularly in synovial osteoclasts., Conclusion: Signaling through mTOR is an important link between synovitis and structural damage in inflammatory arthritis. Current pharmacologic inhibitors of mTOR could be effective in protecting joints against structural damage.

Objective: Immune activation triggers bone loss. Activated T cells are the cellular link between immune activation and bone destruction. The aim of this study was to determine whether immune regulatory mechanisms, such as naturally occurring Treg cells, also extend their protective effects to bone homeostasis in vivo., Methods: Bone parameters in FoxP3-transgenic (Tg) mice were compared with those in their wild-type (WT) littermate controls. Ovariectomy was performed in FoxP3-Tg mice as a model of postmenopausal osteoporosis, and the bone parameters were analyzed. The bones of RAG-1(-/-) mice were analyzed following the adoptive transfer of isolated CD4+CD25+ T cells. CD4+CD25+ T cells and CD4+ T cells isolated from FoxP3-Tg mice and WT mice were cocultured with monocytes to determine their ability to suppress osteoclastogenesis in vitro., Results: FoxP3-Tg mice developed higher bone mass and were protected from ovariectomy-induced bone loss. The increase in bone mass was found to be the result of impaired osteoclast differentiation and bone resorption in vivo. Bone formation was not affected. Adoptive transfer of CD4+CD25+ T cells into T cell-deficient RAG-1(-/-) mice also increased the bone mass, indicating that Treg cells directly affect bone homeostasis without the need to engage other T cell lineages., Conclusion: These data demonstrate that Treg cells can control bone resorption in vivo and can preserve bone mass during physiologic and pathologic bone remodeling.

We previously demonstrated the suppressive effects of regulatory T cells (Treg cells) on osteoclast differentiation in vitro. In this article, we show that blood markers of bone resorption inversely correlate with the amount of circulating Treg cells in healthy controls and rheumatoid arthritis patients, further suggesting that Treg cells may control bone destruction in vivo. Indeed, bone marrow from Foxp3-transgenic (Foxp3tg) mice fully protected human TNF transgenic (hTNFtg) mice from TNF-alpha-induced bone destruction, whereas Foxp3-deficient bone marrow enhanced local and systemic bone loss. The same protective effect was also obtained by treating hTNFtg mice with the CD28 superagonist mAb (CD28 SA), which increased Treg cell numbers. In both models, bone protection by Treg cells was associated with reduced osteoclast numbers, resulting in less bone-resorbing activity. Reduced osteoclast numbers were not caused by an intrinsic defect in osteoclast differentiation because osteoclast precursors from hTNFtg/Foxp3tg chimeras responded normally to M-CSF and receptor activator of NF-kappaB ligand. Although a decrease in the clinical signs of arthritis was observed in Foxp3tg bone marrow-transferred and CD28 SA-treated hTNFtg mice, the bone-protective effect of Treg cells was independent of the suppression of inflammation, as demonstrated by the increased systemic bone density observed in wild-type mice treated with CD28 SA. This work demonstrated that increasing Treg cell numbers improved clinical signs of arthritis and suppressed local and systemic bone destruction. Thus, enhancing the activity of Treg cells would be beneficial for the treatment of inflammation-induced bone loss observed in rheumatoid arthritis.

Objective: To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA)., Methods: We performed bone marrow transplantations in human TNF-transgenic mice using hematopoietic cells from wild-type, TNFRI(-/-), TNFRII(-/-), and TNFRI/II(-/-) mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays., Results: Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full-blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF-responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis., Conclusion: Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

