Systemic Bone Loss Research Articles

Toll-like receptor 9 deficiency induces osteoclastic bone loss via gut microbiota-associated systemic chronic inflammation

Toll-like receptors (TLRs) play pivotal roles in inflammation and provide important links between the immune and skeletal systems. Although the activation of TLRs may affect osteoclast differentiation and bone metabolism, whether and how TLRs are required for normal bone remodeling remains to be fully explored. In the current study, we show for the first time that TLR9−/− mice exhibit a low bone mass and low-grade systemic chronic inflammation, which is characterized by the expansion of CD4+ T cells and increased levels of inflammatory cytokines, including TNFα, RANKL, and IL1β. The increased levels of these cytokines significantly promote osteoclastogenesis and induce bone loss. Importantly, TLR9 deletion alters the gut microbiota, and this dysbiosis is the basis of the systemic inflammation and bone loss observed in TLR9−/− mice. Furthermore, through single-cell RNA sequencing, we identified myeloid-biased hematopoiesis in the bone marrow of TLR9−/− mice and determined that the increase in myelopoiesis, likely caused by the adaptation of hematopoietic stem cells to systemic inflammation, also contributes to inflammation-induced osteoclastogenesis and subsequent bone loss in TLR9−/− mice. Thus, our study provides novel evidence that TLR9 signaling connects the gut microbiota, immune system, and bone and is critical in maintaining the homeostasis of inflammation, hematopoiesis, and bone metabolism under normal conditions.

Assessment of Systemic and Maxillary Bone Loss in Cancer Patients with Endo-Periodontal Lesions Using Dkk-1 Biomarker and Dental Radiological Examinations

The aim of our study was to correlate systemic bone loss by evaluating human Dickkopf-related protein 1 (Dkk-1) biomarker compared to horizontal bone loss as well as the presence and size of periapical lesions assessed by dental X-ray (ortopantomography—OPT) and cone beam computed tomography (CBCT) in patients with cancer in the ears, nose and throat (ENT) region vs. healthy controls. The study included 63 subjects divided into a study group of 33 cancer patients with ENT cancer (larynx/oropharynx/sinuses) and a control group of 30 healthy individuals. Blood samples were collected from both groups to assess Dkk-1 level using a sandwich enzyme immunoassay. The dental radiological examination consisted of a panoramic X-ray and a CBCT in order to appraise the horizontal bone loss, the presence and size of the periapical lesions in 2D vs. 3D images. The panoramic X-ray showed that in the control group, the maximum bone loss reached 13.2 mm, with an average of 4.930 ± 3.258 mm, while in the study group, the maximum horizontal bone loss was 11.3 mm, with an average of 5.191 ± 2.109 mm. The CBCT 3D investigation, when compared to the OPT, showed increased values for horizontal bone loss, both in the control group and in the study group; in the control group, the maximum bone loss reached 14.10 mm, with an average of 5.736 ± 3.471 mm, and in the study group, the maximum value was 12.40 mm, and the average was again slightly higher (6.152 ± 2.519 mm). The mean value for Dkk-1 in cancer patients was 1.209 ± 0.110 ng/mL, significantly lower than the value observed in healthy patients (1.712 ± 0.100 ng/mL). CBCT revealed higher values for the investigated parameters when compared to panoramic X-rays. Taking into account the preliminary nature of our study, we observed a significant correlation between the level of bone loss recorded by the Dkk-1 biomarker and radiological dental examination in patients with ENT cancer when compared to the control group.

The second decade of anti-TNF-a therapy in clinical practice: new lessons and future directions in the COVID-19 era.

