Cellular plasticity and myeloid inflammation in the adult brain are independent of the transcriptional modulator DREAM.

The downstream regulatory element antagonist modulator (DREAM) modulates ion channel function and gene transcription. Functionally, DREAM is implicated in physiological and pathological processes including cell proliferation, inflammation, and nociception. Despite its multiple functions and robust expression in forebrain tissue, neurons and glial cells, the role of DREAM in regard to cellular plasticity and tumor necrosis factor (TNF)-mediated inflammation is largely unexplored. Here, we demonstrate that adult hippocampal neurogenesis as well as the density and plasticity of glial cells in the hippocampus and thalamus are independent of the presence of DREAM. Further, DREAM deletion does not alter the regional myeloid response and inflammatory gene expression induced by chronic peripheral inflammation in mice overexpressing human TNF. Our data suggest that despite their highly dynamic regulation, neural cell plasticity and adult neurogenesis in the hippocampus do not depend on the multifunctional protein DREAM. Furthermore, TNF-mediated myeloid inflammation in the brain persists in the absence of DREAM.

Using Alendronic Acid Coupled Fluorescently Labelled SM Liposomes as a Vehicle for Bone Targeting.

BACKGROUND: We recently developed a liposomal nanoparticle system that can be used for drug delivery and simultaneously be monitored by optical or photoacoustic imaging devices. Here we tested the efficacy of alendronate as a homing molecule in SM-liposomes for bone targeting. METHODS: Alendronate was immobilized covalently on the liposomal surface and the fluorescent dye indocyanine green was used as a payload in the liposomes. The indocyanine green delivery was analyzed by 3D optical tomography, optical fluorescence scanner, photoacoustic imaging, and by ex-vivo biodistribution studies. RESULTS: The results show that the alendronate, coupled to the liposomal surface, increases sphingomyelin containing liposome targeting up to several-folds. CONCLUSION: The alendronate targeted liposomes open possibilities for an application in active bone targeting.

Chronic Peripheral Inflammation Causes a Region-Specific Myeloid Response in the Central Nervous System.

Systemic immune dysregulation contributes to the development of neuropsychiatric and neurodegenerative diseases. The precise effect of chronic peripheral immune stimulation on myeloid cells across anatomical brain regions is unclear. Here, we demonstrate brain-region-specific differences in myeloid responses induced by chronic peripheral inflammation. This shift in the myeloid compartment is associated with the appearance of an inflammatory myeloid subpopulation in the cortex, striatum, and thalamus accompanied by regional transcriptomic fingerprints that include induction of chemokines, complement factors, and endothelial adhesion molecules. In contrast, myeloid immune responses within the hippocampus and cerebellum are subtle or absent. Treatment with the anti-tumor necrosis factor α (anti-TNF-α) antibody infliximab ablates the region-specific inflammatory response. A region-specific myeloid cell response to chronic peripheral inflammation is observed in postmortem brains from individuals with rheumatoid arthritis. Our data suggest that chronic peripheral inflammation has heterogeneous effects on the brain, as evidenced by the spectrum of myeloid cell responses observed across brain regions.

A network of trans-cortical capillaries as mainstay for blood circulation in long bones.

Closed circulatory systems (CCS) underlie the function of vertebrate organs, but in long bones their structure is unclear, although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil emigration from BM, we studied the vascular system of murine long bones. Here we show that hundreds of capillaries originate in BM, cross murine cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical-vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% arterial and 59% venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the CCS in long bones and may represent an important route for immune cell export from the BM.

Glucocorticoid receptor in stromal cells is essential for glucocorticoid-mediated suppression of inflammation in arthritis.

