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Rheumatoid arthritis (RA) is an immune mediated inflammatory disease (IMID), characterized by chronic inflammation and irreversible bone loss. Studies have shown that fibroblast-like synoviocytes (FLS), a key cell population in the pathogenesis of RA, have an impact on balancing bone-forming osteoblasts and bone-destroying osteoclasts towards joint damage. Once activated, RA-FLS are able to destroy cartilage and subchondral bone through the release of RANKL, members of the metalloproteinase family and many more cytokines, chemokines and growth factors. Additionally, RA-FLS are responsible for the perpetuation and chronicity of the disease due the interaction with immune cells supporting the influx of T and B lymphocytes, monocytes, macrophages neutrophils and dendritic cells from the blood stream into the inflamed synovial tissue. In this review we highlight the direct and indirect impact of synovial fibroblasts in RA on joint damage and disease progression. Moreover, we describe mechanisms of synovitis and regulators of bone homeostasis in further inflammatory joint diseases such as ankylosing spondylitis (AS) and psoriatic arthritis (PsA) and compare them to RA.

ObjectiveThe 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.MethodsLevels 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.ResultsIn 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 (ActβAd/dColVI-Cre) but not in myeloid cells (ActβAd/dLysM-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, ActβAd/dLysM-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.ConclusionsThe 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.

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/β-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., Fibroblast-like synoviocytes are important mediators of joint pathology in rheumatoid arthritis (RA). Here the authors show that Lasp1 is epigenetically regulated and highly expressed by these cells in RA and its deletion can limit joint pathology in a mouse model of inflammatory arthritis.

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.

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.

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.

Glucocorticoids (GCs) are used in the treatment of chronic inflammatory diseases due to their potent anti-inflammatory actions. Their use is limited due to major systemic side effects including osteoporosis and muscle wasting. This is further complicated by pre-existing inflammatory muscle wasting and bone loss, driven by pro-inflammatory pathways that are themselves directly suppressed by GCs. Understanding the interaction between these processes and their net effects on bone and muscle metabolism in vivo is critical in informing our approach to managing these patients. We investigated this in the TNF-tg model of chronic inflammation receiving the GC corticosterone (100 μg/ml) at therapeutic doses in drinking water over 3 weeks. Arthritis severity and clinical parameters were scored, front paws and tibias assessed by μCT analysis and markers of bone metabolism and inflammation determined by serum ELISA. Muscles were weighed and taken for histological analysis. Metabolic and inflammatory gene expression were determined by RT qPCR. Corticosterone potently suppressed clinical scores of inflammation and circulating pro-inflammatory cytokines including IL-6 in TNF-tg mice. A decrease in pro-inflammatory gene expression was seen in bone and muscle. Analysis of trabecular bone volume (BV/TV) and trabecular number (Tb.N) by μCT revealed that TNF-tg mice receiving corticosterone were protected from bone loss relative to controls (BV/TV: TNF-tg 2.19% ± 0.2 vs TNF-tg CORT 4.25% ± 0.2, P≤0.001; Tb.N: TNF-tg 0.0004 1/μm ± 0.00004 vs TNF-tg CORT 0.0008 1/μm ± 0.00003, P≤0.001). Whilst serum markers of bone formation (P1NP) and osteoblast gene expression were significantly suppressed in mice receiving corticosterone, an overwhelming suppression of osteoclast activity determined by serum CTX-1 and osteoclast numbers appeared to mediate the protective actions of GCs in the TNF-tg mouse (CTX-1: TNF-tg 81.6 ng/ml ± 10.7 vs TNF-tg CORT 36 ng/ml ± 2.7, P≤0.01). In contrast, in TNF-tg mice receiving corticosterone muscle wasting was further exacerbated. Muscle weights were significantly reduced in TNF-tg mice receiving corticosterone, with muscle fibre size markedly suppressed (Quad: TNF-tg 0.15 g ± 0.012 vs TNF-tg CORT 0.10 g ± 0.009, P≤0.01; TA: TNF-tg 0.06 g ± 0.005 vs TNF-tg CORT 0.03 g ± 0.004, P≤0.01). This was driven by a marked upregulation of anti-anabolic and catabolic gene expression in TNF-tg mice receiving corticosterone relative to vehicle treated controls (FBXO32; 4.7 fold, p

