Bone Erosion In Collagen-induced Arthritis Research Articles

F4/80+CD206+ M2-like macrophages contribute to bone erosion in collagen-induced arthritis by differentiating into osteoclasts

Objective Rheumatoid arthritis is an autoimmune disease characterized by synovial inflammation-driven cartilage and bone destruction, a process mainly mediated by osteoclasts. In recent years M2-like macrophages have been found to play an important role in the pathological process of RA by mediating pro-inflammatory effects, but their roles in the bone destruction of autoimmune arthritis have not been reported. Methods and Results In this study we identified that an abundant cell population of CD45+CD11b+Gr-1-F4/80+CD206+ cells, which were normally classified as M2-like macrophages, was present in synovium of collagen-induced arthritis (CIA) mice, and these cells had the potential to differentiate into osteoclasts. These M2-like macrophages sorted from CIA synovium highly expressed RANK and could be activated by RANKL and M-CSF to acquire osteoclast markers and bone resorption function both in vitro and in vivo. Furthermore, in vitro differentiated M2 macrophages from both CIA mouse bone marrow and RA patient peripheral blood mononuclear cells were also able to differentiate into osteoclasts, confirming the general osteoclastogenesis capability of M2 subtype macrophages. Conclusion All these results suggest that synovial F4/80+CD206+ M2-like macrophages in RA may be novel osteoclast precursors and contribute significantly to bone erosive changes seen in RA. Our studies provided new directions and targets for the diagnosis and treatment of rheumatoid arthritis.

POS0442 GLPG4399: SELECTIVE SIK3 INHIBITION AS A NOVEL MODE OF ACTION FOR THE TREATMENT OF INFLAMMATORY ARTHRITIC DISEASES (PRECLINICAL)

BackgroundSalt-inducible kinases (SIKs) is a family of kinases with immunomodulatory function identified using a proprietary adenoviral shRNA knockdown target discovery platform. Moreover, SIK inhibition has previously been shown to have a role in inflammatory signalling.1–3 These findings suggest a therapeutic potential for SIK inhibition in inflammatory indications. A medicinal chemistry effort resulted in the development of a first-in-class, oral, selective SIK3 inhibitor: GLPG4399. This compound may be beneficial in inflammatory diseases such as rheumatoid and psoriatic arthritis which are chronic disorders characterized by impaired joint synovial inflammation.ObjectivesOur research aimed to characterize GLPG4399 and explore its impact in arthritis-relevant inflammatory in vitro phenotypic cell assays, and to evaluate the therapeutic potential of selective SIK3 inhibition in in vivo experimental models of arthritis.MethodsThe selectivity and potency of GLPG4399 was profiled using biochemical and target-based cell assays. The mode of action of selective SIK3 inhibition in inflammation was explored in an in vitro panel of innate (monocytes, macrophages, dendritic cells) and adaptive (B and T lymphocytes) immune phenotypic assays and in a lipopolysaccharide (LPS)-stimulated human whole blood assay by measuring the production of inflammatory cytokines. In vivo target engagement was evaluated in an acute LPS-stimulated cytokine release mouse model by measuring plasma tumour necrosis factor (TNF) α levels. The therapeutic efficacy of GLPG4399 was evaluated in vivo in collagen-induced arthritis (CIA) and IL-23-induced psoriatic arthritis mouse models by assessing disease activity endpoints.ResultsGLPG4399 was shown to be a SIK3 inhibitor with high selectivity against a panel of 370 kinases. The wide effect of SIK3 inhibition on key immune cell types was demonstrated by GLPG4399’s reduction of pro-inflammatory cytokines produced by monocytes, macrophages, dendritic cells, B and T lymphocytes in a panel of in vitro innate and adaptive immune phenotypic assays. The biological activity and target engagement of GLPG4399 was further demonstrated by dose-dependent inhibition of TNFα production in vitro in LPS-stimulated human whole blood and in vivo in the blood of LPS-challenged mice. Oral treatment with GLPG4399 in mice resulted in a significant and dose-dependent improvement of disease activity score in both CIA and the psoriatic arthritis disease model. Moreover, bone erosion in CIA and new bone formation in the psoriatic arthritis disease model were significantly reduced.ConclusionOur preclinical findings demonstrate the strong immunomodulatory effect of SIK3 inhibition in arthritis-relevant inflammatory cell assays and highlight the significant preclinical efficacy of GLPG4399 in two experimental arthritis mouse models. The novel mechanisms of action of GLPG4399 represents a promising approach for the treatment of arthritis.