BACKGROUND: Glucocorticoid (GC) therapy is frequently used to treat rheumatoid arthritis due to potent anti-inflammatory actions of GCs. Direct actions of GCs on immune cells were suggested to suppress inflammation. OBJECTIVES: Define the role of the glucocorticoid receptor (GR) in stromal cells for suppression of inflammatory arthritis. METHODS: Bone marrow chimeric mice lacking the GR in the hematopoietic or stromal compartment, respectively, and mice with impaired GR dimerisation (GRdim) were analysed for their response to dexamethasone (DEX, 1 mg/kg) treatment in serum transfer-induced arthritis (STIA). Joint swelling, cell infiltration (histology), cytokines, cell composition (flow cytometry) and gene expression were analysed and RNASeq of wild type and GRdim primary murine fibroblast-like synoviocytes (FLS) was performed. RESULTS: GR deficiency in immune cells did not impair GC-mediated suppression of STIA. In contrast, mice with GR-deficient or GR dimerisation-impaired stromal cells were resistant to GC treatment, despite efficient suppression of cytokines. Intriguingly, in mice with impaired GR function in the stromal compartment, GCs failed to stimulate non-classical, non-activated macrophages (Ly6Cneg, MHCIIneg) and associated anti-inflammatory markers CD163, CD36, AnxA1, MerTK and Axl. Mice with GR deficiency in FLS were partially resistant to GC-induced suppression of STIA. Accordingly, RNASeq analysis of DEX-treated GRdim FLS revealed a distinct gene signature indicating enhanced activity and a failure to reduce macrophage inflammatory protein (Mip)-1α and Mip-1ß. CONCLUSION: We report a novel anti-inflammatory mechanism of GC action that involves GR dimerisation-dependent gene regulation in non-immune stromal cells, presumably FLS. FLS control non-classical, anti-inflammatory polarisation of macrophages that contributes to suppression of inflammation in arthritis.

Upper zone of growth plate and cartilage matrix associated protein protects cartilage during inflammatory arthritis.

BACKGROUND: ADAMTS aggrecanases play a major role in cartilage degeneration during degenerative and inflammatory arthritis. The cartilage-specific secreted protein Upper zone of growth plate and cartilage matrix associated protein (Ucma) has been shown to block ADAMTS-triggered aggrecanolysis in experimental osteoarthritis. Here we aimed to investigate whether and how Ucma may affect cartilage destruction and osteophyte formation in the context of inflammatory arthritis. METHODS: Ucma-ADAMTS5 protein interactions were studied using slot blot and solid phase binding assays. Chondrocyte cultures were stimulated with ADAMTS5 or IL-1ß in the presence or absence of Ucma and aggrecanolysis was assessed by neoepitope formation. Arthritis was induced by transfer of K/BxN serum into wild-type (WT), Ucma-deficient and WT mice treated with recombinant Ucma. Cartilage proteoglycan loss and cartilage damage was assessed by safranin-O stain, aggrecanase-induced neoepitope formation and histomorphometry, respectively. Osteophytes were assessed by histomorphometry, micro-computed tomography, RNA in-situ hybridisation for collagen10a1 and osteocalcin, and staining for TRAP activity. Gene expression analyses were performed using real-time RT-PCR. RESULTS: Ucma physically interacted with ADAMTS5 and blocked its aggrecanase activity in chondrocyte cultures. Ucma was highly expressed in the articular cartilage and in osteophytes during arthritis. Ucma had no effect on inflammation and bone erosion. In contrast, Ucma-deficient mice developed significantly more severe cartilage proteoglycan loss and cartilage destruction. Conversely, treatment with Ucma inhibited cartilage degeneration in arthritis. Ucma effectively inhibited ADAMTS5-triggered or IL-1ß-triggered aggrecanolysis in vitro and in vivo. Furthermore, osteophyte formation was reduced in Ucma-deficient mice. CONCLUSIONS: These results indicate that Ucma inhibits aggrecanolysis by physical interaction with ADAMTS5 and protects from cartilage degeneration in inflammatory arthritis. Ucma therefore represents an interesting novel and specific target for preventing cartilage degradation in the context of inflammatory arthritis.

Neurodegeneration Enhances the Development of Arthritis.