Career situation of first and presenting author Post-doctoral fellow. Introduction Osteocytes derive from bone-forming osteoblasts and are located deep inside the bone matrix. They are encysted in cavities (lacunae) and form dendritic extensions to develop a dense sentinel network inside the bone. Osteocytes are not passive cells. They modulate bone remodelling through regulation of both osteoclast and osteoblast activity. Immature osteocytes express the transmembrane glycoprotein podoplanin (PDPN/gp38)1 which is important for dendrite elongation and osteocyte function.2 Moreover it has been reported that PDPN expression is regulated by inflammatory cytokines including TNFα, IL-6, IL-22, TGF-β1, IFN-γ3 and is highly expressed in synovial tissues from RA patients.4 However the role of PDPN in osteocytes during inflammatory disease has not previously been investigated. Objectives In this study we investigated the effect of osteocyte-specific deletion of PDPN in the KBxN serum transfer (ST) mouse model of rheumatoid arthritis. Methods Dmp1 Cre mice were crossed with PDPNflox/flox mice to generate osteocyte specific conditional knockout mice as well as appropriate PDPNflox/fox controls. KBxN ST arthritis was used to study the effect of PDPN deletion on mouse arthritis progression. PDPN expression was assessed by immunohistochemistry. Osteoclast numbers were calculated by TRAP staining. Loss of cartilage and pannus formation was evaluated by Safranin-O and H and E staining. Standard microCT and synchrotron microCT analysis was used to assess bone density parameters, osteocyte location and bone erosions. Results PDPN is expressed in osteocytes at sites of bone erosions in inflamed joints of KBxN ST mouse model. Osteocyte-specific PDPN deletion has no effect on trabecular and cortical bone density parameters in mouse tibiae under resting conditions, as has been previously reported.2 However, loss of PDPN on osteocytes leads to significantly more bone erosions in the resolving phase of the KBxN ST model compared to non-deleted controls. Deletion of osteocyte PDPN has no effect on pannus formation, cartilage loss and osteoclast numbers. Conclusions Osteocyte-derived PDPN has been suggested as an important sensor for bone damage and this study demonstrates its bone-protective function during inflammatory arthritis. References Zhang KQ, et al. Molecular and Cellular Biology 2006;26:4539–4552. Staines KA, et al. J Cell Physiol 2017;232:3006–3019. Honma M, Minami-Hori M, Takahashi H, Iizuka HP. J Dermatol Sci 2012;65:134–140. Del Rey MJ, et al. Plos One 2014;9:e99607. Disclosure of Interest None declared.

Career situation of first and presenting author Student for a master or a PhD. Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Sclerostin loss-of-function leads to a higher bone mass and bone strength. Therefore, inhibition of sclerostin is currently considered as a promising treatment for osteoporosis. Surprisingly, in a TNFα-dependent arthritis mouse model (hTNFtg) the genetic deficiency of sclerostin caused a deterioration of disease severity. hTNFtg mice lacking sclerostin displayed enhanced and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives In order to understand the underlying mechanisms, we aimed to investigate the direct and indirect impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost-/-) mice were crossbred with hTNFtg mice to obtain sost-/-/hTNFtg mice, from which synovial fibroblasts (SF) were isolated. Cocultures of synovial fibroblasts and green fluorescent protein (GFP+) bone marrow-derived macrophages (BMM) were performed and osteoclastogenesis was analysed. Receptor activator of NF-kB ligand (RANKL) and macrophage colony stimulating factor (MCSF) expression was measured by ELISA and IL-1a expression by Western Blot. Viability of osteoclasts precursors was measured by MTT Assay. Results In cocultures of SF and GFP+ BMM, osteoclast formation was enhanced by sost-/-/hTNFtg SF compared to hTNFtg SF. Expression of RANKL and MCSF, two crucial factors for osteoclast differentiation, was not different between the genotypes. Interestingly, stimulation of wildtype BMM with conditioned media (CM) from hTNFtg or sost-/-/hTNFtg synovial fibroblasts showed no TRAP+ cells at all. However, CM supplemented with RANKL lead to an elevated number of osteoclasts using sost-/-/hTNFtg CM compared to hTNFtg CM, pointing to a secreted factor promoting osteoclast development. In this regard, the osteogenic factor IL-1α, was higher expressed in sost-/-/hTNFtg SF than in hTNFtg SF. Moreover, the treatment of cocultures with recombinant sclerostin lead to a decreased number of osteoclasts in both genotypes. Accordingly, sclerostin inhibited osteoclastogenesis in monocultures when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin mainly on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg SF promotes RANKL-mediated osteoclast differentiation, which is likely dependent on the inhibitory effect of sclerostin itself and/or on the promoting effect of higher levels of IL-1α. Disclosure of Interest None declared.