Targeted inhibition of ATP5B gene prevents bone erosion in collagen-induced arthritis by inhibiting osteoclastogenesis

Bone resorption by osteoclasts is an energy consuming activity, which depends on mitochondrial ATP. ATP5B, a mitochondrial ATP synthase beta subunit, is a catalytic core involved in producing ATP. Here, we investigated the contribution of ATP5B in osteoclast differentiation and joint destruction. ATP5B (LV-ATP5B) targeting or non-targeting (LV-NC) siRNA containing lentivirus particles were transduced into bone marrow macrophage derived osteoclasts or locally administered to arthritic mouse joints. Inhibition of ATP5B reduced the expression of osteoclast related genes and proteins, suppressed bone resorption by significantly impairing F-actin formation and decreased the levels of adhesion-associated proteins. In addition, ATP5B deficiency caused osteoclast mitochondrial dysfunction and, impaired the secretion of vacuole protons and MMP9. Importantly, inhibition of ATP5B expression, protected arthritis mice from joint destructions although serum levels of inflammatory mediators (TNF-α, IL-1β) and IgG2α antibodies were unaffected. These results demonstrate an essential function of ATP5B in osteoclast differentiation and bone resorption, and suggest it as a potential therapeutic target for protecting bones in RA.

Adipose-derived mesenchymal stromal cells suppress osteoclastogenesis and bone erosion in collagen-induced arthritis.

Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA), and adipose-derived mesenchymal stromal cells (ADSCs) can inhibit experimental collagen-induced arthritis model. This study aims to determine whether ADSCs also suppresses osteoclastogenesis and bone erosion in collagen-induced arthritis (CIA). Osteoclasts were induced from bone marrow-derived CD11b+ cells with receptor activator of nuclear factor-κ B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) stimulation and assessed with tartrate-resistant acid phosphatase (TRAP) staining. For human cells, osteoclasts were produced from human CD14+ cells. ADSCs were generated and added to cultures with different ratios with CD11b+ cells. Transwell and antibody blockade experiments were performed to define the mechanism of action. NF-κB and RANKL expression were determined by Western blotting and RT-qPCR. About 2×106 ADSCs or fibroblast cells were adoptively transferred to DBA1/J mice on day 14 after immunization with type II collagen/complete Freund's adjuvant (CII/CFA) while the onset and severity of the CIA were monitored. Adipose-derived mesenchymal stromal cells but not fibroblast cells completely suppressed osteoclastogenesis in vitro for human and mice. ADSCs injected after immunization and before of onset of CIA significantly suppressed disease development. Treatment with ADSCs dramatically decreased the levels of NF-κB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo. We have demonstrated that ADSCs can inhibit RANKL-induced osteoclasts genesis via CD39 signals. Our findings also suggest that ADSCs can inhibit osteoclasts genesis without the involvement of regulatory T cells. ADSCs might represent a promising strategy for stem cell-based therapies for RA. Thus, manipulation of ADSCs may have therapeutic effects on RA and other bone erosion-related diseases.

An herbal formula inhibits STAT3 signaling and attenuates bone erosion in collagen-induced arthritis rats

