Human Osteoclastogenesis Research Articles

Anti-rheumatic property and physiological safety of KMU-11342 in in vitro and in vivo models

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder characterized by joint destruction due to synovial hypertrophy and the infiltration of inflammatory cells. Despite substantial progress in RA treatment, challenges persist, including suboptimal treatment responses and adverse effects associated with current therapies. This study investigates the anti-rheumatic capabilities of the newly identified multi-protein kinase inhibitor, KMU-11342, aiming to develop innovative agents targeting RA. In this study, we synthesized the novel multi-protein kinase inhibitor KMU-11342, based on indolin-2-one. We assessed its cardiac electrophysiological safety using the Langendorff system in rat hearts and evaluated its toxicity in zebrafish in vivo. Additionally, we examined the anti-rheumatic effects of KMU-11342 on human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), THP-1 cells, and osteoclastogenesis in RAW264.7 cells. KMU-11342 demonstrated the ability to inhibit LPS-induced chemokine inhibition and the upregulation of pro-inflammatory cytokines, cyclooxygenase-2, inducible nitric oxide synthase, p-IKKα/β, p-NF-κB p65, and the nuclear translocation of NF-κB p65 in RA-FLS. It effectively suppressed the upregulation of NLR family pyrin domain containing 3 (NLRP3) and caspase-1 cleavage. Furthermore, KMU-11342 hindered the activation of osteoclast differentiation factors such as RANKL-induced TRAP, cathepsin K, NFATc-1, and c-Fos in RAW264.7 cells. KMU-11342 mitigates LPS-mediated inflammatory responses in THP-1 cells by inhibiting the activation of NLRP3 inflammasome. Notably, KMU-11342 exhibited minimal cytotoxicity in vivo and electrophysiological cardiotoxicity ex vivo. Consequently, KMU-11342 holds promise for development as a therapeutic agent in RA treatment.

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

Erianin serves as an NFATc1 inhibitor to prevent breast cancer-induced osteoclastogenesis and bone destruction

IntroductionBreast cancer-related bone metastasis can lead to skeletal-related events (SREs), which decrease patient quality of life. Inhibition of osteoclastogenesis is a key treatment for SREs; however, the availability of clinical drugs remains limited, and all existing ones disrupt physiological bone formation, while exhibiting no effect on patient survival time. ObjectivesThis study aimed to identify a novel osteoclast inhibitor for the treatment of breast cancer-induced SREs. MethodsThe MDA-MB-231 breast cancer cell-induced bone loss model was used to investigate the therapeutic effects of erianin in vivo. Then, we evaluated the inhibitory effects of erianin on osteoclastogenesis and signalling in bone marrow-derived macrophages (BMMs) induced by conditioned medium from MDA-MB-231 breast cancer cells (231 CM) and receptor activator of nuclear factor-κB ligand (RANKL) in vitro. Next, a Cellular Thermal Shift Assay and siRNA-mediate knockdown were performed, to investigate the target of erianin during osteoclast formation. The effects of erianin on human osteoclastogenesis were evaluated using CD14+ monocytes obtained from patients with breast cancer. ResultsErianin effectively improved breast cancer cells-induced bone destruction at doses of 2 and 20 mg/kg/day in vivo, while suppressing osteoclastogenesis and the upregulation of SRC-NFATc1, INTEGRIN β3-MMP9 signals induced by 231 CM and RANKL in vitro. Furthermore, erianin interacted with NFATc1 but not SRC, and Nfatc1 knockdown eliminated the inhibitory effects of erianin on osteoclastogenesis. Notably, lower expression of NFATc1 positively correlated with longer survival in patients with cancer and a high risk of bone metastasis. We further revealed that 62.5–250 nM erianin suppresses NFATc1 and excessive osteoclastogenesis in CD14+ monocytes from patients with breast cancer. ConclusionErianin acts as an NFATc1 inhibitor that attenuates breast cancer-induced osteoclastogenesis and bone destruction.

Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity

Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis, which is characterized by increased bone resorption and inadequate bone formation. As novel antiosteoporotic therapeutics are needed, understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets. This study aimed to provide an overview of transcriptional reprogramming during human osteoclast differentiation. Osteoclasts were differentiated from CD14+ monocytes from eight female donors. RNA sequencing during differentiation revealed 8 980 differentially expressed genes grouped into eight temporal patterns conserved across donors. These patterns revealed distinct molecular functions associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies and bone mineral density SNPs. Network analyses revealed mutual dependencies between temporal expression patterns and provided insight into subtype-specific transcriptional networks. The donor-specific expression patterns revealed genes at the monocyte stage, such as filamin B (FLNB) and oxidized low-density lipoprotein receptor 1 (OLR1, encoding LOX-1), that are predictive of the resorptive activity of mature osteoclasts. The expression of differentially expressed G-protein coupled receptors was strong during osteoclast differentiation, and these receptors are associated with bone mineral density SNPs, suggesting that they play a pivotal role in osteoclast differentiation and activity. The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5 A receptor 1 (C5AR1), somatostatin receptor 2 (SSTR2), and free fatty acid receptor 4 (FFAR4/GPR120). Activating C5AR1 enhanced osteoclast formation, while activating SSTR2 decreased the resorptive activity of mature osteoclasts, and activating FFAR4 decreased both the number and resorptive activity of mature osteoclasts. In conclusion, we report the occurrence of transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as antiresorptive G-protein coupled receptors and FLNB and LOX-1 as potential molecular markers of osteoclast activity. These data can help future investigations identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel antiosteoporotic targets.

The Notch1 signaling pathway directly modulates the human RANKL-induced osteoclastogenesis

Notch signaling is an evolutionary conserved pathway with a key role in tissue homeostasis, differentiation and proliferation. It was reported that Notch1 receptor negatively regulates mouse osteoclast development and formation by inhibiting the expression of macrophage colony-stimulating factor in mesenchymal cells. Nonetheless, the involvement of Notch1 pathway in the generation of human osteoclasts is still controversial. Here, we report that the constitutive activation of Notch1 signaling induced a differentiation block in human mononuclear CD14+ cells directly isolated from peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation to osteoclasts. Additionally, using a combined approach of single-cell RNA sequencing (scRNA-Seq) simultaneously with a panel of 31 oligo-conjugated antibodies against cell surface markers (AbSeq assay) as well as unsupervised learning methods, we detected four different cell stages of human RANKL-induced osteoclastogenesis after 5 days in which Notch1 signaling enforces the cell expansion of specific subsets. These cell populations were characterized by distinct gene expression and immunophenotypic profiles and active Notch1, JAK/STAT and WNT signaling pathways. Furthermore, cell–cell communication analyses revealed extrinsic modulators of osteoclast progenitors including the IL7/IL7R and WNT5a/RYK axes. Interestingly, we also report that Interleukin-7 receptor (IL7R) was a downstream effector of Notch1 pathway and that Notch1 and IL7R interplay promoted cell expansion of human RANKL-induced osteoclast progenitors. Taken together, these findings underline a novel cell pattern of human osteoclastogenesis, outlining the key role of Notch1 and IL-7R signaling pathways.

L-arginine metabolism inhibits arthritis and inflammatory bone loss

ObjectivesTo investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss.MethodsL-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation...

Jagged1 Acts as an RBP-J Target and Feedback Suppresses TNF-Mediated Inflammatory Osteoclastogenesis.