Objective: To investigate the efficacy of a murine anti-interleukin-6 receptor (anti-IL-6R) antibody in directly blocking tumor necrosis factor (TNF)- and RANKL-mediated osteoclastogenesis in vitro and in vivo., Methods: The efficacy of a murine antibody against IL-6R in blocking osteoclast differentiation of mononuclear cells stimulated with RANKL was tested. In addition, arthritic human TNFalpha-transgenic mice were treated with anti-IL-6R antibody, and osteoclast formation and bone erosion were assessed in arthritic paws., Results: Blockade of IL-6R dose dependently reduced osteoclast differentiation and bone resorption in monocyte cultures stimulated with RANKL or RANKL plus TNF. In human TNFalpha-transgenic mice, IL-6R blockade did not inhibit joint inflammation, but it strongly reduced osteoclast formation in inflamed joints as well as bone erosion in vivo. Neither the cell influx into joints nor the synovial expression of IL-6 and RANKL changed with IL-6R blockade, while the synovial expression of IL-1 was significantly reduced. In contrast, TNF-mediated systemic bone loss was not inhibited by IL-6R blockade., Conclusion: These data suggest that blockade of IL-6R directly affects osteoclast formation in vitro and in vivo, suggesting a direct and specific effect of anti-IL-6R therapy on osteoclasts independently of its antiinflammatory effects. This effect adds significantly to the structure-sparing potential of pharmacologic blockade of IL-6R in arthritis.

Although 12/15-lipoxygenase (12/15-LO) has been implicated as negative regulator of systemic bone mass in mice and humans, the underlying mechanisms remain elusive. Here, we show that 12/15-LO is a positive regulator of osteoclast (OC) development. Enzymatic inhibition as well as genetic ablation of 12/15-LO significantly impaired osteoclastogenesis. Conversely, addition of the 12/15-LO-derived eicosanoids 12- and 15-HETE augmented differentiation of precursors into fully matured OCs. Together these data point towards a crucial role of 12/15-LO in the regulation of OC development. Therefore, 12/15-LO and its human homologue 15-LO may display novel targets for the treatment of diseases such as osteoporosis and rheumatoid arthritis.

CTLA-4 is a regulator of co-stimulation and inhibits the activation of T cells through interfering with the interaction of CD80/86 on antigen-presenting cells with CD28 on T cells. CTLA-4 binds to the surface of antigen-presenting cells, such as dendritic cells and monocytes through CD80/86. Monocytes can differentiate in osteoclasts, the primary bone resorbing cells. Herein, we investigated whether the binding of CTLA-4 affects the differentiation of monocytes into osteoclasts in vitro and vivo. We show that CTLA-4 dose-dependently inhibits RANKL- as well as tumour necrosis factor (TNF)-mediated osteoclastogenesis in vitro without the presence of T cells. Furthermore, CTLA-4 was effective in inhibiting TNF-induced osteoclast formation in a non-T cell dependent TNF-induced model of arthritis as well as the formation of inflammatory bone erosion in vivo. These data suggest that CTLA-4 is an anti-osteoclastogenic molecule that directly binds osteoclast precursor cells and inhibits their differentiation. These findings are an attractive explanation for the anti-erosive effect of abatacept, a CTLA-4 immunoglobulin fusion protein used for the treatment of rheumatoid arthritis.

Objective: Osteoclast-associated receptor (OSCAR) is a newly identified osteoclast-specific receptor and is of key importance in the process of osteoclast costimulation. This study was undertaken to define the role of costimulation in osteoclast differentiation during inflammatory arthritis., Methods: OSCAR expression was assessed in the synovium and peripheral blood monocytes of patients with rheumatoid arthritis (RA), and associations with disease activity were assessed. Serum levels of OSCAR were determined by enzyme-linked immunosorbent assay. In vitro osteoclast assays were performed to characterize the role of OSCAR in human osteoclastogenesis. Cytokine regulation of OSCAR was investigated by reverse transcriptase-polymerase chain reaction., Results: OSCAR was expressed by osteoclasts at the erosion front and by mononuclear cells around synovial microvessels. Flow cytometry revealed enhanced expression of OSCAR in peripheral blood monocytes of RA patients as compared with healthy controls. OSCAR expression was correlated with disease activity and acute-phase reactant concentrations. Serum levels of soluble OSCAR were lower in RA patients than in healthy controls. Monocytes with high OSCAR expression exhibited an enhanced potential to differentiate into osteoclasts. Tumor necrosis factor alpha was identified as the main inducer of OSCAR expression in monocytes., Conclusion: These data suggest that the osteoclast costimulation pathway is activated in RA. OSCAR is induced in monocytes of RA patients, facilitating their differentiation into osteoclasts and bone resorption.