The prevalence of neurodegenerative disease and arthritis increases with age. Despite both processes being associated with immune activation and inflammation, little is known about the mechanistic interactions between neurodegenerative disease and arthritis. In this article, we show that tau-transgenic (tau-tg) mice that develop neurodegenerative disease characterized by deposition of tau tangles in the brain are highly susceptible to developing arthritis. Already at steady-state conditions, tau-tg mice exhibit peripheral immune activation that is manifested by higher numbers of granulocytes, plasmablasts, and inflammatory Ly6Chi CCR2+ monocytes, as well as increased levels of proinflammatory cytokines, such as TNF-α and IL-17. Upon induction of collagen-induced arthritis (CIA), tau-tg mice displayed an increased incidence and an earlier onset of CIA that was associated with a more pronounced inflammatory cytokine response. Furthermore, induction of CIA led to significantly elevated numbers of Iba-1-expressing cells in the brain, indicative of microglia activation, and the formation of anti-tau Abs in tau-tg mice. These changes were accompanied by the resolution of tau tangles and significantly decreased neurodegenerative pathology. In summary, these data show that neurodegenerative disease enhances the development of arthritis. In addition, arthritis, once induced, triggers innate immune responses in the brain, leading to resolution of neurodegenerative changes.

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness.

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.

Hippocampal structure and function are maintained despite severe innate peripheral inflammation.

Chronic peripheral inflammation mediated by cytokines such as TNFα, IL-1ß, and IL-6 is associated with psychiatric disorders like depression and anxiety. However, it remains elusive which distinct type of peripheral inflammation triggers neuroinflammation and affects hippocampal plasticity resulting in depressive-like behavior. We hypothesized that chronic peripheral inflammation in the human TNF-α transgenic (TNFtg) mouse model of rheumatoid arthritis spreads into the central nervous system and induces depressive state manifested in specific behavioral pattern and impaired adult hippocampal neurogenesis. TNFtg mice showed severe erosive arthritis with increased IL-1ß and IL-6 expression in tarsal joints with highly elevated human TNF-α levels in the serum. Intriguingly, IL-1ß and IL-6 mRNA levels were not altered in the hippocampus of TNFtg mice. In contrast to the pronounced monocytosis in joints and spleen of TNFtg mice, signs of hippocampal microgliosis or astrocytosis were lacking. Furthermore, locomotion was impaired, but there was no locomotion-independent depressive behavior in TNFtg mice. Proliferation and maturation of hippocampal neural precursor cells as well as survival of newly generated neurons were preserved in the dentate gyrus of TNFtg mice despite reduced motor activity and peripheral inflammatory signature. We conclude that peripheral inflammation in TNFtg mice is mediated by chronic activation of the innate immune system. However, severe peripheral inflammation, though impairing locomotor activity, does not elicit depressive-like behavior. These structural and functional findings indicate the maintenance of hippocampal immunity, cellular plasticity, and behavior despite peripheral innate inflammation.

Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss.

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

Combined inhibition of tumor necrosis factor α and interleukin-17 as a therapeutic opportunity in rheumatoid arthritis: development and characterization of a novel bispecific antibody.

OBJECTIVE: Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. METHODS: Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested. RESULTS: TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice. CONCLUSION: Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.

Influence of antiTNF-alpha antibody treatment on fracture healing under chronic inflammation.

