The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0753-8) contains supplementary material, which is available to authorized users.     

Erosive arthritis

Inflammation and degradation of bone are two closely linked processes. Chronic inflammatory arthritis not only leads to inflammatory bone loss but it also involves local erosion of articular bone. This osteo-destructive feature of chronic inflammatory arthritis is a major cause of disability in patients with rheumatoid arthritis. Osteoclasts are essential for the resorption of mineralized cartilage and subchondral bone in chronic arthritis. The observed up-regulation of osteoclast differentiation factors (receptor activator of nuclear factor-kappaB ligand [RANKL]) in the synovial membrane of chronically inflamed joints indicates that osteoclasts are abundant in this setting, leading to rapid degradation of mineralized tissue. Blockade of osteoclast formation is thus a key strategy in preventing structural damage in arthritis. Denosumab, a humanized antibody that neutralizes RANKL, is an attractive candidate agent to inhibit inflammatory bone loss.     

Inflammatory cells and bone loss in rheumatoid arthritis

Pathogenic bone erosion is often associated with inflammation. The destructive bone erosion that is often seen in rheumatoid arthritis is probably due to the close proximity of inflamed tissues to bone. Over the past decade, major advances have been made in our understanding of the factors that are crucial in regulating osteoclast bone resorption. It is not surprising that these factors are expressed by inflammatory cells that are present in the rheumatoid joint. It now appears that we can add neutrophils to the list of inflammatory cells found in the inflamed rheumatoid joint that express factors that regulate bone erosion.     

Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications

Rheumatoid arthritis represents an excellent model in which to gain insights into the local and systemic effects of joint inflammation on skeletal tissues. Three forms of bone disease have been described in rheumatoid arthritis. These include: focal bone loss affecting the immediate subchondral bone and bone at the joint margins; periarticular osteopenia adjacent to inflamed joints; and generalized osteoporosis involving the axial and appendicular skeleton. Although these three forms of bone loss have several features in common, careful histomorphometric and histopathological analysis of bone tissues from different skeletal sites, as well as the use of urinary and serum biochemical markers of bone remodeling, provide compelling evidence that different mechanisms are involved in their pathogenesis. An understanding of these distinct pathological forms of bone loss has relevance not only with respect to gaining insights into the different pathological mechanisms, but also for developing specific and effective strategies for preventing the different forms of bone loss in rheumatoid arthritis.     

Immune regulation of bone loss by Th17 cells

A significant macrophage and T-cell infiltrate commonly occurs in inflammatory joint conditions such as rheumatoid arthritis that have significant bone destruction. Cytokines produced by activated macrophages and T cells are implicated in arthritis pathogenesis and are involved in osteoclast-mediated bone resorption. The scope of the present review is to analyze current knowledge and to provide a better understanding of how macrophage-derived factors promote the differentiation of a novel T-helper subset (Th17) that promotes osteoclast formation and activation.     

Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications

Rheumatoid arthritis represents an excellent model in which to gain insights into the local and systemic effects of joint inflammation on skeletal tissues. Three forms of bone disease have been described in rheumatoid arthritis. These include: focal bone loss affecting the immediate subchondral bone and bone at the joint margins; periarticular osteopenia adjacent to inflamed joints; and generalized osteoporosis involving the axial and appendicular skeleton. Although these three forms of bone loss have several features in common, careful histomorphometric and histopathological analysis of bone tissues from different skeletal sites, as well as the use of urinary and serum biochemical markers of bone remodeling, provide compelling evidence that different mechanisms are involved in their pathogenesis. An understanding of these distinct pathological forms of bone loss has relevance not only with respect to gaining insights into the different pathological mechanisms, but also for developing specific and effective strategies for preventing the different forms of bone loss in rheumatoid arthritis.     