Background Due to the potent immunomodulatory and anti-inflammatory nature of glucocorticoids (GCs) they are routinely used in the treatment of inflammatory diseases such as rheumatoid arthritis. However their therapeutic potential is limited due to the prevalence of adverse side effects associated with long term GC exposure such as osteoporosis, insulin resistance and obesity. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily converts inactive GCs to their active counterparts. Previously, local reactivation of GCs by 11β-HSD1 has been shown to play a major role in the metabolic side effects associated with GC excess. Objectives We aim to assess whether local reactivation of GCs by 11β-HSD1 mediates the adverse effects of therapeutic GCs on bone. Methods Wild-type (WT) mice and transgenic mice with a global 11β-HSD1 knockout (11βKO) were treated with the active murine GC corticosterone (CORT) (100 mg/ml) for 4 weeks. Tibia and humerus bones were excised post-mortem for micro-CT analysis, gene expression analysis and three point flexure strength (TFS) tests. Serum was collected from mice for ELISA analysis of TRAcP-5b and P1NP. Results Micro-CT analysis of bone volume to tissue volume (BV/TV), trabecular thickness (TT) and trabecular number (TN) found no significant differences between untreated WT and 11βKO mice (BV/TV: WT 8.5%±0.66 vs 11βKO 7.5%±0.76, NS; TT: WT 96.5 µm±3.8 vs 11βKO 95.8 µm±6.4, NS; TN: WT 0.0009 1/µm±0.00004 vs 11βKO 0.0008 1/μm±0.00004, NS). Humerus TFS tests of WT and 11βKO animals also showed no significant differences (WT 51.2 MPa±15.1 vs 11βKO 49.2 MPa±4.9, NS). All bone parameters were decreased in CORT fed WT mice indicating the development of osteoporosis, whilst 11βKO mice were protected against many of the detrimental effects of CORT (BV/TV: WT 4.2%±0.38 vs 11βKO 7.2%±0.71, p≤0.05; TN: WT 0.0006 1/µm±0.00004 vs 11βKO 0.0009 1/µm±0.00008, p≤0.001; HBS: WT 27.1 MPa±5.6 vs 11βKO±50 MPa±5.1, p≤0.05). ELISA analysis of mouse serum showed no significant differences in the bone resorbing osteoclast marker TRAcP-5b amongst the groups, whereas analysis of the bone forming osteoblast marker P1NP revealed a significant increase in CORT fed 11βKO mice compared with CORT fed WT mice (11βKO 158.6 ng/ml±53.1 vs WT 31.4 ng/ml±7.4, p≤0.05). Gene expression of the mature osteoblast markers ALP (alkaline phosphatase) and BGLAP (osteocalcin) showed significant increases in CORT fed 11βKO animals compared to CORT fed WT animals (ALP: 11βKO 0.0074 AU ±0.0012 vs WT 0.0022 AU ±0.0007, p≤0.01; BGLAP: 11βKO 0.27 AU ±0.04 vs WT 0.02 AU ±0.01, p≤0.001). No significant differences were observed between untreated WT and 11βKO animals. Conclusions These data suggest that local reactivation of GCs by 11β-HSD1 mediates the development of glucocorticoid-induced osteoporosis by inhibiting osteoblastic bone formation. Reference [1] Morgan SA, McCabe EL, Gathercole LL, Hassan-Smith ZK, Larner DP, Bujalska IJ, et al. 11beta-HSD1 is the major regulator of the tissue-specific effects of circulating glucocorticoid excess. Proceedings of the National Academy of Sciences of the United States of America2014;111(24):E2482–91. Disclosure of Interest None declared

Summary Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation, local and systemic bone loss and a lack of compensatory bone repair. Fibroblast-like synoviocytes (FLS) are the most abundant cells of the stroma and a key population in autoimmune diseases such as RA. An increasing body of evidence suggests that these cells play not only an important role in chronic inflammation and synovial hyperplasia, but also impact bone remodelling. Under inflammatory conditions FLS release inflammatory cytokines, regulate bone destruction and formation and communicate with immune cells to control bone homeostasis. Other stromal cells, such as osteoblasts and terminally differentiated osteoblasts, termed osteocytes, are also involved in the regulation of bone homeostasis and are dysregulated during inflammation. This review highlights our current understanding of how stromal cells influence the balance between bone formation and bone destruction. Increasing our understanding of these processes is critical to enable the development of novel therapeutic strategies with which to treat bone loss in RA.

Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. The lack of sclerostin in humans and mice leads to a higher bone mass and bone strength known as sclerosteosis. Therefore, inhibition of sclerostin is currently investigated as a treatment against osteoporosis. Surprisingly, the genetic deficiency or pharmacological inhibition of sclerostin causes a deterioration of disease severity in a TNFα-dependent arthritis mouse model (hTNFtg). hTNFtg mice lacking sclerostin displayed enhanced joint inflammation, cartilage loss and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives We want to investigate the impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost -/- ) mice were crossbred with hTNFtg mice to obtain sost -/- /hTNFtg synovial fibroblasts. Co-cultures of synovial fibroblasts and wildtype bone marrow macrophages were analysed by TRAP staining. RANKL expression was measured by ELISA and cytokine expression by array analysis and Western Blot. Moreover, the influence of sclerostin on osteoclastogenesis was additionally analysed in mono-cultures. Results In our co-culture system of synovial fibroblasts and bone marrow derived macrophages, fibroblasts from sost -/- /hTNFtg mice strongly promote osteoclastogenesis in comparison to hTNFtg synovial fibroblasts. Notably, no increased expression of receptor activator of NF-kB ligand (RANKL) was detectable in sost -/- /hTNFtg fibroblasts even after stimulation with inflammatory cytokines. Interestingly, basal secretion of IL-1α, which is known to stimulate osteoclastogenesis, was higher in sost -/- /hTNFtg compared to hTNFtg fibroblasts. Accordingly, sclerostin inhibited osteoclastogenesis when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg synovial fibroblasts promotes RANKL-mediated osteoclastogenesis, which is most likely dependent on IL-1α. Disclosure of interest None declared

Introduction In patients with rheumatoid arthritis, synovial fibroblasts (SF) highly up-express the surface protein Podoplanin (PDPN) while its ligand, CLEC-2, is brought into the synovial membrane by platelets.1,2 PDPN is also up-expressed by synovial Th17 T cells from arthritic mice.3,4 Interestingly, IL-17 secretion by human Th17 T cells is triggered by direct cellular contacts with SF in a PDPN-dependent manner in vitro.5 PDPN is then an excellent biomarker and a potential regulator of joint inflammation. Despite these observations, in vivo experimental approaches that explore the therapeutic opportunity of targeting PDPN during the disease are missing. Objectives We aimed at refining the understanding of PDPN expression patterns inside the mouse synovium. We explored the therapeutic benefits from an anti-PDPN antibody in mice with auto-immune arthritis. Methods PDPN expression patterns were investigated from freshly isolated TNFa-overexpressing mouse synoviocytes by histology and FACS. The functions of PDPN expressing synoviocytes were sought by cell sorting and quantitative PCR analysis. An anti-PDPN antibody was administrated to collagen-induced arthritis (CIA) mice from day 26 post-immunisation. The CIA mice disease activity was monitored daily until day 42 and their tissues (plasma, synovium, bones, lymph nodes) analysed by ELISA, FACS, histology, micro-CT, T cell in vitro stimulation and multiplex cytokine assays. Results Joint inflammation triggered PDPN up-expression on a pro-inflammatory SF subset with concurrent accumulation of PDPN +anti inflammatory macrophages. These populations disappeared with the resolution of inflammation. Anti-PDPN treated CIA mice were efficiently protected from arthritis as demonstrated by their clinical features, their reduced leucocyte and non-haematopoietic cell accumulations into the joints as well as their attenuated bone erosion and remodelling. The T cell cytokine expression profile was normal in these mice. The anti-collagen auto-antibody plasma titres were significantly reduced in the anti-PDPN treated CIA mice compare to the control group. Conclusions We demonstrated for the first time that PDPN is expressed by pro-inflammatory and anti-inflammatory cell subsets during joint inflammation. Moreover, we are providing strong evidences that an anti-PDPN antibody restrains auto-immune arthritis in mice. This therapeutic benefit provided by the anti-PDPN antibody correlates with a reduction of circulating auto-antibody titres. This observation suggests that the anti-PDPN antibody might interfere with the micro-environment supporting B cell activation and/or plasma cell survival. References . Ekwall AK, et al. Arthritis Res. Ther2011;13:R40. . Del Rey MJ, et al. PLoS One2014;9:e0099607. . Miyamoto Y, et al. Molecular Immunology2013;54:199. . Jones GW, et al. J. Exp. Med2015;212:1793. . Noack M, et al. Arthritis Res. Ther2016;18:148. Disclosure of interest None declared