BackgroundReceptor activator of NF-κB ligand (RANKL) facilitates differentiation of osteoclast precursors into osteoclasts, resulting in bone erosion in rheumatoid arthritis (RA) patients. Fibroblast-like synoviocytes (FLS) are the main cells for producing RANKL. Signal transducer and activator of transcription 3 (STAT3) signaling is activated in FLS of RA patients (RA-FLS), which has been linked to RANKL production. A two-herb formula (RL) comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos is traditionally used for treating RA in China. We have found that a standardized ethanolic extract of RL (RLE for short) alleviates bone erosion in collagen-induced arthritis (CIA) rats. PurposeThis study aimed to determine whether RLE inhibits RANKL production and osteoclastogenesis in cell and rat models, and to explore the involvement of the STAT3 pathway in this inhibition. Study Design and MethodsA CIA rat model, interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS and a co-culture system (IL-6/sIL-6R-stimulated RA-FLS/peripheral blood mononuclear cells) were used to evaluate the effects of RLE. Micro-computed tomography analysis was used to observe bone erosion in CIA rats. Tartrate-resistant acid phosphatase staining was used to evaluate osteoclastogenesis. Western blotting and ELISA assays were employed to examine protein levels. RT-qPCR was used to detect mRNA levels. STAT3-over-activated RA-FLS were used to investigate the involvement of STAT3 signaling in the anti-osteoclastogenic effects of RLE. ResultsRLE alleviated bone erosion in joints of CIA rats. In both synovial tissues of CIA rats and IL-6/sIL-6R-stimulated RA-FLS, RLE downregulated the protein level of RANKL. In the co-culture system, RLE significantly and dose-dependently inhibited IL-6/sIL-6R-induced osteoclastogenesis. Mechanistic studies revealed that RLE lowered the protein level of phospho-STAT3 (Tyr705) in synovial tissues of CIA rats. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited the activation/phosphorylation of a STAT3 upstream kinase Janus kinase 2 (Tyr1007/1008) and STAT3 (Tyr705), decreased the nuclear localization of STAT3, lowered mRNA levels of STAT3-transcriptionally regulated genes IL-1β and TNF-α. RLE's inhibitory effects on RANKL production in RA-FLS gradually decreased when IL-6/sIL-6R doses increased. Over-activation of STAT3 diminished the inhibitory effects of RLE on RANKL production in IL-6/sIL-6R-stimulated RA-FLS, and attenuated the anti-osteoclastogenic effects of RLE in the co-culture system. ConclusionWe, for the first time, demonstrated that suppressing STAT3 signaling contributes to the inhibition of RANKL production and osteoclastogenesis, and thereby supports the mechanisms responsible for the reduction in bone erosion in RLE-treated CIA rats. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug, and supports the notion that targeting STAT3 signaling is a viable strategy for managing bone erosion.

Bone marrow mesenchymal stem cells improve bone erosion in collagen-induced arthritis by inhibiting osteoclasia-related factors and differentiating into chondrocytes

BackgroundRheumatoid arthritis (RA) is characterized by joint inflammation and damage to the cartilage and bone in collagen-induced arthritis (CIA). Mesenchymal stem cells (MSCs) can improve articular symptoms and reduce bone erosion in CIA rats; however, the underlying mechanism remains unknown. This study aimed to investigate the mechanism underlying MSC-induced improvement of bone destruction in CIA.MethodsWistar rats were divided into a normal group, CIA control group, MTX intervention group, and BMSC intervention group, each comprising 8 rats. Serum RANKL, OPG, and CXCL10 levels of all groups were determined via flow cytometry after 42 days of interventions. RANKL, OPG, TRAF6, CXCL10, and CXCR3 were detected on the synovial membrane via immunohistochemistry, and their relative mRNA levels were determined via RT-PCR analysis. BMSCs were labeled with GFP and administered to CIA rats via the tail vein. At different time points, the distribution of implanted GFP-MSCs in synovial tissues was observed using a fluorescence microscope, and the potential of GFP-MSCs to differentiate into chondrocytes was assessed via immunofluorescence analysis.ResultsBMSC transplantation improved joint inflammation and inhibited bone destruction in CIA rats. BMSCs inhibited the expression of serum CXCL10 and CXCL10 and CXCR3 expression at the synovial membrane. Moreover, protein and mRNA expression analyses revealed that BMSCs potentially regulated RANKL/OPG expression levels in the serum and synovial tissue. Upon implantation into CIA rats, GFP-MSCs were traced in the joints. GFP-positive cells were observed in the cartilage tissue from day 11 and until 42 days after transplantation. Anti-type II collagen/GFP double-positive cells were observed in the articular cartilage (especially damaged cartilage) upon immunofluorescence staining of anti-type II collagen.ConclusionsBMSCs improve bone destruction in CIA by inhibiting the CXCL10/CXCR3 chemotactic axis, regulating the RANKL/OPG ratio, and directly differentiating into chondrocytes.