TNF plays a crucial role in inflammation and bone resorption in various inflammatory diseases, including rheumatoid arthritis (RA). However, its direct ability to drive macrophages to differentiate into osteoclasts is limited. Although RBP-J is recognized as a key inhibitor of TNF-mediated osteoclastogenesis, the precise mechanisms that restrain TNF-induced differentiation of macrophages into osteoclasts are not fully elucidated. In this study, we identified that the Notch ligand Jagged1 is a previously unrecognized RBP-J target. The expression of Jagged1 is significantly induced by TNF mainly through RBP-J. The TNF-induced Jagged1 in turn functions as a feedback inhibitory regulator of TNF-mediated osteoclastogenesis. This feedback inhibition of osteoclastogenesis by Jagged1 does not exist in RANKL-induced mouse osteoclast differentiation, as RANKL does not induce Jagged1 expression. The Jagged1 level in peripheral blood monocytes/osteoclast precursors is decreased in RA compared with the nonerosive inflammatory disease systemic lupus erythematosus, suggesting a mechanism that contributes to increased osteoclast formation in RA. Moreover, recombinant Jagged1 suppresses human inflammatory osteoclastogenesis. Our findings identify Jagged1 as an RBP-J direct target that links TNF and Notch signaling pathways and restrains TNF-mediated osteoclastogenesis. Given that Jagged1 has no effect on TNF-induced expression of inflammatory genes, its use may present a new complementary therapeutic approach to mitigate inflammatory bone loss with little impact on the immune response in disease conditions.

DCIR suppresses osteoclastic proliferation and resorption by downregulating M-CSF and RANKL signaling.

Dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lectin receptor that acts as a negative regulator in the immune system and bone metabolism. We previously revealed that DCIR deficiency enhanced osteoclastogenesis and antigen presentation of dendritic cells, and that asialo-biantennary N-glycan (NA2) functions as a ligand for DCIR. NA2 binding to DCIR suppressed murine and human osteoclastogenesis that occurs in the presence of M-CSF and RANKL. The DCIR-NA2 axis, therefore, plays an important role in regulating osteoclastogenesis in both mice and humans, although the underlying mechanisms remain unclear. Here we found that Dcir -/- bone marrow-derived macrophages (BMMs) exhibited greater proliferative and differentiation responses to M-CSF and RANKL, respectively, than wild-type (WT) BMMs. Moreover, Dcir -/- osteoclasts (OCs) increased resorptive activity and cell fusion more significantly than WT OCs. DCIR deficiency affects gene expression patterns in OCs, and we found that the expression of neuraminidase 4 was increased in Dcir -/- OCs. Furthermore, DCIR-NA2 interaction in WT BMMs, but not Dcir -/- BMMs, decreased Akt phosphorylation in response to M-CSF and RANKL. These data suggest that DCIR regulates osteoclastogenesis by downregulating M-CSF and RANKL signaling, and that DCIR-mediated signaling may contribute to the terminal modification of oligosaccharides by controlling the expression of glycosylation enzymes.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases.In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution ofinflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

A Vitronectin-Derived Peptide Restores Ovariectomy-Induced Bone Loss by Dual Regulation of Bone Remodeling.

Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3weeks. Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.

Interspecies Single-Cell RNA-Seq Analysis Reveals the Novel Trajectory of Osteoclast Differentiation and Therapeutic Targets.

ABSTRACTBone turnover is finely tuned by cells in the bone milieu, including osteoblasts, osteoclasts, and osteocytes. Osteoclasts are multinucleated giant cells with a bone‐resorbing function that play a critical role in regulating skeletal homeostasis. Osteoclast differentiation is characterized by dramatic changes in morphology and gene expression following receptor activator of nuclear factor‐kappa‐Β ligand (RANKL) stimulation. We performed single‐cell RNA‐sequencing analyses of human and murine osteoclast‐lineage cells (OLCs) and found that OLCs in the mitotic phase do not differentiate into mature osteoclasts. We also identified a guanosine triphosphatase (GTPase) family member, RAB38, as a highly expressed molecule in both human and murine osteoclast clusters; RAB38 gene expression is associated with dynamic changes in histone modification and transcriptional regulation. Silencing Rab38 expression by using short hairpin RNA (shRNA) inhibited osteoclast differentiation and maturation. In summary, we established an integrated fate map of human and murine osteoclastogenesis; this will help identify therapeutic targets in bone diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Role of Lysine-Specific Demethylase 1 in Metabolically Integrating Osteoclast Differentiation and Inflammatory Bone Resorption Through Hypoxia-Inducible Factor 1α and E2F1.