BACKGROUND: The overexpression of tumor necrosis factor (TNF)-α leads to systemic as well as local loss of bone and cartilage and is also an important regulator during fracture healing. In this study, we investigate how TNF-α inhibition using a targeted monoclonal antibody affects fracture healing in a TNF-α driven animal model of human rheumatoid arthritis (RA) and elucidate the question whether enduring the anti TNF-α therapy after trauma is beneficial or not. METHODS: A standardized femur fracture was applied to wild type and human TNF-α transgenic mice (hTNFtg mice), which develop an RA-like chronic polyarthritis. hTNFtg animals were treated with anti-TNF antibody (Infliximab) during the fracture repair. Untreated animals served as controls. Fracture healing was evaluated after 14 and 28 days of treatment by clinical assessment, biomechanical testing and histomorphometry. RESULTS: High levels of TNF-α influence fracture healing negatively, lead to reduced cartilage and more soft tissue in the callus as well as decreased biomechanical bone stability. Blocking TNF-α in hTNFtg mice lead to similar biomechanical and histomorphometrical properties as in wild type. CONCLUSIONS: High levels of TNF-α during chronic inflammation have a negative impact on fracture healing. Our data suggest that TNF-α inhibition by an anti-TNF antibody does not interfere with fracture healing.

Sclerostin inhibition reverses systemic, periarticular and local bone loss in arthritis.

OBJECTIVE: To test whether inhibition of sclerostin by a targeted monoclonal antibody (Scl-Ab) protects from bone and cartilage damage in inflammatory arthritis. Sclerostin is a potent inhibitor of bone formation and may be responsible for the low level of bone repair in patients with rheumatoid arthritis. METHODS: Human tumour necrosis factor transgenic mice (hTNFtg mice) developing inflammatory arthritis and local and bone loss were administered either vehicle, anti-TNF antibody, Scl-Ab, or a combination of both agents. Inflammation, systemic and periarticular bone loss, bone erosion and cartilage damage were evaluated at baseline (week 8) and after 3 weeks of treatment by clinical assessment, micro-CT and histology. RESULTS: Scl-Ab did not affect joint swelling or synovitis. Systemic bone loss in the spine and periarticular bone loss in the proximal tibia were completely blocked and partially reversed by inhibition of sclerostin but not by inhibition of TNF. Moreover, Scl-Ab completely arrested the progression of bone erosion in hTNFtg mice and in combination with TNF inhibition even led to significant regression of cortical bone erosions. Protective effects of Scl-Ab were also observed for the articular cartilage. CONCLUSIONS: These data suggest that sclerostin inhibition is a powerful tool to enhance bone repair in inflammatory arthritis.

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin.

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Blockade of the hedgehog pathway inhibits osteophyte formation in arthritis.

BACKGROUND: Osteophyte formation is a common phenomenon in arthritis. Bone formation by endochondral ossification is considered a key pathophysiological process in the formation of osteophytes. OBJECTIVE: To examine the hypothesis that inhibition of smoothened (Smo), a key component of the hedgehog pathway inhibits osteophyte formation as the hedgehog pathway mediates endochondral ossification. METHODS: Arthritis was induced in 8-week-old C57/BL6 mice by serum transfer (K/BxN model). Mice were then treated by daily administration of either vehicle or LDE223, a specific small molecule inhibitor for Smo, over 2 weeks starting at the onset of disease. Clinical course of arthritis, histological and molecular changes of bone in the affected joints as well as systemic bone changes were assessed. RESULTS: Serum transfer-induced arthritis led to severe osteophyte formation within 2 weeks of onset. Blockade of Smo inhibited hedgehog signalling in vivo and also significantly inhibited osteophyte formation, whereas the clinical and histopathological signs of arthritis were not affected. Also, systemic bone mass did not change. Smo inhibitor particularly blocked the formation of hypertrophic chondrocytes and collagen type X expression. CONCLUSIONS: The data indicate that blockade of hedgehog signalling by targeting Smo specifically inhibits osteophyte formation in arthritis without affecting inflammation and without eliciting bone destruction at the local and systemic level. Blockade of Smo may thus be considered as a strategy to specifically influence the periosteal bone response in arthritis associated with bone apposition.

Anti IL-17A therapy inhibits bone loss in TNF-α-mediated murine arthritis by modulation of the T-cell balance.