Since the late 1990s, tumor necrosis factor alpha (TNF-α) inhibitors (anti-TNFs) have revolutionized the therapy of immune-mediated inflammatory diseases (IMIDs) affecting the gut, joints, skin and eyes. Although the therapeutic armamentarium in IMIDs is being constantly expanded, anti-TNFs remain the cornerstone of their treatment. During the second decade of their application in clinical practice, a large body of additional knowledge has accumulated regarding various aspects of anti-TNF-α therapy, whereas new indications have been added. Recent experimental studies have shown that anti-TNFs exert their beneficial effects not only by restoring aberrant TNF-mediated immune mechanisms, but also by de-activating pathogenic fibroblast-like mesenchymal cells. Real-world data on millions of patients further confirmed the remarkable efficacy of anti-TNFs. It is now clear that anti-TNFs alter the physical course of inflammatory arthritis and inflammatory bowel disease, leading to inhibition of local and systemic bone loss and to a decline in the number of surgeries for disease-related complications, while anti-TNFs improve morbidity and mortality, acting beneficially also on cardiovascular comorbidities. On the other hand, no new safety signals emerged, whereas anti-TNF-α safety in pregnancy and amid the COVID-19 pandemic was confirmed. The use of biosimilars was associated with cost reductions making anti-TNFs more widely available. Moreover, the current implementation of the “treat-to-target” approach and treatment de-escalation strategies of IMIDs were based on anti-TNFs. An intensive search to discover biomarkers to optimize response to anti-TNF-α treatment is currently ongoing. Finally, selective targeting of TNF-α receptors, new forms of anti-TNFs and combinations with other agents, are being tested in clinical trials and will probably expand the spectrum of TNF-α inhibition as a therapeutic strategy for IMIDs.

Personalized Therapeutic Strategies in the Management of Osteoporosis in Patients with Autoantibody-Positive Rheumatoid Arthritis.

Bone mineral density (BMD) reduction and fragility fractures still represent a major source of morbidity in rheumatoid arthritis (RA) patients, despite adequate control of the disease. An increasing number of clinical and experimental evidence supports the role of autoantibodies, especially anti-citrullinated protein antibodies (ACPAs), in causing localized and generalised bone loss in ways that are both dependent on and independent of inflammation and disease activity. The human receptor activator of nuclear factor kappa B and its ligand—the so-called RANK-RANKL pathway—is known to play a key role in promoting osteoclasts’ activation and bone depletion, and RANKL levels were shown to be higher in ACPA-positive early untreated RA patients. Thus, ACPA-positivity can be considered a specific risk factor for systemic and periarticular bone loss. Through the inhibition of the RANK-RANKL system, denosumab is the only antiresorptive drug currently available that exhibits both a systemic anti-osteoporotic activity and a disease-modifying effect when combined with conventional synthetic or biologic disease-modifying anti-rheumatic drugs (DMARDs). Thus, the combination of DMARD and anti-RANKL therapy could be beneficial in the prevention of fragility fractures and structural damage in the subset of RA patients at risk of radiographic progression, as in the presence of ACPAs.