Hypoxia occurs in tumors, infections, and sites of inflammation, such as in the affected joints of patients with rheumatoid arthritis (RA). It alleviates inflammatory responses and increases bone resorption in inflammatory arthritis by enhancing osteoclastogenesis. The mechanism by which the hypoxia response is linked to osteoclastogenesis and inflammatory bone resorption is unclear. This study was undertaken to evaluate whether the protein lysine-specific demethylase 1 (LSD1) metabolically integrates inflammatory osteoclastogenesis and bone resorption in a state of inflammatory arthritis. LSD1-specific inhibitors and gene silencing with small interfering RNAs were used to inhibit the expression of LSD1 in human osteoclast precursor cells derived from CD14-positive monocytes, with subsequent assessment by RNA-sequencing analysis. In experimental mouse models of arthritis, inflammatory osteolysis, or osteoporosis, features of accelerated bone loss and inflammatory osteolysis were analyzed. Furthermore, in blood samples from patients with RA, cis-acting expression quantitative trait loci (cis-eQTL) were analyzed for association with the expression of hypoxia-inducible factor 1α (HIF-1α), and associations between HIF-1α allelic variants and extent of bone erosion were evaluated. In human osteoclast precursor cells, RANKL induced the expression of LSD1 in a mechanistic target of rapamycin-dependent manner. Expression of LSD1 was higher in synovium from RA patients than in synovium from osteoarthritis patients. Inhibition of LSD1 in human osteoclast precursors suppressed osteoclast differentiation. Results of transcriptome analysis identified several LSD1-mediated hypoxia and cell-cycle pathways as key genetic pathways involved in human osteoclastogenesis. Furthermore, HIF-1α protein, which is rapidly degraded by the proteasome in a normoxic environment, was found to be expressed in RANKL-stimulated osteoclast precursor cells. Induction of LSD1 by RANKL stabilized the expression of HIF-1α protein, thereby promoting glycolysis, in conjunction with up-regulation of the transcription factor E2F1. Analyses of cis-eQTL revealed that higher HIF-1α expression was associated with increased bone erosion in patients with RA. Inhibition of LSD1 decreased pathologic bone resorption in mice, both in models of accelerated osteoporosis and models of arthritis and inflammatory osteolysis. LSD1 metabolically regulates osteoclastogenesis in an energy-demanding inflammatory environment. These findings provide potential new therapeutic strategies targeting osteoclasts in the management of inflammatory arthritis, including in patients with RA.

The Neuropeptide VIP Limits Human Osteoclastogenesis: Clinical Associations with Bone Metabolism Markers in Patients with Early Arthritis.

We aimed to evaluate the direct action of VIP on crucial molecules involved in human osteoclast differentiation and function. We also investigated the relationship between VIP serum levels and bone remodeling mediators in early arthritis patients. The expression of VIP receptors and osteoclast gene markers in monocytes and in vitro differentiated osteoclasts was studied by real-time PCR. NFATc1 activity was measured using a TransAM® kit. Osteoclastogenesis was confirmed by quantification of tartrate-resistant acid phosphatase positive multinucleated cells. OsteoAssay® Surface Multiple Well Plate was used to evaluate bone-resorbing activity. The ring-shaped actin cytoskeleton and the VPAC1 and VPAC2 expression were analyzed by immunofluorescence. We described the presence of VIP receptors in monocytes and mature osteoclasts. Osteoclasts that formed in the presence of VIP showed a decreased expression of osteoclast differentiation gene markers and proteolytic enzymes involved in bone resorption. VIP reduced the resorption activity and decreased both β3 integrin expression and actin ring formation. Elevated serum VIP levels in early arthritis patients were associated with lower BMD loss and higher serum OPG concentration. These results demonstrate that VIP exerts an anti-osteoclastogenic action impairing both differentiation and resorption activity mainly through the negative regulation of NFATc1, evidencing its bone-protective effects in humans.

Effect of metal ions released from orthodontic mini-implants on osteoclastogenesis.