Ubiquitin ligase Smurf1 mediates tumor necrosis factor-induced systemic bone loss by promoting proteasomal degradation of bone morphogenetic signaling proteins

Chronic inflammatory disorders, such as rheumatoid arthritis, are often accompanied by systemic bone loss, which is thought to occur through inflammatory cytokine-mediated stimulation of osteoclast resorption and inhibition of osteoblast function. However, the mechanisms involved in osteoblast inhibition remain poorly understood. Here we test the hypothesis that increased Smad ubiquitin regulatory factor 1 (Smurf1)-mediated degradation of the bone morphogenetic protein pathway signaling proteins mediates reduced bone formation in inflammatory disorders. Osteoblasts derived from bone marrow or long bone samples of adult tumor necrosis factor (TNF) transgenic (TNF-Tg) mice were used in this study. TNF decreased the steady-state levels of Smad1 and Runx2 protein similarly to those in long bones of TNF-Tg mice. In the presence of the proteasome inhibitor MG132, TNF increased accumulation of ubiquitinated Smad1 protein. TNF administration over calvarial bones caused decreases in Smad1 and Runx2 protein levels and mRNA expression of osteoblast marker genes in wild-type, but not in Smurf1(-/-) mice. Vertebral bone volume and strength of TNF-Tg/Smurf1(-/-) mice were examined by a combination of micro-CT, bone histomorphometry, and biomechanical testing and compared with those from TNF-Tg littermates. TNF-Tg mice had significantly decreased bone volume and biomechanical properties, which were partially rescued in TNF-Tg/Smurf1(-/-) mice. We conclude that in chronic inflammatory disorders where TNF is increased, TNF induces the expression of ubiquitin ligase Smurf1 and promotes ubiquitination and proteasomal degradation of Smad1 and Runx2, leading to systemic bone loss. Inhibition of ubiquitin-mediated Smad1 and Runx2 degradation in osteoblasts could help to treat inflammation-induced osteoporosis.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Probiotics ameliorate alveolar bone loss by regulating gut microbiota

ObjectivesOestrogen deficiency is an aetiological factor of postmenopausal osteoporosis (PMO), which not only decreases bone density in vertebrae and long bone but also aggravates inflammatory alveolar bone loss. Recent evidence has suggested the critical role of gut microbiota in osteoimmunology and its influence on bone metabolisms. The present study aimed to evaluate the therapeutic effects of probiotics on alveolar bone loss under oestrogen‐deficient condition.Materials and MethodsInflammatory alveolar bone loss was established in ovariectomized (OVX) rats, and rats were daily intragastrically administered with probiotics until sacrifice. Gut microbiota composition, intestinal permeability, systemic immune status and alveolar bone loss were assessed to reveal the underlying correlation between gut microbiota and bone metabolisms.ResultsWe found administration of probiotics significantly prevented inflammatory alveolar bone resorption in OVX rats. By enriching butyrate‐producing genera and enhancing gut butyrate production, probiotics improved intestinal barrier and decreased gut permeability in the OVX rats. Furthermore, the oestrogen deprivation‐induced inflammatory responses were suppressed in probiotics‐treated OVX rats, as reflected by reduced serum levels of inflammatory cytokines and a balanced distribution of CD4⁺IL‐17A⁺ Th17 cells and CD4⁺CD25⁺Foxp3⁺ Treg cells in the bone marrow.ConclusionsThis study demonstrated that probiotics can effectively attenuate alveolar bone loss by modulating gut microbiota and further regulating osteoimmune response and thus represent a promising adjuvant in the treatment of alveolar bone loss under oestrogen deficiency.     

Anti-RANKL therapy for inflammatory bone disorders: Mechanisms and potential clinical applications

Focal bone loss around inflamed joints in patients with autoimmune disease, such as rheumatoid arthritis, remains a serious clinical problem. The recent elucidation of the RANK/RANK-ligand/OPG pathway and its role as the final effector of osteoclastogenesis and bone resorption has brought a tremendous understanding of the pathophysiology of inflammatory bone loss, and has heightened expectation of a novel intervention. Here, we review the etiology of inflammatory bone loss, the RANK/RANK-ligand/OPG pathway, and the clinical development of anti-RANK-ligand therapy.     

A combination of methotrexate and zoledronic acid prevents bone erosions and systemic bone mass loss in collagen induced arthritis

Introduction  

Osteoclasts play a key role in the pathogenesis of bone erosion and systemic bone mass loss during rheumatoid arthritis (RA). In this study, we aimed to determine the effect of methotrexate (MTX) and zoledronic acid (ZA), used alone or in combination, on osteoclast-mediated bone erosions and systemic bone mass loss in a rat model of collagen induced arthritis (CIA). We hypothesized that MTX and ZA could have an additive effect to prevent both bone erosion and systemic bone loss.     