Cytokine expression in the inflamed synovial membrane of patients with rheumatoid arthritis and other forms of chronic inflammatory arthritis and other forms of chronic inflammatory arthritis leads to formation of osteoclasts. These cells are primarily involved in the resorption of mineralized cartilage and bone and thus participate in joint damage in the course of chronic arthritides. Osteoclastogenesis in the synovial membrane is driven by cytokines such as RANKL, MCSF but also classical proinflammatory mediators such as TNF, IL-1 and IL-6. Inhibition of osteoclast formation has proven as an effective approach to inhibit structural damage in experimental arthritis and preliminary data suggest that such approaches are also effective in human RA.

Objective: To investigate whether Treg cells can suppress osteoclast differentiation, and to define a new potential link between the immune system and the skeleton., Methods: Regulatory CD4+,CD25+,Foxp3+ T cells were isolated and purified from the spleen and cocultured with CD11b+ osteoclast precursor cells isolated from bone marrow. Osteoclastogenesis and bone erosion were assessed by tartrate-resistant acid phosphatase staining and pit resorption assay, respectively. In addition, Transwell experiments and cytokine-blocking experiments were performed to define the mechanisms of interaction between Treg cells and osteoclasts., Results: CD4+,CD25+,Foxp3+ T cells, but not CD4+,CD25- T cells, dose dependently inhibited macrophage colony-stimulating factor- and RANKL-dependent osteoclast formation. Pit formation was inhibited by up to 80% when Treg cells were added. The blockade of osteoclast formation was not based on the alteration of RANKL/osteoprotegerin balance but was essentially dependent on direct cell-cell contact via CTLA-4. Treg cell-mediated expression of transforming growth factor beta, interleukin-4 (IL-4), and IL-10 contributed but was not essential to the inhibitory effect on osteoclastogenesis., Conclusion: These data show that CD4+,CD25+,Foxp3+ Treg cells suppress osteoclast formation, provide a new link between the immune system and bone, and extend our knowledge on regulation of bone homeostasis by the immune system.

Osteoclasts are multinucleated cells of hematopoietic origin and are the primary bone resorbing cells. Numerous osteoclasts are found within the synovial tissue at sites adjacent to bone, creating resorption pits and local bone destruction. They are equipped with specific enzymes and a proton pump that enable them to degrade bone matrix and solubilize calcium, respectively. The synovial tissue of inflamed joints has a particularly high potential to accumulate osteoclasts because it harbors monocytes/macrophages, which function as osteoclast precursors, as well as cells that provide the specific molecular signals that drive osteoclast formation. Osteoclasts thus represent a link between joint inflammation and structural damage since they resorb mineralized tissue adjacent to the joint and destroy the joint architecture.

Objective: To determine the nature of the initial changes of joint inflammation occurring before, at the time of, and shortly after onset of clinically apparent arthritis., Methods: Human tumor necrosis factor (TNF)-transgenic mice were assessed for clinical, histologic, immunophenotypic, serologic, and molecular changes at the preclinical phase of arthritis, at the onset of disease, and at the stage of early clinical disease. In addition, the effects of a genetic osteoclast deficiency and pharmacologic inhibition of TNF were studied in these initial phases of disease., Results: Initial articular changes were observed even before the start of clinical symptoms. Infiltration of the tendon sheaths by granulocytes and macrophages as well as formation of osteoclasts next to the inflamed tendon sheaths were the first pathologic events. Tenosynovitis rapidly led to remodeling of the sheaths into pannus-like tissue, which formed osteoclasts that invaded the adjacent mineralized cartilage. Early lesions were associated with up-regulation of interleukin-1 (IL-1) and IL-6 as well as activation of p38 MAPK and ERK. In contrast, absence of osteoclasts led to uncoupling of tenosynovitis from invasion into cartilage and bone. TNF blockade also attenuated the pathologic changes associated with tenosynovitis., Conclusion: Structural damage begins even before the onset of clinical symptoms of arthritis and involves the tendon sheaths as well as adjacent cartilage and bone. These results suggest that tenosynovitis is an initiating feature of arthritis and that joint destruction starts right from the onset of disease. Our findings thus underscore the importance of immediate initiation of an effective therapy in patients with rheumatoid arthritis.