Orthodontic mini-implants can undergo corrosion and the release of metal ions can affect cellular behavior. Osteoclasts are involved in orthodontic tooth movement and implant stability. Osteoclasts and their precursors can be exposed to metal ions released from orthodontic mini-implants. This study aimed to investigate the effect of metal ions released from orthodontic miniimplants on human osteoclastogenesis. Stainless steel and titanium alloy mini-implants were separately immersed in culture media for 14 days (days 1-14), and then moved to new media for a further 14 days (days 15-28). The concentration of the released metal ions was measured. Osteoclast precursors derived from human CD14+ monocytes were cultured in these media and in a control medium without mini-implant immersion. Cell viability, the number of osteoclasts and the area of resorption were investigated. A higher concentration of metal ions was detected during the first 14 days as compared to the control. The concentration of these metal ions then declined after this period. The viability of osteoclast precursors was not affected by the released metal ions. There was a significant reduction in the number of osteoclasts when cultured in the medium with the titanium alloy mini-implants immersed for days 1-14. The area of resorption was also significantly reduced in this group. The media with the titanium alloy mini-implants immersed for days 15-28 and with the stainless steel mini-implants immersed for both study periods did not show statistically significant changes in the number of osteoclasts. Metal ions were released from orthodontic mini-implants in the early period and declined thereafter. Metal ions released from titanium mini-implants in the early period inhibited osteoclastogenesis, while metal ions from stainless steel mini-implants had no effect on osteoclast differentiation.

The Possible Role of Human Mesenchymal Stem Cells on Human Osteoclast Differentiation from CD34+ Bone Marrow Hematopoietic Progenitors.

Hematopoietic progenitors differentiate into osteoclasts through the influence of systemic hormones and factors produced within the bone marrow microenvironment. Beyond the differentiation to nonhematopoietic tissues, including bone, cartilage, muscle, of pluripotent mesenchymal stem cells (MSCs) are components of this microenvironment and have been shown to produce extracellular matrix, cytokines and growth factors that regulate the formation, activity, and survival of osteoclasts in vitro. In this study, we sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human MSCs and CD34+ hematopoietic stem cells (HSCs). Fifty milliliters of bone marrow were harvested from breast cancer patients to whom planning autologous BMT. Bone marrow cell suspensions were centrifuged on Ficoll-Paque to isolate mononuclear cells. The mononuclear cell fraction was cultured in standard medium to obtain MSCs. After 4–5 passage mesenchymal differentiation was determined by morphologically, Vimentin, and CD54 and CD58 positivity. Ostoeogenic differentiaiton of MSCs was induced with an osteogenic supplement (OS) containing dexamethasane, B-glycerophosphate, L-ascorbic acid for 12 days incubation. CD34+ HSCs were purified from bone marrow mononuclear cells by positive selection using CliniMACS. Aliquots of DAPI-stained HSCs were added to monolayers of MSCs or OS-treated MSCs, and the coculture was maintained at 37°C in 5 % CO2 for up to 3 weeks. To assess the effect of vitamin D3 on osteoclast formation 1,25(OH)2D3 was added to the coculture. Human fibroblasts were used as negative control instead of MSCs. To determine the role of cell contact in osteoclast formation, experiments were performed in which HSCs and MSCs were cultured in either the same chamber or in chambers separated by 0.45-μm porous membrane for 3 weeks. After 3 weeks, this coculture system yielded that MSCs promote differentiation of DAPI-stained HSCs into TRAP+ multinucleated cells in the absence of added hormones, cytokines, and growth factors. These cells showed CD14, CD11b, CD11c, CD56 positivity, whereas CD34, CD45, HLA-DR, CD13, CD71, CD42 were negative after 3 weeks of culture. In contrast, human fibroblasts failed to support the formation of MNCs, the majority of HSCs degenerated within 2 weeks of culture. Separation of MSCs and HSCs reduced osteoclast formation by 80%, suggesting that cell contact is important, but not absolutely required, for osteoclast formation. OS-treated MSCs actually inhibited the formation of osteoclasts from HSCs. The inhibitory effect of OS-treated MSCs on osteoclast formation is dictated by the differentiated state of MSCs. Osteoclast formation was increased if the coculture was performed in the presence of 1,25(OH)2D3. The data show that MSCs support multilineage differentiation of hematopoietic cells and suggest that our model provides opportunities to understand the mechanisms by which MCSs regulate human osteoclastogenesis.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