MACHINE LEARNING AND RADIOMICS IDENTIFY NOVEL BIOMARKERS OF BONE LOSS

Purpose: Altered bone turnover is a factor in many diseases including osteoarthritis (OA), osteoporosis, inflammation, and viral infection. The absence of obvious symptoms and insufficiently sensitive biomarkers in the early stages of bone loss limits early diagnosis and treatment. Therefore, it is urgent to identify novel, more sensitive, and easy-to-detect biomarkers which can be used in the diagnosis and prognosis of bone health. Our previous data using standard micro-computed tomography (μCT) measurements showed that SARS-CoV-2 infection in mice significantly decreased trabecular bone volume at the lumbar spine, suggesting that decreased bone mass, increased fracture risk, and OA may be underappreciated long-haul comorbidities for COVID patients. In this study, we applied integrated state-of-the-art radiomics and machine learning models to identify more sensitive image-based biomarkers of SARS-CoV-2-induced bone loss from μCT images. These radiomic biomarkers can potentially provide a non-invasive way of quantifying and monitoring systemic bone loss and evaluating treatment efficacy in both research and clinical practices. Methods: All animal use was performed with approval of the Institutional Animal Care and Use Committee. To quantify SARS-CoV-2-induced bone loss, 6-week-old transgenic mice (16 male, 16 female) expressing humanized ACE2 receptors were inoculated with a 2020 strain of SARS-CoV-2 or phosphate-buffered saline (Control) [Fig. A]. Viral infection was confirmed by detection of infectious SARS-CoV-2 in throat swabs and histological identification of SARS-CoV-2 labeled cells. At 6-14 days post-infection, lumbar vertebral bodies (L5) were scanned with μCT (μCT 35, SCANCO Medical AG; 6 μm nominal voxel size). The open-source research platform 3D Slicer v2020 with a built-in Python console v3.8 was used for medical image computing and fully automated segmentation of cortical and trabecular bone. Standard μCT assessment of bone microstructure was performed. Radiomic feature extraction and data processing were performed using python based PyRadiomics v3.0.1. A total of 120 radiographic features were extracted from the segmented images [Fig. B]. Principle component analysis ( PCA ) for feature selection, a support vector machine learning (SVML) predictive model for classification, holdback method for model validation, and all statistical analyses (significance at p<0.05) were performed using JMP Pro v15 ( SAS ). Results: Using standard μCT methods, SARS-CoV-2 infection significantly reduced the bone volume fraction (BV/TV) by 10 and 10.5% (p= 0.04) and trabecular thickness (Tb.Th) by 8 and 9% (p= 0.02) in male and female mice, respectively, compared to PBS control mice [Fig. C]. Radiomics detected a 20-fold greater magnitude in change over standard methods. SARS-CoV-2 infection significantly changed radiographic parameters with the largest change being a 300% increase in the second-order parameter: cluster shade [Fig. D]. The 45 radiomic features comprising the first 3 principal components were selected for inclusion in the SVML model. The SVML Model (radial basis function kernel; cost = 4.8; gamma = 0.46) produced an area under the receiver operating characteristic curve (AUC) of 1.0 which reflects a perfectly accurate test [Fig. E]. Conclusions: SARS-CoV-2 infection of humanized ACE2 expressing mice caused significant bone changes, suggesting that decreased bone mass, increased fracture risk, OA, and other musculoskeletal complications could be long-term comorbidities for people infected with COVID-19. We developed an open-source, fully automated segmentation and radiomics system to assess systemic bone loss using μCT images. When coupled with machine learning, this system was able to identify novel radiographic biomarkers of bone loss that better discriminate differences in bone microstructure between SARS-CoV-2 infected and control mice than standard bone morphometric indices. The high accuracy of the SVML model in classifying SARS-CoV-2 infected mice opens the possibility of translating these biomarkers to the clinical setting for early detection of skeletal changes associated with long-haul COVID. The methods presented here were demonstrated using SARS-CoV-2 as a model system and can also be adapted to other diseases associated with altered bone turnover. Development of machine-learning methods for radiomic applications is a crucial step toward clinically relevant radiomic biomarkers of bone health and provides a non-invasive way of quantifying and monitoring systemic bone loss and evaluating treatment efficacy.

Osteoporosis and periodontal diseases - An update on their association and mechanistic links.

Periodontitis and osteoporosis are prevalent inflammation-associated skeletal disorders that pose significant public health challenges to our aging population. Both periodontitis and osteoporosis are bone disorders closely associated with inflammation and aging. There has been consistent intrigue on whether a systemic skeletal disease such as osteoporosis will amplify the alveolar bone loss in periodontitis. A survey of the literature published in the past 25years indicates that systemic low bone mineral density (BMD) is associated with alveolar bone loss, while recent evidence also suggests a correlation between clinical attachment loss and other parameters of periodontitis. Inflammation and its influence on bone remodeling play critical roles in the pathogenesis of both osteoporosis and periodontitis and could serve as the central mechanistic link between these disorders. Enhanced cytokine production and elevated inflammatory response exacerbate osteoclastic bone resorption while inhibiting osteoblastic bone formation, resulting in a net bone loss. With aging, accumulation of oxidative stress and cellular senescence drive the progression of osteoporosis and exacerbation of periodontitis. Vitamin D deficiency and smoking are shared risk factors and may mediate the connection between osteoporosis and periodontitis, through increasing oxidative stress and impairing host response to inflammation. With the connection between systemic and localized bone loss in mind, routine dental exams and intraoral radiographs may serve as a low-cost screening tool for low systemic BMD and increased fracture risk. Conversely, patients with fracture risk beyond the intervention threshold are at greater risk for developing severe periodontitis and undergo tooth loss. Various Food and Drug Administration-approved therapies for osteoporosis have shown promising results for treating periodontitis. Understanding the molecular mechanisms underlying their connection sheds light on potential therapeutic strategies that may facilitate co-management of systemic and localized bone loss.

