Fc Gamma Receptors as Regulators of Bone Destruction in Inflammatory Arthritis.

Bone erosion is one of the primary features of inflammatory arthritis and is caused by excessive differentiation and activation of osteoclasts. Fc gamma receptors (FcγRs) have been implicated in osteoclastogenesis. Our recent studies demonstrate that joint-deposited lupus IgG inhibited RANKL-induced osteoclastogenesis. FcγRI is required for RANKL-induced osteoclastogenesis and lupus IgG-induced signaling transduction. We reviewed the results of studies that analyzed the association between FcγRs and bone erosion in inflammatory arthritis. The analysis revealed the dual roles of FcγRs in bone destruction in inflammatory arthritis. Thus, IgG/FcγR signaling molecules may serve as potential therapeutic targets against bone erosion.

Isolation and Identification of Antiarthritic Constituents from Glycine tabacina and Network Pharmacology-Based Prediction of Their Protective Mechanisms against Rheumatoid Arthritis.

Glycine tabacina (Labill.) Benth is an edible medicinal herb for rheumatoid arthritis (RA) treatment in folk medicine. Current phytochemical research on this dried herb led to the isolation of eight new coumestans, named glytabastan A-H (1-8), and twenty-three known compounds 9-31. Their structures were elucidated using spectroscopic methods. The antiarthritic activities of all isolates were evaluated, and the results showed that coumestans 1-6 and 8-10 could inhibit arthritic inflammation in vitro, while coumestans 1, 2, 9, and 10 significantly blocked the osteoclastogenesis induced by receptor activator of nuclear factor (NF) κB ligand (RANKL). Moreover, network pharmacological analysis revealed that the anti-RA effect of G. tabacina involved multitargets, multipathways such as PI3K/Akt and MAPK signaling pathways, and various biological processes such as inflammatory response and cytokine-mediated signaling pathways. These results suggested that this species and its novel coumestans could serve as potential antiarthritic agents for functional food or medicinal use.

Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis.

BACKGROUND: Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). METHODS: AIA was induced in knee joints of wild-type (WT), FcγRI,II,III-/-, and FcγRI,II,III,IV-/- mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. RESULTS: FcγRI,II,III,IV-/- mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV-/- mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV-/- and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV-/- mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV-/- mice, AIA induction in knee joints of FcγRI,II,III-/- mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. CONCLUSIONS: FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

Bruton's tyrosine kinase inhibitor BMS-986142 in experimental models of rheumatoid arthritis enhances efficacy of agents representing clinical standard-of-care.

Bruton's tyrosine kinase (BTK) regulates critical signal transduction pathways involved in the pathobiology of rheumatoid arthritis (RA) and other autoimmune disorders. BMS-986142 is a potent and highly selective reversible small molecule inhibitor of BTK currently being investigated in clinical trials for the treatment of both RA and primary Sjögren's syndrome. In the present report, we detail the in vitro and in vivo pharmacology of BMS-986142 and show this agent provides potent and selective inhibition of BTK (IC50 = 0.5 nM), blocks antigen receptor-dependent signaling and functional endpoints (cytokine production, co-stimulatory molecule expression, and proliferation) in human B cells (IC50 ≤ 5 nM), inhibits Fcγ receptor-dependent cytokine production from peripheral blood mononuclear cells, and blocks RANK-L-induced osteoclastogenesis. Through the benefits of impacting these important drivers of autoimmunity, BMS-986142 demonstrated robust efficacy in murine models of rheumatoid arthritis (RA), including collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). In both models, robust efficacy was observed without continuous, complete inhibition of BTK. When a suboptimal dose of BMS-986142 was combined with other agents representing the current standard of care for RA (e.g., methotrexate, the TNFα antagonist etanercept, or the murine form of CTLA4-Ig) in the CIA model, improved efficacy compared to either agent alone was observed. The results suggest BMS-986142 represents a potential therapeutic for clinical investigation in RA, as monotherapy or co-administered with agents with complementary mechanisms of action.

Inflammatory arthritis and systemic bone loss are attenuated by gastrointestinal helminth parasites.

Infections with different helminth species have been observed to ameliorate a variety of chronic inflammatory diseases. Herein, we show that the natural murine helminth species, Heligmosomoides polygyrus bakeri (Hp) is capable of attenuating disease severity in two different inflammatory arthritis models. Furthermore, we show that excretory-secretory (ES) products from Hp directly suppress osteoclast differentiation in vitro. Taken together, these results demonstrate that helminth infections can dampen autoimmune diseases and highlight a previously unrecognized and important role for ES products, by directly impacting on bone destruction.