Background The type 14 family of C-type lectins has four members, two of which have very similar structures: Endosialin is expressed on stromal cells (including osteoblasts, fibroblasts and pericytes) and upregulated in rheumatoid arthritis;1 clec14a is expressed on endothelial cells and osteoblasts. Both endosialin and clec14a have been extensively studied in cancer where they are expressed on tumour pericytes and endothelial cells respectively and are required for sprouting angiogenesis.2–5 Here we have investigated the effect of genetic deletion of endosialin or clec14a on inflammatory arthritis. Materials and methods KBxN serum-transfer arthritis was used to study the effect of genetic deletion of either endosialin or clec14a on mouse arthritis progression. Staining for tartrate-resistant alkaline phosphatase was used to quantify osteoclast number. MicroCT and histology were used to assess bone erosion. Osteoblast-osteoclast co-cultures were used to identify the relative roles for each of these cell types in bone erosion. RANKL and OPG were measured at the mRNA and protein levels. Results Both endosialin and clec14a-deficient (-/-) mice show an increase in arthritis severity (increased duration of disease and increased swelling) compared to wildtype. MicroCT data demonstrated that, despite their increased paw swelling, clec14a-/- mice had levels of bone erosion similar to wildtype mice. In contrast, bone erosion in endosialin-/- mice was severe and extensive and histology identified a 3-fold increase in osteoclast number compared to wildtype. Co-culture experiments identified that endosialin-deficient osteoblasts (but not osteoclasts) stimulated osteoclastogenesis. No effect on the known osteoblast-osteoclast coupling factors RankL and OPG was identified. Conclusions Here we have identified two members of a protein family, with the same ligand but different cellular expression, which appear to protect against inflammation. However, underlying these apparently similar phenotypes we saw a severe bone-erosion defect in endosialin-/- animals that was not present in the clec14a-/- animals. In vitro experiments confirmed a role for endosialin in osteoblast-mediated osteoclastogenesis. These data demonstrate an unexpected disconnection between inflammation and bone damage and imply a novel osteoblast-osteoclast coupling mechanism that warrants further investigation. References Maia, et al. CD248 and its cytoplasmic domain. A therapeutic target for arthritis. Arthritis and Rheumatism.2010;62: 3595-606. Mura, et al. Identification and angiogenic role of the novel tumour endothelial marker CLEC14A. Oncogene, 2012;31, 293-305. Noy, et al. Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth. Oncogene, 2015;34, 5821-31. Noy, et al. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalysed by rhomboid-like 2 protein (RHBDL2). FASEB J. 2016;30, 2311-23. Naylor, et al. A Differential Role for CD248 (Endosialin) in PDGF-Mediated Skeletal Muscle Angiogenesis. PLOS One. 2014;9: e107146

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)-1β 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-1β 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.

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.

Item does not contain fulltext Sclerostin, an inhibitor of the Wnt/beta-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 TNFalpha (tumor necrosis factor alpha)-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 TNFalpha 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 TNFalpha-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 TNFalpha signaling. Sclerostin effectively blocked TNFalpha- 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 TNFalpha-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 TNFalpha-dependent comorbidities.

Myostatin is shown to directly promote osteoclast differentiation, and its inhibition improves arthritic bone loss in two mouse models. Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) 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 regeneration1,2,3,4,5. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone6,7,8,9,10,11. 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 RA12. 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.