The inhibitory effect of tocilizumab on systemic bone loss and tendon inflammation in a juvenile Collagen-Induced arthritis rat model

ABSTRACT Purpose of the study Reduced Bone Mineral Density (BMD) is a prevalent comorbidity in Juvenile Idiopathic Arthritis (JIA). Enthesitis and other tendon abnormalities, such as tenosynovitis, tendinitis and tendon ruptures are, also, common extra-articular manifestations of the disease. The aim of the present study was to investigate the effect of tocilizumab, an antibody that binds the Interleukin-6 (IL-6) Receptor, on inflammation-related bone loss and tendon inflammation in an animal model of JIA. Materials and Methods The Collagen-Induced Arthritis (CIA) model was induced in male rats followed by intraperitoneal administration of tocilizumab for 8 weeks. Methotrexate, the most widely used Disease-Modifying Antirheumatic Drug in the management of JIA, was, also, administered, either as a monotherapy or as an add-on therapy to tocilizumab. BMD was evaluated with Micro-Computed Tomography (Micro-CT) and histopathological examination. Tendon damage was, also, assessed histologically. Finally, two pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNF-a) and Interleukin-23 (IL-23) were quantified in tendon tissues by ELISA analysis. Results Tocilizumab-treated animals exhibited a significantly improved trabecular microarchitecture on micro-CT analysis and histological examination. Tendon morphology was also improved. Anti-IL-6 treatment led to a significant decrease in TNF-a and IL-23 expression in tendon tissue. Conclusions The results of the present study provide evidence that tocilizumab reduces inflammation-related bone loss and suppresses tendon inflammation in a juvenile CIA rat model. These findings offer perspectives for the management of osteoporosis and enthesitis in JIA.

A CD40 variant is associated with systemic bone loss among patients with rheumatoid arthritis.

Little is known about genes predisposing to systemic bone loss (SBL) in rheumatoid arthritis (RA). Therefore, we examined the association between SBL and variants of genes playing a critical role in both immune response and bone homeostasis among patients with RA. IRAK-1 rs3027898, IRAK-2 rs3844283, IRAK-2 rs708035, IFIH1 rs1990760, CD40 rs48104850, TNFAIP3 rs2230926, and miR146-a rs2910164 were genotyped in 176 adult RA patients. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DXA). Low BMD was observed in 116 (65.9%) patients. Among them, 60 (34.1%) had low femoral neck (FN) Z score, 72 (40.9%) had low total femur (TF) Z score, and 105 (59.6%) had low lumbar spine (LS) Z score. Among all the SNPs assessed, only CD40 rs4810485 was found to be associated with reduced TF Z score with the CD40 rs4810485 T allele protecting against reduced TF Z score (OR = 0.40, 95% CI = 0.23-0.68, p = 0.0005). This association was confirmed in the multivariate logistic regression analysis (OR = 0.31, 95% CI = 0.16-0.59, p = 3.84 × 10-4). Moreover, median FN BMD was reduced among RA patients with CD40 rs4810485 GG genotype compared to RA patients harbouring CD40 rs4810485 TT and GT genotypes (0.788 ± 0.136 versus 0.826 ± 0.146g/cm2, p = 0.001). IRAK-1 rs3027898, IRAK-2 rs3844283, rs708035, IFIH rs1990760, TNFAIP3 rs2230926, and miR146-a rs2910164 were not found to be associated with SBL. This study for the first time ever demonstrated an association between aCD40 genetic variant and SBL among patients with RA. • CD40 rs4810485 GG genotype is associated with decreased BMD among patients with RA. • CD40 rs4810485 might serve as a genetic marker for SBL in RA. • CD40 genetic variations might be integrated in future development of more effective therapeutic interventions for prevention of SBL in RA.