Tumour necrosis factor alpha (TNF-α) is a major inducer for inflammation and bone loss. Here, we investigated whether interleukin (IL)-17 plays a role in TNF-α-mediated inflammation and bone resorption. Human TNF-α transgenic (hTNFtg) mice were treated with a neutralizing anti-IL-17A antibody and assessed for inflammation, cartilage and bone damage. T-cell transcription factors and lymphokine patterns were measured in the LNs. IL-17A inhibition in the absence of IL-1 was also evaluated by treating hTNFtg/IL-1(-/-) mice with an IL-17A neutralizing antibody. IL-17A neutralization had only minor effects on TNF-α-induced inflammation but effectively reduced local and systemic bone loss by blocking osteoclast differentiation in vivo. Effects were based on a shift to bone-protective T-cell responses such as enhanced Th2 differentiation, IL-4 and IL-12 expression and Treg cell numbers. Whereas inflammation in hTNFtg/IL-1(-/-) mice was highly sensitive to IL-17A blockade, no shift in the T-cell lineages and no additional benefit on bone mass were observed in response to IL-17A neutralization. We thus conclude that IL-17A is a key mediator of TNF-α-induced bone loss by closely interacting with IL-1 in blocking bone protective T-cell responses.

Proteasome inhibition aggravates tumor necrosis factor-mediated bone resorption in a mouse model of inflammatory arthritis.

OBJECTIVE: The proteasome inhibitor bortezomib has potent anti-myeloma and bone-protective activity. Recently, bortezomib was shown to directly inhibit osteoclastogenesis. The aim of this study was to analyze the influence and therapeutic effect of bortezomib in a mouse model of inflammatory arthritis. METHODS: Heterozygous human tumor necrosis factor α (hTNFα)-transgenic mice and their wild-type (WT) littermates were intravenously injected with 0.75 mg/kg of bortezomib or phosphate buffered saline twice weekly. The mice were assessed for clinical signs of arthritis. After 6 weeks of treatment, mice were analyzed for synovial inflammation, cartilage damage, bone erosions, and systemic bone changes. Osteoclast precursors from WT and hTNF-transgenic mice were isolated from bone marrow, treated with bortezomib, and analyzed for osteoclast differentiation, bone resorption, and expression of osteoclast-specific genes as well as apoptosis and ubiquitination. RESULTS: Bortezomib-treated hTNF-transgenic mice showed moderately increased inflammatory activity and dramatically enhanced bone erosions associated with a significant increase in the number of synovial osteoclasts. Interestingly, bortezomib did not alter systemic bone turnover in either hTNF-transgenic mice or WT mice. In vitro, treatment with therapeutically relevant concentrations of bortezomib resulted in increased differentiation of monocytes into osteoclasts and more resorption pits. Molecularly, bortezomib increased the expression of TNF receptor-associated factor 6, c-Fos, and nuclear factor of activated T cells c1 in osteoclast precursors. CONCLUSION: In TNF-mediated bone destruction, bortezomib treatment increased synovial osteoclastogenesis and bone destruction. Hence, proteasome inhibition may have a direct bone-resorptive effect via stimulation of osteoclastogenesis during chronic arthritis.

Vitamin D receptor regulates TNF-mediated arthritis.

OBJECTIVE: Reduced vitamin D intake has been linked to increased susceptibility to develop rheumatoid arthritis (RA) and vitamin D deficiency is associated with increased disease activity in RA patients. The pathophysiological role of vitamin D in joint inflammation is, however, unclear. METHODS: To determine the influence of absent vitamin D signalling in chronic arthritis, vitamin D receptor (VDR)-deficient mice were crossed with human tumour necrosis factor (TNF) transgenic mice (hTNFtg), which spontaneously develop chronic arthritis. RESULTS: Clinical signs and symptoms of chronic arthritis were aggravated in hTNFtg mice lacking functional VDR signalling. Moreover, synovial inflammation was clearly increased in VDR(-/-)hTNFtg mice as compared to hTNFtg mice and was associated with an increased macrophage influx in inflamed joints. In vitro, VDR-deficient monocytes were proinflammatory and hyper-responsive to TNF stimulation associated with prolonged mitogen-activated protein kinase activation and cytokine secretion. Also, VDR(-/-) monocytes showed enhanced potential to differentiate into bone resorbing osteoclasts in vitro. In line, VDR(-/-)hTNFtg mice had significantly increased cartilage damage and synovial bone erosions. CONCLUSIONS: VDR plays an important role in limiting the inflammatory phenotype in a mouse model of RA. Absent VDR signalling causes a proinflammatory monocyte phenotype associated with increased inflammation, cartilage damage and bone erosion.