CARD6 protects against collagen-induced rheumatoid arthritis in mice through attenuating the inflammatory response and joint destruction via suppression of TNFR1/TRAF2 signaling

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by chronic inflammation and bone destruction. However, the pathogenesis that contributes to RA is still unclear. Caspase recruitment domain protein 6 (CARD6) is a typical member of CARD domain-containing proteins, and shows regulatory effects on nuclear factor-κB (NF-κB) activation to meditate inflammation. In the present study, the role of CARD6 in the progression of inflammatory bone erosion in RA was investigated using the in vitro and in vivo experiments. In vitro results indicated that CARD expression was markedly down-regulated in the activated macrophages induced by lipopolysaccharide (LPS), accompanied with time-dependently increased expression of pro-inflammatory cytokines. Notably, over-expressing CARD6 in macrophages by adenoviral (Ad) vector significantly abolished the expression levels of pro-inflammatory cytokines and chemokines. We found that CARD6 over-expression-suppressed inflammatory response was associated with the blockage of tumor necrosis factor receptor-1/tumor necrosis factor receptor-associated factor-2 (TNFR1/TRAF2) signaling, inhibiting NF-κB pathway subsequently. In addition, LPS-induced apoptosis in macrophages was also blunted due to AdCARD6 infection. CARD6-alleviated inflammatory response and apoptotic cell death were further confirmed in TNF-α-stimulated macrophages. Then, the in vivo studies showed that promoting CARD6 expression using adeno-associated virus (AAV) effectively attenuated the severity of arthritis, improved histopathological damage, and hindered the bone erosion in collagen-induced arthritis (CIA) mice. Moreover, pro-inflammatory factors in the joint samples were also markedly decreased in CIA mice with CARD6 over-expression, which was related to the down-regulation of TNFR1/TRAF2/NF-κB signaling pathway. Meanwhile, apoptosis in joint of CIA mice was also ameliorated by AAV-CARD6, as evidenced by the obviously reduced expression of cleaved Caspase-3. These results clearly suggested that CARD6 might have anti-inflammatory and anti-apoptotic effects during RA progression, and thus could be defined as a novel therapeutic target for RA treatment in future.

Intraarticular injection of dexamethasone promotes bone erosion in collagen-induced arthritis in mice through up-regulation of RANKL expression.

Dexamethasone (DEX) is an effective therapeutic option commonly used in the treatment of many inflammatory diseases. However, DEX could impair proliferation or differentiation of osteoblasts, suggesting a pivotal role of DEX in bone destruction. To investigate whether intraarticular injection of DEX could exacerbate bone erosion during CIA development. Collagen-induced arthritis (CIA) mice were divided into PBS-treated and DEX-treated groups (n = 5/group). Negative control group: DBA/1 mice (n = 5) were used as age-matched, healthy, untreated controls. CIA was induced in male DBA/1 mice. Intraarticular injected DEX (0.01mg/Kg, 10μl) into the knee joint of CIA on Day 28, Day 35, Day 42 and Day 49 post the 1st immunization. The severity of the arthritic disease was ameliorated in DEX-treated mice, accompanied by the decreased expression of IL-6, IL-8 and TNF-α. However, DEX treatment accelerates bone erosion and osteoporosis during CIA development and triggers higher expression of RANKL, IL-17 in vitro and vivo. The effect of DEX on bone structure was analyzed using Haematoxylin & Eosin (H&E) staining and Micro-CT. The levels of receptor activator for nuclear factor-κ B ligand (RANKL) and osteoprotegerin (OPG) were investigated by real-time PCR, Western Blot and immunohistochemical analysis. RASFs were stimulated with Interleukin (IL)-1β and then treated with different concentrations of DEX for 72h. Intraarticular injection of DEX could exacerbate bone erosion in CIA model via up-regulation of RANKL expression.