The calcitonin receptor protects against bone loss and excessive inflammation in collagen antibody-induced arthritis

SummaryPharmacological application of teleost calcitonin (CT) has been shown to exert chondroprotective and anti-resorptive effects in patients with rheumatoid arthritis (RA). However, the role of endogenous CT that signals through the calcitonin receptor (CTR) remains elusive. Collagen II antibody-induced arthritis (CAIA) was stimulated in wild type (WT) and CTR-deficient (Calcr−/−) mice. Animals were monitored over 10 or 48 days. Joint inflammation, cartilage degradation, and bone erosions were assessed by clinical arthritis score, histology, histomorphometry, gene expression analysis, and μ-computed tomography. CAIA was accompanied by elevated systemic CT levels and CTR expression in the articular cartilage. Inflammation, cartilage degradation, and systemic bone loss were more pronounced in Calcr−/− CAIA mice. Expression of various pro-inflammatory, bone resorption, and catabolic cartilage markers were exclusively increased in Calcr−/− CAIA mice. Endogenous CT signaling through the mammalian CTR has the potential to protect against joint inflammation, cartilage degradation, and excessive bone remodeling in experimental RA.

Different Effects of Biologics on Systemic Bone Loss Protection in Rheumatoid Arthritis: An Interim Analysis of a Three-Year Longitudinal Cohort Study.

ObjectiveTo compare changes in bone mineral density (BMD) in rheumatoid arthritis (RA) patients receiving three-year conventional synthetic disease-modifying anti-rheumatic drugs (csDMARD), tumor necrosis factor-α inhibitors (TNFi), and abatacept.MethodsPatients with RA were recruited from September 2014 to February 2021. Dual-energy X-ray absorptiometry was used to measure BMD at the femoral neck (FN), total hip (TH), and lumbar spine (L1-4) at enrollment and three years later. Changes in the BMD of each regimen group were analyzed. Multiple ordinary least squares regression was used with the dependent variables to develop a model to predict the change in BMD.ResultsA total of 752 participants were enrolled and 485 completed the three-year follow-up period. Of these, 375 (Group I), 84 (Group II), and 26 (Group III) participants received csDMARDs, TNFi, and abatacept therapy, respectively. Considering both type of therapy and completion of the follow-up period, participants were divided into groups A (csDMARDs, n = 104), B (TNFi, n = 52), and C (abatacept, n = 26). Compared to baseline, BMD decreased significantly at FN (p = 0.003) and L1-4 (p = 0.002) in Group A and at L1-4 (p = 0.005) in Group B, but remained stable at all sites in Group C. In terms of regression-adjusted percent change in BMD, there was a significant difference seen at all measured sites between group C compared to both groups A and B (+0.8%, -2.7%, -1.8% at FN; +0.5%, -1.1%, -1.0% at TH; +0.8%, -2.0%, -3.5% at L1-4, respectively; all p < 0.05). Anti-osteoporosis therapy had a BMD-preserving effect in RA.ConclusionCompared with csDMARDs and TNFi, abatacept may have a better BMD-preserving effect in RA. Anti-osteoporosis therapy can prevent systemic bone loss irrespective of RA therapy.

Yi Shen Juan Bi Pill Regulates the Bone Immune Microenvironment via the JAK2/STAT3 Signaling Pathway in Vitro.

Rheumatoid arthritis (RA) is characterized by an impaired articular bone immune microenvironment, which is associated with regulatory T cells (Tregs) hypofunction and osteoclasts (OCs) hyperfunction and leads to articular bone erosion and systemic bone loss. Studies have shown that Tregs slow bone loss in RA by regulating the bone resorption function of OCs and the JAK/STAT signaling pathway can regulate the immunosuppressive function of Tregs and reduce the bone erosion function of OCs. Yi Shen Juan Bi Pill (YSJB) is a classic Chinese herbal compound for the treatment of RA. However, whether YSJB regulates bone immune microenvironment homeostasis through JAK/STAT signaling pathway remains unclear. Based on in vitro OC single culture, Treg single culture and OC-Treg coculture systems, treatments were performed using drug-containing serum, AG490 and JAK2 siRNA to explore whether YSJB-containing serum regulates the homeostasis of the bone immune microenvironment through the JAK/STAT signaling pathway. In vitro, YSJB treatment decreased the number of TRAP+ cells and the areas of bone resorption and inhibited the expression of RANK, NFATc1, c-fos, JAK2, and STAT3 in both the OC single culture system and the OC-Treg coculture system. Tregs further reduced the number of TRAP+ cells and the areas of bone resorption in the coculture system. YSJB promoted the secretion of IL-10 while inhibiting the expression of JAK2 and STAT3 in Tregs. Moreover, inhibiting the expression of JAK2 with the JAK2 inhibitor AG490 and JAK2 siRNA improved the immunosuppressive functions of Treg, inhibited OC differentiation and bone resorption. Our study demonstrates that YSJB can regulate OC-mediated bone resorption and Treg-mediated bone immunity through the JAK2/STAT3 signaling pathway. This study provides a new strategy for regulating the bone immune microenvironment in RA with traditional Chinese medicine.