Smac-mimetics reduce numbers and viability of human osteoclasts

Elevated activity of bone-degrading osteoclasts (OC) contributes to pathological bone degradation in diseases such as multiple myeloma. Several proinflammatory cytokines, including TNF, contribute to osteoclastogenesis. The receptor-interacting protein kinase 1 (RIPK1) regulates inflammation and cell death. It is recruited to the TNF-receptor complex, where it is ubiquitinated, and activates transcription factor NF-κB and mitogen-activated protein kinases (MAPK). Smac-mimetics (SM) is a group of drugs that block RIPK1 ubiquitination and shifts RIPK1 to activation of apoptosis or necroptosis. In this manuscript, we show that the two SM birinapant and LCL-161 reduced the number and viability of primary human OC, and induced TNF-dependent cell death in OC precursors (pre-OC). Birinapant was more cytotoxic than LCL-161 and induced predominantly apoptosis and to some degree necroptosis. Both inhibitors restrained osteoclastogenesis induced by myeloma patient bone-marrow aspirates. SM has gained attention as novel treatment strategies both for cancer and chronic inflammatory pathologies, but limited information has been available on interactions with primary human immune cells. As LCL-161 is in phase 2 clinical studies for multiple myeloma, we propose that SM might possess additional benefits in reducing bone degradation in myeloma patients. Taken together, we show that SM reduces human osteoclastogenesis, and that these compounds may represent promising drug candidates for pathological bone degradation.

Anti-Myeloma Drug Melflufen Inhibits RANKL-Stimulated Osteoclastogenesis By Suppressing Proliferation of CD14+ Precursor Cells

Background Melflufen (melphalan flufenamide) is a first-in-class peptide-drug conjugate (PDC) that targets aminopeptidases and rapidly releases alkylating agents into tumor cells. Osteolytic lesions are a hallmark of multiple myeloma and occur in almost all MM patients, causing severe bone pain and pathological fractures. Previous reports indicate that melphalan enhances osteoclastogenesis. On other other hand, melflufen is able to suppress proliferation of malignant myeloid cells including osteoclast precursors, monocytes. In the present study we therefore evaluated the direct effects of melflufen and melphalan on human osteoclastogenesis using cultured peripheral blood CD14+ monocytes. Methods CD14+ osteoclast precursors were isolated from peripheral blood. Osteoclastogenesis was stimulated by adding RANKL and M-CSF to CD14+ osteoclast precursors and assessed by tartrate-resistant acid phosphatase (TRAP) staining using para-nitrophenyl phosphate (PNPP). Osteoclastic activity was assessed by two assays. First, TRAP activity was measured by quantification of resorption pits on osteo assay synthetic surface made of crystalline calcium phosphate coating mimicking living bone material. Second, collagen degradation was quantified by release of C-terminal telopeptide of type I collagen (CTx) from mineralized bone slices. The proliferation of mature osteoclasts and their precursors in the presence of melflufen and melphalan was assessed using cytotoxicity assays. RNA isolated from osteoclast precursors and mature osteoclasts was subject to gene expression analysis by microarray. Results Melflufen inhibited RANKL-induced osteoclast formation at 1 µM concentration, while 25 µM melphalan was needed to initiate osteoclastogenesis inhibition. However, only extremely high concentrations of melflufen (10 µM) and melphalan (100 µM) affected mature osteoclasts and their TRAP activity. Cytotoxicity assays have demonstrated that melflufen, but not melphalan, can inhibit proliferation of monocytes, osteoclast progenitor cells, at clinically relevant concentrations. When added during osteoclastogenesis, melflufen has significantly inhibited TRAP phosphatase activity on crystalline calcium phosphate-coated surface at 1 µM, whereas 25 µM of melphalan was required to achieve the same effect. As for collagen degradation, the addition of 1 µM melflufen completely suppressed the CTx fragment release. In a parallel assay, 10 µM of melphalan was necessary to produce the same effect. Noteworthy, we did not observe any positive effect of melphalan on osteoclastogenesis or proliferation of osteoclasts. Gene expression analysis showed that mature osteoclasts express higher levels of bone lytic genes ACP5/TRAP, CTSK, MMP2, MMP7, and MMP9. In addition, CD276 and PDCD1LG2 mRNAs encoding B7 family immune checkpoint molecules B7-H3 and PD-L2 are also strongly up-regulated upon osteoclastogenesis. Discussion Myeloma patients often suffer from severe bone pain associated with osteolytic bone lesions. These lesions are the result of excessive osteoclastogenesis induced by multiple myeloma in the bone marrow microenvironment. Moreover, this process creates an immunosuppressive microenvironment since osteoclasts express immune checkpoint molecules known to negatively control activity of T lymphocytes (PD-L2, B7-H3) and NK cells (B7-H3). In this study, we have shown that a novel anti-myeloma PDC melflufen efficiently inhibits osteoclastogenesis and associated collagen degradation and bone resorption when used at clinically relevant concentrations. Melphalan could also induce a reduction of bone resorption and collagen release, however high and clinically irrelevant concentrations were necessary to obtain these effects. Given these results, it is plausible that melflufen-treated MM patients might achieve a reduction in bone pain, and this should be assessed in more detail in the follow-up studies. Also, elimination of immunosuppressive osteoclasts by melflufen may provide a synergistic effect between melflufen and immunotherapy while treating MM patients. This hypothesis is currently being tested in clinical trials. Disclosures Byrgazov: Oncopeptides AB: Current Employment. Gullbo:Theradex: Current Employment; Oncopeptides AB: Consultancy, Current Employment, Current equity holder in publicly-traded company. Slipicevic:Oncopeptides AB: Current Employment. Lehmann:Oncopeptides AB: Current Employment, Current equity holder in publicly-traded company. Larsson:Oncopeptides AB: Current equity holder in publicly-traded company. Fryknäs:Oncopeptides AB: Research Funding.