Neutralisation of Dkk-1 protects from systemic bone loss during inflammation and reduces sclerostin expression.

UNLABELLED: Introduction Inflammation is a major risk factor for systemic bone loss. Proinflammatory cytokines like tumour necrosis factor (TNF) affect bone homeostasis and induce bone loss. It was hypothesised that impaired bone formation is a key component in inflammatory bone loss and that Dkk-1, a Wnt antagonist, is a strong inhibitor of osteoblast-mediated bone formation. METHODS: TNF transgenic (hTNFtg) mice were treated with neutralising antibodies against TNF, Dkk-1 or a combination of both agents. Systemic bone architecture was analysed by bone histomorphometry. The expression of ß-catenin, osteoprotegerin and osteocalcin was analysed. In vitro, primary osteoblasts were stimulated with TNF and analysed for their metabolic activity and expression of Dkk-1 and sclerostin. Sclerostin expression and osteocyte death upon Dkk-1 blockade were analysed in vivo. RESULTS: Neutralisation of Dkk-1 completely protected hTNFtg mice from inflammatory bone loss by preventing TNF-mediated impaired osteoblast function and enhanced osteoclast activity. These findings were accompanied by enhanced skeletal expression of ß-catenin, osteocalcin and osteoprotegerin. In vitro, TNF rapidly increased Dkk-1 expression in primary osteoblasts and effectively blocked osteoblast differentiation. Moreover, blockade of Dkk-1 not only rescued impaired osteoblastogenesis but also neutralised TNF-mediated sclerostin expression in fully differentiated osteoblasts in vitro and in vivo. CONCLUSIONS: These findings indicate that low bone formation and expression of Dkk-1 trigger inflammatory bone loss. Dkk-1 blocks osteoblast differentiation, induces sclerostin expression and leads to osteocyte death. Inhibition of Dkk-1 may thus be considered as a potent strategy to protect bone from inflammatory damage.

The bisphosphonate zoledronic acid has antimyeloma activity in vivo by inhibition of protein prenylation.

Nitrogen-containing bisphosphonates (N-BPs) are effective antiosteolytic agents in patients with multiple myeloma. Preclinical studies have also demonstrated that these agents have direct antitumor effects in vitro and can reduce tumor burden in a variety of animal models, although it is not clear whether such effects are caused by direct actions on tumor cells or by inhibition of bone resorption. N-BPs prevent bone destruction in myeloma by inhibiting the enzyme farnesyl pyrophosphate synthase in osteoclasts, thereby preventing the prenylation of small GTPase signaling proteins. In this study, utilizing a plasmacytoma xenograft model without complicating skeletal lesions, treatment with zoledronic acid (ZOL) led to significant prolongation of survival in severe combined immunodeficiency mice inoculated with human INA-6 plasma cells. Following treatment with a clinically relevant dose of ZOL, histological analysis of INA-6 tumors from the peritoneal cavity revealed extensive areas of apoptosis associated with poly (ADP-ribose) polymerase cleavage. Furthermore, Western blot analysis of tumor homogenates demonstrated the accumulation of unprenylated Rap1A, indicative of the uptake of ZOL by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase. These studies provide, for the first time, clear evidence that N-BPs have direct antitumor effects in plasma cell tumors in vivo and this is executed by a molecular mechanism similar to that observed in osteoclasts.