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1β-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

Multi-Targeting DKK1 and LRP6 Prevents Myeloma-Induced Bone Disease

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodeling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting their quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. Therapeutic strategies aimed at promoting bone formation are therefore required to overcome the loss of skeletal integrity and subsequent fractures in MM patients.Therapeutic agents that target the canonical Wnt signaling pathway, a potent regulator of bone formation, have the potential to address these skeletal complications, where they could rebuild lost bone and improve bone strength in affected individuals. We have demonstrated a novel anti-LRP6 agent, which potentiates Wnt signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. However, since MM patients present with both increased bone resorption and decreased bone formation, we hypothesised that combining anti-LRP6 with the bone anabolic anti-DKK1 (100mg/kg twice weekly intravenously) would lead to more robust improvements in bone structure than single treatment approaches.MicroCT analysis demonstrated a 74% increase in femoral trabecular bone volume per tissue volume (BV/TV) in naïve, non-tumour bearing mice given the combination treatment compared to control agents (p<0.0001). Mice injected with 5TGM1eGFP murine myeloma cells had a 34% reduction in femoral BV/TV compared to naïve controls (p<0.0001). Combination treatment drastically improved BV/TV in 5TGM1-bearing mice by 111% (p<0.0001), compared to control, and this improvement with the combination treatment strategy was 25% greater than anti-LRP6 single treatment approaches (p<0.001). MicroCT analysis in L4 lumbar vertebrae demonstrated similar bone structural changes in 5TGM1-bearing mice treated with the combination strategy. Consequently, this combination significantly improved resistance to fracture in L4 vertebrae in 5TGM1eGFP-bearing mice compared to their controls (p<0.001), and it provided greater protection against fracture compared to anti-LRP6 single agent treatment. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1eGFP-bearing mice treated with the combination strategy (p<0.001) compared to control. Importantly, tumour activity was not altered with either single or combination Wnt-promoting treatment strategies.This study defines a novel therapeutic strategy, which will reduce fractures and improve quality of life in patients with MM when used in combination with tumour-targeted treatments. [Display omitted] DisclosuresCong: Novartis Institutes for Biomedical Research: Current Employment. Daley: Novartis Institutes of Biomedical Research: Current Employment.

Urolithin A suppresses RANKL-induced osteoclastogenesis and postmenopausal osteoporosis by, suppresses inflammation and downstream NF-κB activated pyroptosis pathways

Osteoporosis (OP) is characterized by decreased trabecular bone volume and microarchitectural deterioration in the medullary cavity. Urolithin A (UA) is a biologically active metabolite generated by the gut microbiota. UA is the measurable product considered the most relevant urolithin as the final metabolic product of polyphenolic compounds. Considering that catabolic effects mediated by the intestinal microbiota are highly involved in pathological bone disorders, exploring the biological influence and molecular mechanisms by which UA alleviates OP is crucial. Our study aimed to investigate the effect of UA administration on OP progression in the context of estrogen deficiency-induced bone loss. The in vivo results indicated that UA effectively reduced ovariectomy-induced systemic bone loss. In vitro, UA suppressed Receptor Activator for Nuclear Factor-κB Ligand (RANKL)-triggered osteoclastogenesis in a concentration-dependent manner. Signal transduction studies and sequencing analysis showed that UA significantly decreased the expression of inflammatory cytokines (e.g., IL-6 and TNF-α) in osteoclasts. Additionally, attenuation of inflammatory signaling cascades inhibited the NF-κB-activated NOD-like receptor signaling pathway, which eventually led to decreased cytoplasmic secretion of IL-1β and IL-18 and reduced expression of pyroptosis markers (NLRP3, GSDMD, and caspase-1). Consistent with this finding, an NLRP3 inflammasome inhibitor (MCC950) was employed to treat OP, and modulation of pyroptosis was found to ameliorate osteoclastogenesis and bone loss in ovariectomized (OVX) mice, suggesting that UA suppressed osteoclast formation by regulating the inflammatory signal-dependent pyroptosis pathway. Conceivably, UA administration may be a safe and promising therapeutic strategy for osteoclast-related bone diseases such as OP.

