A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes.

BACKGROUND: Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. RESULTS: Human monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates. CONCLUSION: Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

Deletion of activin A in mesenchymal but not myeloid cells ameliorates disease severity in experimental arthritis.

OBJECTIVE: The aim of this study was to assess the extent and the mechanism by which activin A contributes to progressive joint destruction in experimental arthritis and which activin A-expressing cell type is important for disease progression. METHODS: Levels of activin A in synovial tissues were evaluated by immunohistochemistry, cell-specific expression and secretion by PCR and ELISA, respectively. Osteoclast (OC) formation was assessed by tartrat-resistant acid phosphatase (TRAP) staining and activity by resorption assay. Quantitative assessment of joint inflammation and bone destruction was performed by histological and micro-CT analysis. Immunoblotting was applied for evaluation of signalling pathways. RESULTS: In this study, we demonstrate that fibroblast-like synoviocytes (FLS) are the main producers of activin A in arthritic joints. Most significantly, we show for the first time that deficiency of activin A in arthritic FLS (ActßAd/d ColVI-Cre) but not in myeloid cells (ActßAd/d LysM-Cre) reduces OC development in vitro, indicating that activin A promotes osteoclastogenesis in a paracrine manner. Mechanistically, activin A enhanced OC formation and activity by promoting the interaction of activated Smad2 with NFATc1, the key transcription factor of osteoclastogenesis. Consistently, ActßAd/d LysM-Cre hTNFtg mice did not show reduced disease severity, whereas deficiency of activin A in ColVI-Cre-expressing cells such as FLS highly diminished joint destruction reflected by less inflammation and less bone destruction. CONCLUSIONS: The results highly suggest that FLS-derived activin A plays a crucial paracrine role in inflammatory joint destruction and may be a promising target for treating inflammatory disorders associated with OC formation and bone destruction like rheumatoid arthritis.

A myostatin-CCL20-CCR6 axis regulates Th17 cell recruitment to inflamed joints in experimental arthritis.

The interactions of fibroblast-like synoviocyte (FLS)-derived pro-inflammatory cytokines/chemokines and immune cells support the recruitment and activation of inflammatory cells in RA. Here, we show for the first time that the classical myokine myostatin (GDF-8) is involved in the recruitment of Th17 cells to inflammatory sites thereby regulating joint inflammation in a mouse model of TNFalpha-mediated chronic arthritis. Mechanistically, myostatin-deficiency leads to decreased levels of the chemokine CCL20 which is associated with less infiltration of Th17 cells into the inflamed joints. In vitro, myostatin alone or in combination with IL-17A enhances the secretion of CCL20 by FLS whereas myostatin-deficiency reduces CCL20 secretion, associated with an altered transmigration of Th17 cells. Thus, the communication between activated FLS and Th17 cells through myostatin and IL-17A may likely contribute to a vicious cycle of inflammation, accounting for the persistence of joint inflammation in chronic arthritis. Blockade of the CCL20-CCR6 axis by inhibition of myostatin may, therefore, be a promising treatment option for chronic inflammatory diseases such as arthritis.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Importance of osteocyte-mediated regulation of bone remodelling in inflammatory bone disease.

Although the impact of osteoblast-osteoclast crosstalk in bone remodelling has been intensively studied, the importance of osteocytes, descendants of osteoblasts, in this process has for a long time been neglected. During their embedding phase, osteocytes undergo considerable phenotypic transformation, from a cuboidal, highly metabolically active osteoblast secreting extracellular matrix to a small, stellate, quiescent osteocyte with numerous long dendrites. Osteocytes are encysted in cavities (lacunae) and their dendritic extensions are located in tunnels (canaliculi) forming a remarkable, highly branched, lacunar-canalicular signalling network that spans the entire bone matrix. Osteocytes and their dendrites can communicate directly with each other and through the release of effector proteins such as sclerostin and nuclear factor κB ligand (RANKL), influence osteoblast and osteoclast formation. This allows osteocytes embedded within the bone matrix to communicate and coordinate activity of cells on the bone surface to adapt to mechanical needs and hormonal changes. Besides their importance in sustaining physiological bone homeostasis, accumulating evidence suggests that dysregulated osteocyte function and alterations in the osteocyte lacunar-canalicular network structure are characteristics of skeletal diseases. This review highlights some aspects of osteocyte communication with osteoclasts and mesenchymal stromal cells, the importance of blood vessel-osteocyte interaction and describes central functions of these cells in rheumatoid arthritis, osteoarthritis, osteomyelitis and osteoporosis. Within the last decade new technologies and tools have facilitated the study of osteocyte biology and the search for therapeutic targets to address bone fragility in the near future.

Synovial fibroblasts and articular tissue remodelling: Role and mechanisms.