Dietary dried plum increases bone mass, suppresses proinflammatory cytokines and promotes attainment of peak bone mass in male mice.

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (<2 weeks) increased bone volume (+32%) and trabecular thickness (+24%). These changes were associated with decreased osteoclast surface (Oc.S/BS) and decreased serum CTX, a marker of bone resorption. The reduction in Oc.S/BS was associated with a reduction in the osteoclast precursor pool. Osteoblast surface (Ob.S/BS) and bone formation rate were also decreased suggesting that the gain in bone in adult mice is a consequence of diminished bone resorption and formation, but resorption is reduced more than formation. The effects of DP on bone were accompanied by a decline in interleukins, TNF and MCP-1, suggesting that DP is acting in part through the immune system to suppress inflammatory activity and reduce the size of the osteoclast precursor pool. Feeding DP was accompanied by an increase in plasma phenolics, some of which have been shown to stimulate bone accrual. In growing and young adult mice DP at levels as low as 5% of diet (w/w) increased bone volume. At higher levels (DP 25%), bone volume was increased by as much as 94%. These data demonstrate that DP feeding dramatically increases peak bone mass during growth.

Epigallocatechin-3-gallate ameliorates autoimmune arthritis by reciprocal regulation of T helper-17 regulatory T cells and inhibition of osteoclastogenesis by inhibiting STAT3 signaling.

The green tea polyphenol epigallocatechin-3-gallate is a potent antioxidant. Here, we describe the effects of epigallocatechin-3-gallate on T cell differentiation and osteoclast differentiation in an animal model of arthritis. Mice with collagen-induced arthritis were injected intraperitoneally with epigallocatechin-3-gallate, 3 times/wk after the primary immunization. Surface markers of T helper 17 cells and regulatory T cells were analyzed by flow cytometry. Flow cytometry, Western blotting, and enzyme-linked immunosorbent assays were used to evaluate the effect of epigallocatechin-3-gallate on cell signaling in the collagen-induced arthritis model. Epigallocatechin-3-gallate decreased the arthritis index and showed protective effects against joint destruction in collagen-induced arthritis mice. The expression of cytokines, oxidative stress proteins, and phosphorylated-signal transducer and activator of transcription-3, 705 and 727, were significantly less in mice treated with epigallocatechin-3-gallate than it was in controls. Epigallocatechin-3-gallate reduced the expression of osteoclast markers in vitro and in vivo relative to the control, and the antiosteoclastic activity was observed in epigallocatechin-3-gallate-treated, interferon-γ knockout mice. The proportion of forkhead box protein 3-positive regulatory T cells was increased in the spleens of mice treated with epigallocatechin-3-gallate compared with control mice, whereas the proportion of T helper 17 cells was reduced. In vitro, the expression of nuclear respiratory factor 2, heme oxygenase-1, and extracellular signal-regulated kinase was increased significantly by epigallocatechin-3-gallate. We demonstrated that the administration of epigallocatechin-3-gallate attenuated the symptoms of arthritis, inhibited osteoclastogenesis and T helper 17 cell activation, and increased the number of regulatory T cells. At the molecular level, the antiarthritic effects of epigallocatechin-3-gallate may be due to induction of phosphorylated-extracellular signal-regulated kinase, nuclear respiratory factor 2, and heme oxygenase-1 and inhibition of signal transducer and activator of transcription-3 activation.

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

AIM: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. METHODS: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. RESULTS: Aconine (0.125, 0.25 µmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, ß3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. CONCLUSION: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis.

BACKGROUND: Wear particle-induced periprosthetic osteolysis that results in aseptic loosening is the most common cause of long-term failure after total joint replacement. MATERIALS AND METHODS: Icariin (ICA), a flavonoid isolated from Epimedium pubescens, inhibits osteoclast formation, but its effects on wear particle-induced inflammatory osteoclastogenesis remains unclear. We investigated the role of ICA in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which is stimulated by titanium (Ti) particles and the receptor activator of NF-κB ligand. RESULTS: ICA effectively inhibited osteoclast formation and bone resorption in the differentiation medium. ICA (10(-7) mol/L) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells compared with the control, and significantly reduced the percentage of the surface covered by resorption lacunae. Quantitative real-time polymerase chain reaction analysis showed that ICA inhibited messenger RNA expression for the receptor activator of nuclear factor-κB, cathepsin K, tartrate-resistant acid phosphatase-positive, and matrix metalloproteinase-9 in RAW264.7 cells stimulated by Ti particles and receptor activator of NF-κB ligand. ICA also reduced pro-inflammatory cytokine expression of interleukin-1ß and tumor necrosis factor-α in RAW264.7 cells cultured with Ti particles. In addition, incubation with cholecystokinin-8 showed that ICA had no toxic effects on RAW264.7 cells. CONCLUSIONS: ICA possibly elicited inhibitory effects on inflammatory osteoclastogenesis induced by Ti particles, indicating that ICA may be useful for the prevention and treatment of wear particle-induced osteolysis.

Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

BACKGROUND AND PURPOSE: Osteoclasts play a pivotal role in diseases such as osteoporosis, rheumatoid arthritis and tumour bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. Here, we examined changes in osteoclastogenesis and LPS-induced osteolysis in response to andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata. EXPERIMENTAL APPROACH: Effects of AP on osteoclast differentiation and bone resorption were measured in vitro. Western blots and RT-PCR techniques were used to examine the underlying molecular mechanisms. The bone protective activity of AP in vivo was assessed in a mouse model of osteolysis. KEY RESULTS: AP concentration-dependently suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro and reduced the expression of osteoclast-specific markers, including tartrate-resistant acid phosphatase, calcitonin receptors and cathepsin K. Further molecular analysis revealed that AP impaired RANKL-induced NF-κB signalling by inhibiting the phosphorylation of TGF-ß-activated kinase 1, suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. AP also inhibited the ERK/MAPK signalling pathway without affecting p38 or JNK signalling. CONCLUSIONS AND IMPLICATIONS: AP suppressed RANKL-induced osteoclastogenesis through attenuating NF-κB and ERK/MAPK signalling pathways in vitro, thus preventing bone loss in vivo. These data indicated that AP is a promising natural compound for the treatment of osteoclast-related bone diseases.

Retinal attenuates inflammatory arthritis by reciprocal regulation of IL-17-producing T cells and Foxp3(+) regulatory T cells and the inhibition of osteoclastogenesis.

Retinoids (e.g., vitamin A and its derivatives) can regulate immune responses. The aim of this study was to determine whether all-trans retinaldehyde (retinal), a vitamin A derivative, can inhibit inflammatory responses and joint destruction in DBA/1J mice with collagen-induced arthritis (CIA). The arthritis score and incidence of arthritis were lower in mice treated with retinal compared to those treated with cottonseed oil. Histopathologic evidence of joint damage was lower in mice treated with retinal, corresponding with a reduction in the infiltration of immune cells in mice treated with retinal type II collagen (CII)-stimulated spleen cells. In addition, the expression of proinflammatory cytokines, oxidative stress proteins, and osteoclast markers were significantly reduced in mice treated with retinal. In vitro, retinal induced increased Foxp3 expression and inhibited Th17 development. The proportion of Foxp3(+) Treg cells was increased in the spleens of mice treated with retinal, whereas the proportion of Th17 cells was reduced. In both mice and a human culture system, tartrate-resistant acid phosphatase (TRAP) positive mononuclear cells and multinucleated cells were significantly reduced after treatment with retinal. The expression of osteoclast differentiation markers was dramatically decreased upon addition of retinal. This is the first study to demonstrate the therapeutic effect of retinal on an autoimmune arthritis model in mice through reciprocal regulation of Th17 and regulatory T cells and protection of differentiation and activation of osteoclasts. Taken together, our findings indicate that retinal has profound immunoregulatory functions and potential value for the treatment of autoimmune inflammatory disorders.

New insights into osteoclastogenic signaling mechanisms.

Bone is continuously renewed through a dynamic balance between bone resorption and formation. This process is the fundamental basis for the maintenance of normal bone mass and architecture. Osteoclasts play a crucial role in both physiological and pathological bone resorption, and receptor activator of nuclear factor-κB ligand (RANKL) is the key cytokine that induces osteoclastogenesis. Here we summarize the recent advances in the understanding of osteoclastogenic signaling by focusing on the investigation of RANKL signaling and RANKL-expressing cells in the context of osteoimmunology. The context afforded by osteoimmunology will provide a scientific basis for future therapeutic approaches to diseases related to the skeletal and immune systems.

The aqueous extract of Angelica sinensis, a popular Chinese herb, inhibits wear debris-induced inflammatory osteolysis in mice.