Extracellular vesicles derived from the periodontal pathogen Filifactor alocis induce systemic bone loss through Toll-like receptor 2.

Periodontitis is an inflammatory disease induced by local infection in tooth‐supporting tissue. Periodontitis is associated with systemic bone diseases, but little is known about the mechanism of the causal effect of periodontitis on systemic bone resorption. Bacteria‐derived extracellular vesicles (EVs) act as natural carriers of virulence factors that are responsible for systemic inflammation. In this study, we investigated the role of EVs derived from Filifactor alocis, a Gram‐positive, anaerobic periodontal pathogen, in systemic bone loss and osteoclast differentiation. F. alocis EVs accumulated in the long bones of mice after intraperitoneal administration. These EVs induced proinflammatory cytokines, osteoclastogenesis, and bone resorption via Toll‐like receptor 2 (TLR2). The phase separation of F. alocis EVs showed that amphiphilic molecules were responsible for the induced bone resorption and osteoclastogenesis. The osteoclastogenic effects of F. alocis EVs were reduced by lipoprotein lipase. Proteomic analysis of the amphiphilic molecules identified seven lipoproteins. Our results indicate that lipoprotein‐like molecules in F. alocis EVs may contribute to systemic bone loss via TLR2.

Effects of Autoantibodies on Osteoclasts

AbstractThe balance between bone forming osteoblasts and bone resorbing osteoclasts can be disturbed in autoimmune diseases resulting in local and systemic bone loss. It was long time believed that autoantibodies only indirectly contribute to bone loss by fueling the overall inflammation. However, in the last decade, more and more evidence emerged that autoantibodies and immune complexes directly activate osteoclasts and pre-osteoclasts by binding to Fcγ receptors (FcγRs) on the (pre)-osteoclast cell surface. This pro-osteoclastogenic effect seems to be dependent on the absence of sialic acid in the IgG Fc glycan, which is a typical feature of many autoantibodies. Clinical studies revealed the importance of autoantibody-mediated bone loss mainly in rheumatoid arthritis, but also in other diseases, such as celiac disease. In summary, the gained knowledge about autoantibody-mediated bone loss helps to better understand bone pathologies of autoimmune diseases. However, studies are still relatively limited and more research is needed to fully understand the impact of autoantibodies on bone and to develop future therapeutic strategies.

Effective Osteogenic Priming of Mesenchymal Stem Cells through LNA-ASOs-Mediated Sfrp1 Gene Silencing.

Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis.

IgA rheumatoid factor is associated with bone mineral density preservation in rheumatoid arthritis

Autoantibodies such as IgM rheumatoid factor (RF) and anti-citrullinated proteins/peptides antibodies (ACPA) have previously been incriminated in systemic bone loss in rheumatoid arthritis (RA). There are, however, no data describing association of IgA RF and IgG RF with systemic bone loss. This study was aimed to investigate the association of RF isotypes with systemic bone loss among patients with RA. RF isotypes and ACPA were measured by enzyme-linked immunosorbent assay among 153 patients with RA. Bone mineral density (BMD) was assessed using dual-energy X-ray absorptiometry. Ninety-four (61.4%) patients had positive IgA RF, 89 (58.2%) had positive IgG RF, 109 (71.2%) had positive IgM RF, whereas 122 (80.3%) RA patients tested positive for ACPA. Compared to the IgA RF-negative patients, IgA RF-positive patients exhibited higher disease activity and had higher RF titers. Seven (4.6%) patients had low BMD at femoral neck, 12 (7.8%) at total femur, and 47 (30.7%) at lumbar spine. IgA RF was found to be associated with protection against low BMD at spine (OR = 0.47, 95% CI = 0.23-0.95, p = 0.034). This association was further confirmed in the multivariate regression analysis taking into account several potential confounding factors(OR = 0.21, 95% CI = 0.06-0.65, p = 0.039). No association between low BMD and the presence of IgG RF or IgM RF or ACPA was found. IgA RF for the first time ever was shown to be associated with BMD preservation at spine in RA. Key points • IgA RF was associated with protection against low spinal BMD. • No association between low BMD and the presence of IgG RF or IgM RF was found.