MiR-145-5p Increases Osteoclast Numbers In Vitro and Aggravates Bone Erosion in Collagen-Induced Arthritis by Targeting Osteoprotegerin.

BackgroundOsteoprotegerin (OPG) inhibits bone resorption and binds with strong affinity to receptor activator of NF κB ligand (RANKL), thereby preventing RANKL from binding to its receptor RANK. Osteoclasts have documented effects on bone erosion of rheumatoid arthritis (RA). The aim of this study was to examine the role of miR-145-5p in the regulation of RA osteoclast differentiation and bone erosion.Material/MethodsExpression of microRNA-145-5p in human peripheral blood mononuclear cells (PBMC) and synovial tissue was assayed by real-time polymerase chain reaction (RT-PCR). OPG, RANK, and RANKL expression in RAW-264.7 cells was examined by RT-PCR and Western blot analysis. Osteoclast formation was detected by tartrate-resistant acid phosphatase (TRAP) staining. The effect of miR-145-5p on predicted target mRNAs was examined by luciferase reporter assays. Collagen-induced arthritis (CIA) was induced by injecting DBA/1 mice with bovine type II collagen (CII), and miR-145-5p agomir was administered by intravenous injection. Morphological changes in the CIA joint were assessed by micro-computed tomography (CT) and histopathology.ResultsmiR-145-5p levels significantly increased in RA PBMC and synovial tissue compared with normal PBMC and osteoarthritis (OA) tissue. After transfection of RAW-264.7 cells with miR-145-5p, RANK and RANKL expression increased significantly, while OPG expression decreased significantly. TRAP staining results showed osteoclast numbers increased. Micro-CT analysis of the arthritic joints showed that the miR-145-5p agomir caused bone erosion in mice, and histopathological analysis revealed that miR-145-5p agomir aggravates cartilage erosion.ConclusionsOur findings indicate that administration of miR-145-5p aggravates joint erosion in CIA mice. This suggests that miR-145-5p is a potential target for the treatment of RA.

Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts

Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP+ osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11–7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.

GMSCs suppress osteoclastogenesis and bone erosion in collagen-induced arthritis through curbing NF-KB and RANKL signals (THER5P.900)

Abstract Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and gingiva-derived mesenchymal stem cells (GMSCs) can inhibit experimental collagen-induced-arthritis (CIA) model. This study aims to determine if GMSCs also suppress osteolastogenesis and bone erosion in CIA. Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. Human osteoclasts were induced from human CD14+ cells. GMSCs were generated and added to cultures with different ratios with CD11b+ or CD14+ cells. NF-κB activation was determined by western blot. 2×106 GMSCs or fibroblast cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by HE staining. We showed that GMSCs but not fibroblast cells completely suppressed osteoclastogenesis in vitro for human and mice cells. GMSCs injected after immunization and before of onset of CIA significantly suppressed disease development. Treatment with GMSCs dramatically decreased the levels of NF-κB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo. These results suggest that the manipulation of GMSCs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.

Digoxin ameliorates autoimmune arthritis via suppression of Th17 differentiation.

Digoxin is a cardiac glycoside that is commonly used to treat heart failure. Based on its known anti-inflammatory effect, this study was undertaken to investigate the effect of digoxin on collagen-induced arthritis (CIA) and to delineate the underlying mechanism. Digoxin or vehicle was injected intraperitoneally thrice weekly in mice with CIA, from day 7 or day 35 after immunization to investigate preventive or therapeutic effect, respectively. The incidence and severity of arthritis was evaluated. Digoxin treatment suppressed the incidence of arthritis and joint inflammation in mice with CIA. The expression of IL-17 and other proinflammatory cytokines, including IL-1β, IL-6, TNF-α and IL-21, were markedly reduced in the arthritic joints of digoxin-treated CIA mice. Th17 cells and CD4(+) pSTAT3(+) cells were less frequently observed in the spleen of digoxin-treated CIA mice than controls. The mRNA expression of IL-17 and ROR γt was consistently lower in total splenocytes or draining lymph node cells obtained from digoxin-treated CIA mice. Digoxin also reduced in vitro Th17 differentiation and LPS-stimulated IgG production. The number of osteoclasts in the arthritic joint was lower in digoxin-treated mice, whereas digoxin treatment did not directly suppress in vitro osteoclastogenesis. Our findings suggest that digoxin can regulate Th17 and reciprocally promote Treg cells and suppress joint inflammation and bone erosion in CIA. Digoxin may be a therapeutic option by targeting pathogenic Th17 and immunoglobulin production, for treatment of autoimmune arthritis and other Th17-related diseases.