Background and objectives Wnt-inhibitor sclerostin has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Loss of sclerostin expression results in high bone mass and bone strength in patients with sclerosteosis and sclerostin knockout mice. Therefore, antibody-mediated inhibition of sclerostin is currently evaluated for the treatment of osteoporosis in humans. Since it has been shown that sclerostin is upregulated by TNFα, we studied its impacton inflammatory arthritis using RA mouse models such as the human TNF transgenic (hTNFtg) model, the G6PI-induced arthritis model and the K/BxN serum transfer-induced arthritis model. Materials and methods Sclerostin knockout ( sost -/- ) mice were crossed with hTNFtg mice to generate sost -/ - /hTNFtg. Mice with serum transfer-induced arthritis were generated by injection of arthritogenic serum collected from K/BxN mice in sost -/- and wild type (WT) mice. Arthritis was induced in DEREG mice by immunisation with recombinant glucose-6-phosphate isomerase (G6PI). To switch to the non-remitting G6PI-induced arthritis, Foxp3 + regulatory T-cells were depleted by diphtheria toxin. hTNFtg and G6PI-induced arthritis mice were treated with a neutralising antibody against human and murine sclerostin. Clinical disease severity, bone erosion, cartilage destruction and pannus formation were evaluated by histomorphometric, x-ray and micro-CT analysis. Sclerostin expression and p38 activation was assessed by immunohistochemistry, western-blot-analysisor RT-PCR. Knockdown of LRP5 and LRP6 was performed by transfection of fibroblast-like synoviocytes (FLS) with siRNA. Results This study showed for the first time that TNFα induces sclerostin expressionin RA-FLS. Surprisingly, the lack of sclerostin and its antibody-mediated inhibition led to deterioration of RA-like disease in hTNFtgmice with enhanced pannus formation and joint destruction. Suggesting a specific role for sclerostin in TNFα signalling, inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent G6PI-induced arthritis, but ameliorated disease severity in K/BxN serum transfer-induced arthritis,in which TNFα plays only a minor role. FLS from sost -/- /hTNFtg mice displayed increased TNFα-mediated p38 activation, a key step in arthritis development. In turn, sclerostin effectively blocked TNFα-induced but not IL-1-induced activation of p38 with participation of the canonical Wnt receptor LRP6. Conclusion Collectively, these data demonstrated that sclerostin appears to have a protective function in TNF-mediated chronic inflammation.

Rheumatoid arthritis (RA) is the prototype for an inflammatory arthritis, characterised by chronic inflammation and progressive joint destruction. In this context, tumour necrosis factor α (TNFα) is a key cytokine in promoting destruction of bone by increasing the number of bone-resorbing osteoclasts and decreasing the number of bone-forming osteoblasts, thereby leading to an overall bias toward bone resorption. Myostatin (GDF-8) is a member of the transforming growth factor β family that is expressed in skeletal muscle, which is also its primary target tissue. Since several lines of evidence suggest a role for myostatin also in bone homeostasis, the authors investigated the specific contribution of myostatin to pathological bone remodelling and damage in RA. Methods In vivo, myostatin expression was studied by immunohistochemistry in synovial tissues of RA and osteoarthritis (OA) patients as well as in arthritic human TNF transgenic (hTNFtg) mice. Effects of myostatin on osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase (TRAP) staining and signalling pathways as well as expression of osteoclast-specific genes were analysed by immunoblotting. To assess the functional role of myostatin in vivo, myostatin knockout ( Mstn −/− ) mice were crossed with hTNFtg mice and the clinical severity of disease was assessed. In addition, histological changes including bone erosion and inflammation were evaluated by histomorphometric analyses. Results Myostatin was highly expressed in RA synovial tissues with most prominent staining of synovial fibroblasts. In contrast, only very few myostatin expressing cells were found in OA synovial tissues. In line with these results, strong expression of myostatin in joints of hTNFtg mice was observed, whereas only negligible staining was found in wild type animals. In vitro, myostatin dramatically accelerated receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast development. TRAP staining revealed the appearance of very large osteoclasts with a huge cytoplasmatic compartment and a very high number of nuclei already after 3 days. In contrast, the conventional setting of osteoclast differentiation (macrophage colony-stimulating factor and RANKL) did not generate osteoclasts during this short time period. In addition, myostatin-deficient BMMs showed a reduced RANKL-induced osteoclast formation, suggesting that myostatin is not only a paracrine stimulator of osteoclastogenesis. Histomorphometric analyses revealed that the lack of myostatin not only ameliorate the clinical severity of arthritis in hTNFtg mice but improve joint damage in this mouse model of RA. Mstn −/− /hTNFtg mice displayed less bone erosion and synovial inflammation compared to hTNFtg mice. Conclusions This data suggest that the expression of myostatin in arthritic joints is associated with an enhanced osteoclast formation and thus, significantly involved in bone destruction during chronic inflammatory arthritis.