Persistent Abnormal Immunocytes Induced Systemic Bone Loss in Locally Irradiated Rats.

Chronic and systemic bone complications frequently occur in patients who undergo radiotherapy; however, the pathological mechanisms underlying these complications remain unclear. This study aimed to observe persistent and systemic changes in locally irradiated rats and to determine the systemic pathological changes that persistently affect bone metabolism. We examined the inflammatory and oxidative stress responses that occurred after local irradiation using enzyme immunoassays and biochemical analyses. Lymphocytes obtained from the blood, spleen, thymus, and bone marrow were evaluated using flow cytometry. The proliferation and apoptosis characteristics of co-cultured bone marrow-derived mesenchymal stem cells (BMSCs) were detected by MTT assay and PI/Annexin V-FITC staining, respectively, and the differentiation of BMSCs was measured according to alkaline phosphatase (ALP) staining, alizarin red staining, and Oil Red O staining and by evaluating the mRNA expression of ALP, osteocalcin (OCN), osteopontin (OPN), collagen I, Runx2, and PPARγ. Our results revealed that no significant or continuous differences were present in the inflammatory response or the oxidative stress response throughout the body after local irradiation. B lymphocyte levels increased continuously in the blood, spleen, and bone marrow after local irradiation. T lymphocyte levels were decreased at 2weeks after local irradiation, and CD8+T lymphocyte levels were increased in the blood, thymus, and bone marrow at 12weeks after local irradiation. The ratio of CD4+/CD8+T lymphocytes began to decrease during the early phase after local irradiation and became significantly decreased at 12weeks after local irradiation. Normal BMSCs co-cultured with lymphocytes derived from irradiated rats exhibited decreased proliferation and increased apoptosis, and the ALP staining intensity, alizarin red staining intensity, and mRNA expression of related genes were all also decreased. Oil Red O staining intensity and mRNA expression of PPARγ were both increased. Lymphocyte levels contribute to chronic and systemic bone complications after radiotherapy by inhibiting the proliferation and osteoblastogenesis of BMSCs.

Sex differences in systemic bone and muscle loss following femur fracture in mice.

Fracture induces systemic bone loss in mice and humans, and a first (index) fracture increases the risk of future fracture at any skeletal site more in men than women. The etiology of this sex difference is unknown, but fracture may induces a greater systemic bone loss response in men. Also sex differences in systemic muscle loss after fracture have not been examined. We investigated sex differences in systemic bone and muscle loss after transverse femur fracture in 3-month-old male and female C57BL/6 J mice. Whole-body and regional bone mineral content and density (BMC and BMD), trabecular and cortical bone microstructure, muscle contractile force, muscle mass, and muscle fiber size were quantified at multiple time points postfracture. Serum concentrations of inflammatory cytokines (IL-1β, IL-6, and TNF-α) were measured 1-day postfracture. One day postfracture, IL-6 and Il-1B were elevated in fracture mice of both sexes, but TNF-α was only elevated in male fracture mice. Fracture reduced BMC, BMD, and trabecular bone microstructural properties in both sexes 2 weeks postfracture, but declines were greater in males. Muscle contractile force, mass, and fiber size decreased primarily in the fractured limb at 2 weeks postfracture and females showed a trend toward greater muscle loss. Bone and muscle properties recovered by 6 weeks postfracture. Overall, postfracture systemic bone loss is greater in men, which may contribute to sex differences in subsequent fracture risk. In both sexes, muscle loss is primarily confined to the injured limb and fracture may induce greater inflammation in males.