Celastrol attenuates bone erosion in collagen-Induced arthritis mice and inhibits osteoclast differentiation and function in RANKL-induced RAW264.7.

Recently, the traditional Chinese medicine Tripterygium wilfordii Hook f (TwHF) of the Celastraceae family has attracted increasing attention for its potential therapeutic application in patients with rheumatoid arthritis (RA). It is well accepted that TwHF exerts the antirheumatic activity and mainly depends on its potent anti-inflammatory property. To further explore the therapeutic potential of the well-defined TwHF-derived single compound - celastrol in RA, we study the therapeutic efficacy of celastrol on bone erosion in collagen-induced arthritis (CIA) mice and delineate its effects on osteoclast differentiation and functions in RANKL-induced osteoclast precursors RAW264.7 cell line. In CIA mice, daily injection of celastrol (beginning on day 28 after arthritis induction) markedly suppressed arthritis, and reduced bone damage in the joints as demonstrated by histology and bone micro-computed tomography (CT). The effects were accompanied by reductions of osteoclast cells in joints, serum tartrate-resistant acid phosphatase (TRAP) 5b, and expression of osteoclastic genes (Trap, Ctsk, Ctr, Mmp-9) and transcriptional factors (c-Fos, c-Jun and NFATc1). When RAW264.7 cells were treated with RANKL, celastrol inhibited the formation of TRAP+ multinucleated cells and the bone-resorbing activity in dose-dependent manners. Furthermore, celastrol reduced the RANKL-induced expression of osteoclastic genes and transcriptional factors, as well as phosphorylation of NF-kB and mitogen-activated protein kinases (MAPK). These findings show that celastrol could directly inhibit osteoclast formation and function, suggesting a novel therapeutic strategy of celastrol for managing RA, especially in preventing bone destruction.

FRI0367 Celastrol Inhibits Osteoclastogenesis and Bone Resorption in Rankl-Induced Raw264.7 via Nf-κB and MAPK Pathways and Attenuates Bone Erosion in Collagen-Induced Arthritis

BackgroundCelastrus have been long used to treat various inflammatory disorders including Rheumatoid arthritis (RA), SLE and Ankylosing Spondylitis in China. Celastrol is one of the main bioactive component of Celastrus....

THU0502 Ad Promotes the Differentiation of TH17 Cell and Exacerbates Disease Progression in CIA Model via Enhancing TH17 Response

BackgroundAdiponectin (AD), produced prevalently by adipose tissues, has suggested playing a significant role in chronic inflammation and immune response. The relation between AD and rheumatoid arthritis (RA) has been a...

Plectranthus amboinicus Attenuates Inflammatory Bone Erosion in Mice with Collagen-induced Arthritis by Downregulation of RANKL-induced NFATc1 Expression

Plectranthus amboinicus has been known to treat inflammatory diseases or swelling symptoms. We investigated whether P. amboinicus exhibited an inhibitory effect on osteoclastogenesis in vitro and inflammatory bone erosion in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. We attempted to identify the active component of P. amboinicus involved in regulation of osteoclastogenesis. We treated M-CSF- and RANKL-stimulated murine bone marrow-derived macrophages (BMM) and RANKL-induced RAW264.7 cells with different concentrations of P. amboinicus or rosmarinic acid, a phytopolyphenol purified from P. amboinicus, to monitor osteoclast formation by TRAP staining. The mechanism of the inhibition was studied by biochemical analysis such as RT-PCR and immunoblotting. CIA mice were administered gavages of P. amboinicus (375 mg/kg) or placebo. Then clinical, histological, and biochemical measures were assessed to determine the effects of P. amboinicus on synovial inflammation and bone erosion by H&E staining of the inflamed joints and ELISA. Rosmarinic acid strongly inhibited RANKL-induced NF-κB activation and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation in BMM, and also inhibited RANKL-induced formation of TRAP-positive multinucleated cells. A pit formation assay and the CIA animal model showed that P. amboinicus significantly inhibited the bone-resorbing activity of mature osteoclasts. We postulated that rosmarinic acid conferred the inhibitory activity on P. amboinicus for inhibition of osteoclastogenesis via downregulation of RANKL-induced NFATc1 expression. Our results indicated the possibility of P. amboinicus as a new remedy against inflammatory bone destruction.