Heme oxygenase 1 (HO-1) plays an important role in vascular disease, transplantation, and inflammation. In animal models of acute and chronic inflammation, induction of HO-1 has anti-inflammatory and cytoprotective properties. Since inflammation is an important trigger of osteoclastogenesis, we hypothesized that HO-1 might influence osteoclastogenesis. We investigated the effects of induction of HO-1 on osteoclast formation in vitro and in vivo. Furthermore, we addressed the role of HO-1 in inflammatory bone loss in humans. When HO-1 was induced by hemin in vitro, a significant dose-dependent inhibition of osteoclastogenesis was observed. Up-regulation of HO-1 was mediated by activation of MAPK and primarily prevented differentiation of osteoclast precursors to osteoclasts, whereas it did not affect mature osteoclasts. Anti-osteoclastogenic properties of hemin were based on a down-regulation of c-fms, RANK, TRAF-6, and c-fos. In addition, induction of HO-1 inhibited TNF-triggered osteoclast differentiation in vitro as well as LPS-driven inflammatory bone loss in vivo. Furthermore, HO-1 induction suppressed osteoclastogenesis and bone destruction in a TNF-mediated arthritis. In line, assessment of synovial tissue from rheumatoid arthritis patients revealed that osteoclasts are usually HO-1 negative. Moreover, serum levels of bilirubin, a metabolite of HO-1, were elevated in rheumatoid arthritis patients without bone damage, suggesting HO-1 affects bone loss in humans. In summary, these data indicate that HO-1 negatively regulates osteoclastogenesis, leading to a positive net balance of bone.

Objective: To analyze the kinetics of osteoclastogenesis in 2 models of chronic immune-mediated arthritis and 1 model of acute arthritis., Methods: Adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in Lewis rats were used as models of chronic arthritis. Acute arthritis was induced in Lewis rats by injecting carrageenan into the hind paw. Osteoclasts were identified by cathepsin K immunohistochemistry at various time points after the onset of arthritis. The location, size, and nucleation of osteoclasts were also analyzed., Results: In both AIA and CIA, multinucleated and cathepsin K-positive osteoclasts first were observed on the day of disease onset. Initially, osteoclasts were localized at the periosteum next to the synovial membrane and in subchondral bone channels. The number, size, and nucleation of osteoclasts rapidly increased, leading to severe bone loss within days after disease onset. In addition, numerous mononucleated cathepsin K-positive osteoclast precursor cells emerged in the synovial membrane. All osteoclasts (cathepsin K-positive, multinucleated, attached to bone) and osteoclast precursors (cathepsin K-positive, mononucleated or multinucleated, within synovial tissue) were also positive for a macrophage-specific marker. Upon induction of acute arthritis with carrageenan, osteoclasts formed transiently in subchondral bone, but regressed 7 days after disease onset., Conclusion: Functional osteoclasts are generated at the earliest stage of arthritis, and new precursors are continuously formed in the synovial membrane to replenish the osteoclast pool. These data indicate that anti-resorptive therapies may provide the most effective bone protection, when treatment is started soon after the onset of arthritis.

Transcription factor, nuclear factor kappaB (NF-kappaB), is required for osteoclast formation in vivo and mice lacking both of the NF-kappaB p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the IkappaB kinase (IKK) complex that mediate NF-kappaB activation, IKKalpha and IKKbeta, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKbeta subunit as a transducer of signals from receptor activator of NF-kappaB (RANK) to NF-kappaB. Although IKKalpha is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKbeta is required for osteoclastogenesis in vitro and in vivo. IKKbeta also protects osteoclasts and their progenitors from tumor necrosis factor alpha-induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.