BACKGROUND: More and more studies have shown Angelica sinensis' (AS) therapeutic action on chronic inflammatory diseases in recent years. We investigated effects of aqueous extract of AS on inflammatory cytokines release and wear debris particles-induced osteolysis. MATERIALS AND METHODS: Ultra high molecular weight polyethylene (UHMWPE) particles were used to induce inflammation in RAW264.7 cell and C57BL/J6 mice. AS extract was obtained through a series of purification steps, and divided into high dose group and low dose group during the research of cell culture, tissue culture, and animal treatment. After 72 h culture with optimal particles, supernatants were collected for cytokine analysis. Calvaria were harvested from the mice model after 10 d treatment with the AS extract. Six calvaria of each group were cultured into medium for 72 h for analyzing cytokine generated in vivo. Histologic analyses and micro-computed tomography (micro-CT) scan were used to determine osteoclastogenesis and inflammatory bone resorption. RESULTS: Concentration of tumor necrosis-alpha (TNF-α) and interleukin-1beta (IL-1ß) was significantly attenuated by AS extract both in vitro and in vivo. The osteolysis area and the osteoclast numbers were decreased from 0.406 ± 0.0799 to 0.117 ± 0.0103 mm(2), and from 22.7 ± 5.0 to 11.3 ± 1.8, respectively (P < 0.01). Compared with the control group, the protection effects of AS extract was further confirmed with data of the more accurate 3-dimension micro-CT reconstruction. CONCLUSIONS: This study suggests a potential resolution of inhibiting wear debris particles-induced inflammatory bone resorption, as well as a possible way of inhibiting aseptic loosening after joint replacement surgery.

Spontaneous and induced osteoclastogenic behaviour of human peripheral blood mononuclear cells and their CD14(+) and CD14(-) cell fractions.

OBJECTIVES: Osteoclasts are descended from the CD14(+) monocyte/macrophage lineage, but influence of other haematopoietic cells on osteoclastic commitment of their precursors has remained poorly understood. In this study, osteoclastogenic behaviour of peripheral blood mononuclear cells (PBMC) and their CD14(+) and CD14(-) subpopulations has been accessed, in the absence or presence of M-CSF and RANKL. MATERIALS AND METHODS: Cell cultures were characterized for presence of actin rings and vitronectin and calcitonin receptors, TRAP activity and calcium phosphate resorbing activity, expression of osteoclast-related genes and secretion of M-CSF and RANKL. RESULTS: In the absence of growth factors, PBMC and CD14(+) cultures had some degree of cell survival, and some spontaneous osteoclastogenesis was observed, only on cultures of the former. Supplementation with M-CSF and RANKL significantly increased osteoclastogenic behaviour of cell cultures, particularly CD14(+) cell cultures. Nevertheless, PBMC derived a higher degree of osteoclastogenesis, either as absolute values or after normalization by protein content. It was observed that unlike CD14(+) cells, PBMC were able to express M-CSF and RANKL, which increased following growth factor treatment. Also, expression of TNF-α, GM-CSF, IL-1ß, IL-6 and IL-17 was higher in PBMC cultures. Finally, CD14(-) cultures exhibited limited cell survival and did not reveal any osteoclast features. CONCLUSIONS: Results show that although osteoclastic precursors reside in the CD14(+) cell subpopulation, other populations (such as CD14(-) cells) derived from PBMC, have the ability to modulate osteoclastogenesis positively.

Regulation of bone by the adaptive immune system in arthritis.

Studies on the immune regulation of osteoclasts in rheumatoid arthritis have promoted the new research field of 'osteoimmunology', which investigates the interplay between the skeletal and immune systems at the molecular level. Accumulating evidence lends support to the theory that bone destruction associated with rheumatoid arthritis is caused by the enhanced activity of osteoclasts, resulting from the activation of a unique helper T cell subset, 'Th17 cells'. Understanding the interaction between osteoclasts and the adaptive immune system in rheumatoid arthritis and the molecular mechanisms of Th17 development will lead to the development of potentially effective therapeutic strategies.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis.

OBJECTIVE: Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS: Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS: Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION: These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

Obesity-mediated inflammatory microenvironment stimulates osteoclastogenesis and bone loss in mice.