IC87114, a Selective Inhibitor of PI3Kδ Suppresses Joint Inflammation and Bone Erosion in Collagen-Induced Arthritis in Rat (50.14)

Abstract PI3Kδ plays an essential role in antigen-receptor signaling, proliferation, activation and function of lymphocytes. Mice deficient in PI3Kδ activity show substantial reduction in immunoglobulin levels, partial impairment in chemoattractant-induced neutrophil migration as well as defects in signaling and function of macrophages. Collagen-induced arthritis (CIA), a commonly used model for studying antirheumatic drugs, requires participation of both B and T cells to initiate disease and reproduces many of the pathogenic mechanisms detected in human rheumatoid arthritis. In this study, we have investigated the ability of IC87114, a selective PI3Kδ inhibitor, to reverse the rheumatoid arthritic like state in the CIA model of arthritis. Clinical arthritis was initiated by immunizing animals with collagen followed by a booster dose on day 7. IC87114 or control treatment was initiated when at least one hind paw was significantly inflamed and continued for an 18-day treatment course. IC87114-treatment of arthritic rats reduced the progression and severity of clinical arthritis that was evident within 6 days after initiation of therapy. IC87114-treatment significantly reduced the level of anti-collagen antibodies. Arthritic rats treated with IC87114 had significantly lower radiographic scores compared with control, indicating that treatment with IC87114 was effective in protecting bone integrity. Histopathological evaluation demonstrated that IC87114 was effective in reducing the histological changes induced by rheumatoid arthritis. The effect of IC87114-treatment on these parameters together suggests a therapeutic potential for PI3Kδ-selective compounds to ameliorate rheumatoid arthritis.

Protective effect of vasoactive intestinal peptide on bone destruction in the collagen-induced arthritis model of rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, characterized by the presence of inflammatory synovitis accompanied by destruction of joint cartilage and bone. Treatment with vasoactive intestinal peptide (VIP) prevents experimental arthritis in animal models by downregulation of both autoimmune and inflammatory components of the disease. The aim of this study was to characterize the protective effect of VIP on bone erosion in collagen-induced arthritis (CIA) in mice. We have studied the expression of different mediators implicated in bone homeostasis, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), receptor activator of nuclear factor-κB (RANK), receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), IL-1, IL-4, IL-6, IL-10, IL-11 and IL-17. Circulating cytokine levels were assessed by ELISA and the local expression of mediators were determined by RT-PCR in mRNA extracts from joints. VIP treatment resulted in decreased levels of circulating IL-6, IL-1β and TNFα, and increased levels of IL-4 and IL-10. CIA-mice treated with VIP presented a decrease in mRNA expression of IL-17, IL-11 in the joints. The ratio of RANKL to OPG decreased drastically in the joint after VIP treatment, which correlated with an increase in levels of circulating OPG in CIA mice treated with VIP. In addition, VIP treatment decreased the expression of mRNA for RANK, iNOS and COX-2. To investigate the molecular mechanisms involved, we tested the activity of NFκB and AP-1, two transcriptional factors closely related to joint erosion, by EMSA in synovial cells from CIA mice. VIP treatment in vivo was able to affect the transcriptional activity of both factors. Our data indicate that VIP is a viable candidate for the development of treatments for RA.

Increase in expression of receptor activator of nuclear factor kappaB at sites of bone erosion correlates with progression of inflammation in evolving collagen-induced arthritis.

The receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL) pathway is critical in osteoclastogenesis and bone resorption and has been implicated in the process of focal bone erosion in arthritis. This study was undertaken to identify in vivo the hitherto-unknown origin and localization of RANK-expressing osteoclast precursor cells at sites of bone erosion in arthritis. DBA-1 mice were immunized with bovine type II collagen/Freund's complete adjuvant and were given an intraperitoneal booster injection of type II collagen on day 21. Arthritis was monitored visually, and joint pathology was examined histologically. RANK and RANKL expression were analyzed using specific immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining was performed. In addition, TRAP and cathepsin K messenger RNA expression were analyzed by in situ hybridization. A marked increase in the number of cells expressing RANK correlated with the progression of synovial inflammation and clinical disease severity in evolving collagen-induced arthritis (CIA). Interestingly, RANK expression demonstrated a gradient pattern with increased numbers of RANK-positive cells within the synovial infiltrate in areas closer to periosteum and cortical bone. Cells expressing RANK included cells in synovial tissue, bone lining cells on the surface of trabecular bone at sites of erosion, and cells in periosteal areas adjacent to synovial inflammation. In areas where RANK-positive cells were abundant, TRAP-positive, multinucleated osteoclast-like cells were also present at sites of focal bone erosion, suggesting differentiation of synovially derived RANK-positive osteoclast precursor cells into osteoclasts. In addition, TRAP- and cathepsin K-double-positive osteoclast-like cells were detected on the synovial side of cortical bone at sites of early and advanced cortical bone erosion. Sites of RANK expression also correlated well with sites of RANKL expression, and there was a close correlation of the temporal expression of the receptor-ligand pair. Cells expressing RANK increased in abundance with the progression of arthritis in evolving CIA, and sites of RANK-expressing cells correlated with sites of TRAP-positive, multinucleated osteoclast-like cells as well as with sites of RANKL expression. These data support the hypothesis that the RANK/RANKL pathway plays an important role in the process of bone erosion in CIA.

Osteoprotegerin Reduces Osteoclast Numbers and Prevents Bone Erosion in Collagen-Induced Arthritis

Rheumatoid arthritis is characterized by progressive synovial inflammation and joint destruction. While matrix metalloproteinases (MMPs) are implicated in the erosion of unmineralized cartilage, bone destruction involves osteoclasts, the specialized cells that resorb calcified bone matrix. RANK ligand (RANKL) expressed by stromal cells and T cells, and its cognate receptor, RANK, were identified as a critical ligand-receptor pair for osteoclast differentiation and survival. A decoy receptor for RANKL, osteoprotegerin, (OPG) impinges on this system and regulates osteoclast numbers and activity. RANKL is also expressed in collagen-induced arthritis (CIA) in which focal collections of osteoclasts are prominent at sites of bone destruction. To determine the role of RANK signaling events in the effector phase of CIA, we investigated effects of Fc-osteoprotegerin fusion protein (Fc-OPG) in CIA. After induction of CIA in Dark Agouti rats, test animals were treated with or without Fc-OPG (3 mg/kg/day) subcutaneously for 5 days, beginning at the onset of disease. Paraffin-embedded joints were then analyzed histologically and the adjacent bone assessed by histomorphometry. Osteoclasts were identified using TRAP staining and expression of the mRNA for OPG and RANKL was identified by in situ hybridization. The results indicated that short-term Fc-OPG effectively prevented joint destruction, even though it had no impact on the inflammatory aspects of CIA. In arthritic joints, Fc-OPG depleted osteoclast numbers by over 75% and diminished bone erosion scores by over 60%. Although cartilage loss was also reduced by Fc-OPG, the effects on cartilage were less striking than those on bone. In arthritic joints OPG mRNA was highly expressed and co-localized with RANK ligand, and treatment with Fc-OPG did not affect the expression of endogenous RANKL or OPG mRNA. These data demonstrate that short term Fc-OPG treatment has powerful anti-erosive effects, principally on bone, even though synovitis is not affected. These findings indicate the potential utility of disrupting RANK signaling to preserve skeletal integrity in inflammatory arthritis.