Syndecan-4 is a transmembrane heparansulfate proteoglycan and one out of four members of the syndecan family described in mammals. Several studies have implicated syndecan-4 in cell-matrix adhesion, cell migration, differentiation and proliferation, but its specific function in inflammatory pathologies remains unclear. Here, we used the human tumour necrosis factor α (TNFα) transgenic (hTNFtg) mouse to analyse the expression and function of syndecan-4 in chronic destructive arthritis and answer the question whether inhibition of syndecan-4 by specific …

Background The Fibroblast Activation Protein alpha (FAPα) is an integral membrane serine protease that plays a major role in migration, wound healing, and metastasis. Based on recent studies that have implicated membrane-bound serine proteases in osteoclast migration, we studied the expression of FAPα in rheumatoid arthritis (RA) and analysed its role in osteoclast development under inflammatory conditions. Methods FAPα expression in vivo was determined by immunohistochemistry in human synovial tissues of patients with RA and osteoarthritis (OA) as well as in hind paws of tumour necrosis factor-alpha transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. In vitro expression and regulation of FAPα was analysed in differentiated osteoclasts cocultured with osteoblasts, RA or OA synovial fibroblasts (SF) by PCR. To scrutinise the role of FAPα in osteoclastogenesis, we analysed the in vitro differentiation of osteoclasts from wildtype (WT) and FAP -/- mice (Oncology Research, Boehringer Ingelheim RCV, Vienna) by TRAP staining. In order to assess the role of FAP in arthritis severity, we crossed FAP -/- and hTNFtg mice and performed TRAP staining. Results RA synovial tissues demonstrated a high expression of FAPα throughout the tissue whereas in OA samples FAPα was expressed only in the lining layer. In vitro, no expression of FAPα was found in differentiated preosteoclasts and osteoclasts, but coculture experiments showed that RASF, but not OASF or osteoblasts, induce the expression of FAPα in preosteoclasts and osteoclasts. Consistent with the selective induction of FAPα in osteoclasts by RASF, FAPα expression was detected in osteoclasts at the invasion front of the hyperplastic synovial tissues in joints of hTNFtg mice. FAP -/- mice show a severely diminished osteoclast formation compared to WT. We also found a lesser amount of osteoclasts in the hind paws of FAP -/- hTNFtg compared to hTNFtg. Conclusions The disease-dependent expression of FAPα by osteoclasts in human RA and hTNFtg mice suggests an important role of FAPα in joint destruction in RA. The selective induction of FAPα in preosteoclasts and osteoclasts by RASF indicate that FAPα may be regulated through the interaction with the pannus tissue. The fact, that under inflammatory conditions the loss of FAP led to a reduced number of osteoclasts in the hind paws and FAP deficient bone marrow derived macrophages showed a reduced osteoclast formation, suggest a role of this serine protease in macrophage/osteoclast precursor migration and differentiation.