Synovial joints are unique functional elements of the body and provide the ability for locomotion and for physical interaction with the environment. They are composed of different connective tissue structures, of which the synovial membrane is one central component. It shows a number of peculiarities that makes it different from other membranes in our body, while several lines of evidence suggest that synovial fibroblasts, also termed fibroblast-like synoviocytes (FLS) critically contribute to these peculiarities. This becomes evident particularly under disease conditions such as in rheumatoid arthritis and osteoarthritis, where the synovium is a key pathophysiological component. Therefore, an in-depth knowledge of FLS biology is not only important for understanding key features of articular function but also provides explanations for important characteristics of both degenerative and inflammatory joint diseases. This article reviews the structure, biochemical composition and functions of the synovial membrane and by focusing on the role of synovial fibroblasts explains key features of articular tissue remodelling particularly under disease conditions.

Distinct fibroblast subsets drive inflammation and damage in arthritis.

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Endogenous Galectin-9 Suppresses Apoptosis in Human Rheumatoid Arthritis Synovial Fibroblasts.

Galectin-9 (Gal9) has been postulated to have anti-inflammatory properties based on the ability of exogenous Gal9 to induce apoptosis in synovial fibroblasts in animal models of rheumatoid arthritis (RA). Here we aimed to assess the potential role of endogenous Galectins, including Gal9, in the inflammatory pathology of the RA synovium in humans. Firstly expression of Galectins 1-9 was determined in synovial fibroblasts (RASF) and dermal fibroblasts (DF) isolated from RA patients, the latter representing a non-inflamed site. We then further challenged the cells with pro-inflammatory TLR agonists and cytokines and assessed Galectin expression. Gal9 was found to be differentially and abundantly expressed in RASF compared to DF. Agonists of TLR3 and TLR4, along with IFNgamma were also found to induce Gal9 expression in RASF. siRNA was then used to knock-down Gal9 expression in RASF and the effects of this on apoptosis and cell viability were assessed. Increased apoptosis was observed in RASF following Gal9 knock-down. We conclude that, unlike exogenous Gal9, endogenous Gal9 is protective against apoptosis and enhances synovial fibroblast viability suggesting that its role in RA is both pathogenic and pro-inflammatory.

Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation.

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction.

Sclerostin, an inhibitor of the Wnt/ß-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFα (tumor necrosis factor α)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFα transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFα signaling. Sclerostin effectively blocked TNFα- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFα-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFα-dependent comorbidities.

Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice.

Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-ß (TGF-ß) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA.

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis.

INTRODUCTION: Inflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA. METHODS: Expression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses. RESULTS: RA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP(-/-) hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro. CONCLUSIONS: These data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.

Cell surface-bound TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival pathways.

BACKGROUND: The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitor of metalloproteinases (TIMPs 1-4) are responsible for the physiological remodeling of the extracellular matrix (ECM). Among all TIMPs, TIMP3 appears to play a unique role since TIMP3 is a secreted protein and, unlike the other TIMP family members, is tightly bound to the ECM. Moreover TIMP3 has been shown to be able to induce apoptotic cell death. As little is known about the underlying mechanisms, we set out to investigate the pro-apoptotic effect of TIMP3 in human mesenchymal cells. METHODOLOGY/PRINCIPAL FINDINGS: Lentiviral overexpression of TIMP3 in mesenchymal cells led to a strong dose-dependent induction of ligand-independent apoptosis as reflected by a five-fold increase in caspase 3 and 7 activity compared to control (pLenti6/V5-GW/lacZ) or uninfected cells, whereas exogenous TIMP3 failed to induce apoptosis. Concordantly, increased cleavage of death substrate PARP and the caspases 3 and 7 was observed in TIMP3 overexpressing cultures. Notably, activation of caspase-8 but not caspase-9 was observed in TIMP3-overexpressing cells, indicating a death receptor-dependent mechanism. Moreover, overexpression of TIMP3 led to a further induction of apoptosis after stimulation with TNF-alpha, FasL and TRAIL. Most interestingly, TIMP3-overexpression was associated with a decrease in phosphorylation of cRaf, extracellular signal-regulated protein kinase (Erk1/2), ribosomal S6 kinase (RSK1) and Akt and serum deprivation of TIMP3-overexpressing cells resulted in a distinct enhancement of apoptosis, pointing to an impaired signaling of serum-derived survival factors. Finally, heparinase treatment of heparan sulfate proteoglycans led to the release of TIMP3 from the surface of overexpressing cells and to a significant decrease in apoptosis indicating that the binding of TIMP3 is necessary for apoptosis induction. CONCLUSION: The results demonstrate that exclusively cell surface-bound endogenous TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival signaling pathways.

Regulation of matrixmetalloproteinase-3 and matrixmetalloproteinase-13 by SUMO-2/3 through the transcription factor NF-κB.

OBJECTIVE: Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs). METHODS: Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082. RESULTS: Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1ß induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1ß mediated MMP-1 expression was not regulated by SUMO-2/3. CONCLUSIONS: Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.