Estradiol ameliorates arthritis and protects against systemic bone loss in Staphylococcus aureus infection in mice

Introduction

Staphylococcus aureus is a common cause of bacterial arthritis, which is associated with progressive bone loss in affected joints. We recently showed that S. aureus infection also induces a significant systemic bone loss in mice. This study was performed to assess the effect of estradiol treatment on the clinical course and outcome of S. aureus arthritis and on infection-induced bone loss in experimental S. aureus infection.

Methods

Mice were ovariectomized, treated with estradiol or placebo, and S. aureus infection was established by intravenous inoculation of bacteria.

Results

Estradiol treatment was found to decrease significantly the frequency and clinical severity of S. aureus arthritis, a finding that was accompanied with significantly higher serum levels of interleukin-10 in estradiol-treated mice. Estradiol was also highly protective against S. aureus-induced systemic trabecular, and cortical bone loss. Lack of endogenous estrogens and S. aureus infection had additive effects on trabecular bone loss. The S. aureus-infected, ovariectomized mice lost as much as 76% of their trabecular bone mass.

Conclusions

Treatment with estradiol ameliorates S. aureus arthritis and is protective against infection-induced systemic bone loss in experimental S. aureus infection.     

Stachydrine prevents LPS‐induced bone loss by inhibiting osteoclastogenesis via NF‐κB and Akt signalling

Osteoclast overactivation‐induced imbalance in bone remodelling leads to pathological bone destruction, which is a characteristic of many osteolytic diseases such as rheumatoid arthritis, osteoporosis, periprosthetic osteolysis and periodontitis. Natural compounds that suppress osteoclast formation and function have therapeutic potential for treating these diseases. Stachydrine (STA) is a bioactive alkaloid isolated from Leonurus heterophyllus Sweet and possesses antioxidant, anti‐inflammatory, anticancer and cardioprotective properties. However, its effects on osteoclast formation and function have been rarely described. In the present study, we found that STA suppressed receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced osteoclast formation and bone resorption, and reduced osteoclast‐related gene expression in vitro. Mechanistically, STA inhibited RANKL‐induced activation of NF‐κB and Akt signalling, thus suppressing nuclear factor of activated T cells c1 induction and nuclear translocation. In addition, STA alleviated bone loss and reduced osteoclast number in a murine model of LPS‐induced inflammatory bone loss. STA also inhibited the activities of NF‐κB and NFATc1 in vivo. Together, these results suggest that STA effectively inhibits osteoclastogenesis both in vitro and in vivo and therefore is a potential option for treating osteoclast‐related diseases.     

Trabecular bone loss in collagen antibody-induced arthritis

IntroductionPostmenopausal women with rheumatoid arthritis (RA) have increased risk of developing osteoporosis due to chronic inflammation and estrogen deprivation. Collagen antibody-induced arthritis (CAIA), an experimental polyarthritis model representing the effector phase of arthritis, is mainly mediated by the innate immune system. Compared to the widely used collagen-induced arthritis model, CAIA is conveniently short and can be used in C57BL/6 mice, enabling studies with knock-out mice. However, the impact on bone of the CAIA model in C57BL/6 mice has not previously been studied. Therefore, the aim of this study was to determine if CAIA can be used to study postmenopausal arthritis-induced osteoporosis.MethodsCAIA was induced by administration of collagen-type II antibodies and lipopolysaccharide to ovariectomized female C57BL/6J mice. Control mice received lipopolysaccharide, but no antibodies. Nine days later, femurs were collected for high-resolution micro-CT and histomorphometry. Serum was used to assess cartilage breakdown and levels of complement. Frequencies of immune cell subsets from bone marrow and lymph nodes were analyzed by flow cytometery.ResultsTrabecular bone mass was decreased and associated with increased number of osteoclasts per bone surface in the CAIA model. Also, the frequency of interleukin-17⁺ cells in lymph nodes was increased in CAIA.ConclusionThe present study show that CAIA, a short reproducible arthritis model that is compatible with C57BL/6 mice, is associated with increased number of osteoclasts and trabecular bone loss.     

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

Hand bone loss as an outcome measure in established rheumatoid arthritis: 2-year observational study comparing cortical and total bone loss

The aim of this 2-year longitudinal observational study was to explore hand bone loss as a disease outcome measure in established rheumatoid arthritis (RA).