Clinical evidence indicates that fat is inversely proportional to bone mass in elderly obese women. However, it remains unclear whether obesity accelerates bone loss. In this report we present evidence that increased visceral fat leads to inflammation and subsequent bone loss in 12-month-old C57BL/6J mice that were fed 10% corn oil (CO)-based diet and a control lab chow (LC) for 6 months. As expected from our previous work, CO-fed mice demonstrated increased visceral fat and enhanced total body fat mass compared to LC. The adipocyte-specific PPARγ and bone marrow (BM) adiposity were increased in CO-fed mice. In correlation with those modifications, inflammatory cytokines (IL-1ß, IL-6, TNF-α) were significantly elevated in CO-fed mice compared to LC-fed mice. This inflammatory BM microenvironment resulted in increased superoxide production in osteoclasts and undifferentiated BM cells. In CO-fed mice, the increased number of osteoclasts per trabecular bone length and the increased osteoclastogenesis assessed ex-vivo suggest that CO diet induces bone resorption. Additionally, the up-regulation of osteoclast-specific cathepsin k and RANKL expression and down-regulation of osteoblast-specific RUNX2/Cbfa1 supports this bone resorption in CO-fed mice. Also, CO-fed mice exhibited lower trabecular bone volume in the distal femoral metaphysis and had reduced OPG expression. Collectively, our results suggest that increased bone resorption in mice fed a CO-enriched diet is possibly due to increased inflammation mediated by the accumulation of adipocytes in the BM microenvironment. This inflammation may consequently increase osteoclastogenesis, while reducing osteoblast development in CO-fed mice.

Efficient osteoclast differentiation requires local complement activation.

Previous studies using blocking antibodies suggested that bone marrow (BM)-derived C3 is required for efficient osteoclast (OC) differentiation, and that C3 receptors are involved in this process. However, the detailed underlying mechanism and the possible involvement of other complement receptors remain unclear. In this report, we found that C3(-/-) BM cells exhibited lower RANKL/OPG expression ratios, produced smaller amounts of macrophage colony-stimulating factor and interleukin-6 (IL-6), and generated significantly fewer OCs than wild-type (WT) BM cells. During differentiation, in addition to C3, WT BM cells locally produced all other complement components required to activate C3 and to generate C3a/C5a through the alter-native pathway, which is required for efficient OC differentiation. Abrogating C3aR/C5aR activity either genetically or pharmaceutically suppressed OC generation, while stimulating WT or C3(-/-) BM cells with exogenous C3a and/or C5a augmented OC differentiation. Furthermore, supplementation with IL-6 rescued OC generation from C3(-/-) BM cells, and neutralizing antibodies to IL-6 abolished the stimulatory effects of C3a/C5a on OC differentiation. These data indicate that during OC differentiation, BM cells locally produce components, which are activated through the alternative pathway to regulate OC differentiation. In addition to C3 receptors, C3aR/C5aR also regulate OC differentiation, at least in part, by modulating local IL-6 production.

Phosphatidylserine-containing liposomes inhibit the differentiation of osteoclasts and trabecular bone loss.

Liposomes containing phosphatidylserine (PS) are engulfed by phagocytes including macrophages, microglia, and dendritic cells. PS liposomes (PSLs) mimic the effects of apoptotic cells on these phagocytes to induce the secretion of anti-inflammatory molecules and to inhibit the maturation of dendritic cells. However, the effects of PSLs on osteoclasts, which are also differentiated from the common myeloid precursors, remain to be determined. This study investigated the effects of PSLs on the osteoclastogenesis. In the rat bone marrow culture system, osteoclast precursors phagocytosed PSLs to secrete TGF-beta1 and PGE(2), which in turn inhibited osteoclastogenesis through the downregulation of receptor activator for NF-kappaB ligand, receptor activator of NF-kappaB, ICAM-1, and CD44. Consistent with these in vitro observations, i.m. injection of PSLs significantly increased the plasma level of TGF-beta1 and PGE(2) and decreased the expression of receptor activator for NF-kappaB ligand, receptor activator of NF-kappaB, and ICAM-1 in the skeletal tissues of ankle joints of rats with adjuvant arthritis (AA). A quantitative analysis using microcomputed tomography revealed that PSLs as well as TGF-beta1 together with PGE(2) significantly inhibited AA-induced trabecular bone loss. These observations strongly suggest that PSLs generate TGF-beta1 and PGE(2) release, leading to inhibit osteoclastogenesis and AA-induced trabecular bone loss. Because PS is a component of the cell membrane, PSLs therefore can be a potentially effective pharmacological intervention against abnormal bone loss, such as osteoporosis without deleterious side effects.

Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation.

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.