The human sialic acid-binding immunoglobulin-like lectin Siglec-9 and its murine homolog Siglec-E control osteoclast activity and bone resorption.

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.

Osteoblast-Osteoclast Coculture Amplifies Inhibitory Effects of FG-4592 on Human Osteoclastogenesis and Reduces Bone Resorption.

ABSTRACTThe link between bone and blood vessels is regulated by hypoxia and the hypoxia‐inducible transcription factor, HIF, which drives both osteogenesis and angiogenesis. The recent clinical approval of PHD enzyme inhibitors, which stabilize HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis, and skeletal fracture and nonunion. However, bone‐resorbing osteoclasts also play a central role in bone remodeling and pathological osteolysis, and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the result of PHD enzyme inhibition in vivo would be mediated by a balance between increased bone formation and increased bone resorption. It is essential that we improve our understanding of the effects of HIF on osteoclast formation and function and consider the potential contribution of inhibitory interactions with other musculoskeletal cells. The PHD enzyme inhibitor FG‐4592 stabilized HIF protein and stimulated osteoclast‐mediated bone resorption, but inhibited differentiation of human CD14+ monocytes into osteoclasts. Formation of osteoclasts in a more physiologically relevant 3D collagen gel did not affect the sensitivity of osteoclastogenesis to FG‐4592, but increased sensitivity to reduced concentrations of RANKL. Coculture with osteoblasts amplified inhibition of osteoclastogenesis by FG‐4592, whether the osteoblasts were proliferating, differentiating, or in the presence of exogenous M‐CSF and RANKL. Osteoblast coculture dampened the ability of high concentrations of FG‐4592 to increase bone resorption. These data provide support for the therapeutic use of PHD enzyme inhibitors to improve bone formation and/or reduce bone loss for the treatment of osteolytic pathologies and indicate that FG‐4592 might act in vivo to inhibit the formation and activity of the osteoclasts that drive osteolysis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.