Background In rheumatoid arthritis (RA), progressive joint destruction is a hallmark of disease and results from both increased bone resorption and the lack of repair mechanisms. tumour necrosis factor α (TNFα) contributes to both aspects of pathologic joint remodelling by increasing the number of bone-resorbing osteoclasts and decreasing the number of bone-forming osteoblasts. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway. Based on recent data that have shown increased expression of sclerostin under inflammatory conditions, the authors studied its expression in human RA and in human TNF transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. Moreover, the authors analysed the effects of sclerostin deficiency on the development and severity of the arthritis in these mice. Methods Expression of sclerostin was determined by immunohistochemistry, western blot and reverse transcriptase PCR. To assess the functional role of sclerostin in vivo, sclerostin knockout ( SOST −/− ) mice were crossed with hTNFtg mice. In addition to determining the clinical severity of disease in SOST −/− /hTNFtg and hTNFtg mice, histological changes including bone erosion, cartilage destruction and inflammation were evaluated by histomorphometric analyses. The number of osteoclasts was quantified using tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed to analyse the expression of molecules of the Wnt pathway. Results Immunohistochemistry and western blot analyses revealed a strong overexpression of sclerostin in synovial tissue of RA compared to osteoarthritis patients. Likewise, ankle joints of hTNFtg mice showed high levels of sclerostin, especially in the infiltrating pannus, whereas only negligible staining was observed in wild-type animals. In vitro, expression of sclerostin was only found in osteoblasts and osteoclasts, but could be induced in RA synovial fibroblasts by TNFα. Surprisingly, the lack of sclerostin not only increased the clinical severity of arthritis in hTNFtg mice but most dramatically accelerated joint damage in this mouse model of RA. SOST −/− /hTNFtg mice displayed significant higher bone erosion, synovial hyperplasia and osteoclast numbers compared to hTNFtg mice. Moreover, immunohistochemistry revealed higher levels of dickkopf 1 (DKK-1) and Wnt-5a in joints of SOST-deficient hTNFtg mice. Conclusions The authors hypothesise that under inflammatory conditions, higher levels of DKK-1 and Wnt-5a in joints of sclerostin-deficient arthritic mice counteract the beneficial effect of sclerostin deficiency by increasing osteoclast development through enhanced blockade of the Wnt-3a pathway by DKK-1 as well as by promoting synovial hyperplasia through Wnt-5a-mediated synoviocyte activation. These results may have an impact on the use of sclerostin inhibitors in inflammatory joint diseases.

Background Several studies have implicated syndecan-4 in cell-matrix-adhesion, cell-migration, differentiation and proliferation, but its specific function in inflammatory pathologies remains unclear. Here, the authors used the human tumour necrosis factor α (TNFα) transgenic (hTNFtg) mouse, to analyse the expression and function of syndecan-4 in chronic-destructive-arthritis and answer the question whether inhibition of syndecan-4 by specific antibodies may prevent cartilage-estruction and/or improve the phenotype after onset of the disease in this animal model of human rheumatoid arthritis. Methods Expression of syndecan-4 was investigated by immunohistochemistry in the hind-paws of 8-/12-week-old hTNFtg mice and wild type controls. In addition, synovial fibroblasts were isolated and analysed for syndecan-4-expression by reverse transcriptase PCR. For functional analyses, the authors generated blocking-antibodies against syndecan-4. To investigate their effect on TNFα mediated-destructive-arthritis, hTNFtg mice were injected with the antibodies or with IgG-control twice weekly for 4-week prophylacticly (age 4–8 weeks) and for disease treatment (age 8–12 weeks) into their hind paws. Evaluation of disease severity included clinical parameters (weight, arthritis-score, grip-strength) as well as histomorphometric analysis of toluidine-blue-stained paraffin sections. Results As seen in immunohistochemistry, there was a strong expression of syndecan-4 in the synovial membranes of hTNFtg mice, whereas only negligible staining for syndecan-4 was found in synovial tissues of wild type animals. In vitro, synovial fibroblasts isolated from hTNFtg mice showed more than 30-fold higher expression of syndecan-4 than wild type controls. Administration of the anti-syndecan-4 antibodies but not of IgG-control in pretreated 8-week-old hTNFtg mice clearly ameliorated the clinical signs of arthritis and protected the treated joints from cartilage damage. At histomorphometric analysis, this was evident for all analysed parameters but seen most prominently for area of distained cartilage. Significantly reduced cartilage damage in the anti-syndecan-4 treated hTNFtg mice was accompanied by a strinking reduction in the expression of matrix metalloproteinase 3 (MMP3). The treatment with anti-syndecan-4 in 12-week-old hTNFtg mice after onset of the arthritic disease prevented the mice from massive joint-destruction, and improved the severe cartilage-damage. The treatment also showed a clear reduction of inflammation in the paws compared to the untreated. Conclusions This findings indicate that syndecan-4 is involved prominently in fibroblast-mediated cartilage-damage in hTNFtg mice by regulating the expression of disease-relevant MMPs. More importantly, My data suggest that inhibition of syndecan-4 can not only prevent cartilage damage, but also reduces the severity after onset of the disease.

Joints affected by rheumatoid arthritis (RA) display signs of bone resorption and usually lack signs of repair which contributes to rapid and progressive loss of joint structures. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway, a key regulator of bone turnover. Since it has been shown that sclerostin is expressed under inflammatory conditions, we analysed the …

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.