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Inflammatory arthritides such as rheumatoid arthritis are a major cause of disability. Pre-clinical murine models of inflammatory arthritis continue to be invaluable tools with which to identify and validate therapeutic targets and compounds. The models used are well-characterised and, whilst none truly recapitulates the human disease, they are crucial to researchers seeking to identify novel therapeutic targets and to test efficacy during preclinical trials of novel drug candidates. The arthritis parameters recorded during clinical trials and routine clinical patient care have been carefully standardised, allowing comparison between centres, trials, and treatments. Similar standardisation of scoring across in vivo models has not occurred, which makes interpretation of published results, and comparison between arthritis models, challenging. Here, we include a detailed and readily implementable arthritis scoring system, that increases the breadth of arthritis characteristics captured during experimental arthritis and supports responsive and adaptive monitoring of disease progression in murine models of inflammatory arthritis. In addition, we reference the wider ethical and experimental factors researchers should consider during the experimental design phase, with emphasis on the continued importance of replacement, reduction, and refinement of animal usage in arthritis research.

Therapeutic glucocorticoids (GCs) are powerful anti-inflammatory tools in the management of chronic inflammatory diseases such as rheumatoid arthritis (RA). However, their actions on bone in this context are complex. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a mediator of the anti-inflammatory actions of therapeutic glucocorticoids (GCs) in vivo. In this study we delineate the role of 11β-HSD1 in the effects of GC on bone during inflammatory polyarthritis. Its function was assessed in bone biopsies from patients with RA and osteoarthritis, and in primary osteoblasts and osteoclasts. Bone metabolism was assessed in the TNF-tg model of polyarthritis treated with oral GC (corticosterone), in animals with global (TNF-tg11βKO), mesenchymal (including osteoblast) (TNF-tg11βflx/tw2cre) and myeloid (including osteoclast) (TNF-tg11βflx/LysMcre) deletion. Bone parameters were assessed by micro-CT, static histomorphometry and serum metabolism markers. We observed a marked increase in 11β-HSD1 activity in bone in RA relative to osteoarthritis bone, whilst the pro-inflammatory cytokine TNFα upregulated 11β-HSD1 within osteoblasts and osteoclasts. In osteoclasts, 11β-HSD1 mediated the suppression of bone resorption by GCs. Whilst corticosterone prevented the inflammatory loss of trabecular bone in TNF-tg animals, counterparts with global deletion of 11β-HSD1 were resistant to these protective actions, characterised by increased osteoclastic bone resorption. Targeted deletion of 11β-HSD1 within osteoclasts and myeloid derived cells partially reproduced the GC resistant phenotype. These data reveal the critical role of 11β-HSD1 within bone and osteoclasts in mediating the suppression of inflammatory bone loss in response to therapeutic GCs in chronic inflammatory disease.

Bone remodelling is a highly active and dynamic process that involves the tight regulation of osteoblasts, osteoclasts, and their progenitors to allow for a balance of bone resorption and formation to be maintained. Ageing and inflammation are risk factors for the dysregulation of bone remodelling. Once the balance between bone formation and resorption is lost, bone mass becomes compromised, resulting in disorders such as osteoporosis and Paget’s disease. Key molecules in the sphingosine-1-phosphate signalling pathway have been identified for their role in regulating bone remodelling, in addition to its more recognised role in inflammatory responses. This review discusses the accumulating evidence for the different, and, in certain circumstances, opposing, roles of S1P in bone homeostasis and disease, including osteoporosis, Paget’s disease, and inflammatory bone loss. Specifically, we describe the current, often conflicting, evidence surrounding S1P function in osteoblasts, osteoclasts, and their precursors in health and disease, concluding that S1P may be an effective biomarker of bone disease and also an attractive therapeutic target for disease.

ObjectivesThe enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays a well-characterised role in the metabolism and activation of endogenous glucocorticoids (GCs). However, despite its potent upregulation at sites of inflammation, its role in peripheral metabolism and action of therapeutic GCs remains poorly understood. We investigated the contribution of 11β-HSD1 to the anti-inflammatory properties of the active GC corticosterone, administered at therapeutic doses in murine models of polyarthritis.MethodsUsing the tumour necrosis factor-tg and K/BxN serum-induced models of polyarthritis, we examined the anti-inflammatory properties of oral administration of corticosterone in animals with global, myeloid and mesenchymal targeted transgenic deletion of 11β-HSD1. Disease activity and joint inflammation were scored daily. Joint destruction and measures of local and systemic inflammation were determined by histology, micro-CT, quantitative RT-PCR, fluorescence activated cell sorting and ELISA.ResultsGlobal deletion of 11β-HSD1 resulted in a profound GC resistance in animals receiving corticosterone, characterised by persistent synovitis, joint destruction and inflammatory leucocyte infiltration. This was partially reproduced with myeloid, but not mesenchymal 11β-HSD1 deletion, where paracrine GC signalling between cell populations was shown to overcome targeted deletion of 11β-HSD1.ConclusionsWe identify an entirely novel component of therapeutic GC action, whereby following their systemic metabolism, they require peripheral reactivation and amplification by 11β-HSD1 at sites of inflammation to deliver their anti-inflammatory therapeutic effects. This study provides a novel mechanistic understanding of the anti-inflammatory properties of therapeutic GCs and their targeting to sites of inflammation in polyarthritis.

Interaction between endothelial cells and osteoblasts is essential for bone development and homeostasis. This process is mediated in large part by osteoblast angiotropism, the migration of osteoblasts alongside blood vessels, which is crucial for the homing of osteoblasts to sites of bone formation during embryogenesis and in mature bones during remodeling and repair. Specialized bone endothelial cells that form “type H” capillaries have emerged as key interaction partners of osteoblasts, regulating osteoblast differentiation and maturation and ensuring their migration towards newly forming trabecular bone areas. Recent revolutions in high-resolution imaging methodologies for bone as well as single cell and RNA sequencing technologies have enabled the identification of some of the signaling pathways and molecular interactions that underpin this regulatory relationship. Similarly, the intercellular cross talk between endothelial cells and entombed osteocytes that is essential for bone formation, repair, and maintenance are beginning to be uncovered. This is a relatively new area of research that has, until recently, been hampered by a lack of appropriate analysis tools. Now that these tools are available, greater understanding of the molecular relationships between these key cell types is expected to facilitate identification of new drug targets for diseases of bone formation and remodeling.

Introduction The Endosialin/CD248/TEM1 protein is expressed in adipose tissue and its expression increases with obesity. Recently, genetic deletion of CD248 has been shown to protect mice against atherosclerosis on a high fat diet. Objectives We investigated the effect of high fat diet feeding on visceral fat pads and circulating lipid profiles in CD248 knockout mice compared to controls. Methods From 10 weeks old, CD248−/− and +/+ mice were fed either chow (normal) diet or a high fat diet for 13 weeks. After 13 weeks the metabolic profiles and relative quantities of circulating lipid species were assessed using ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry (UHPLC–MS) with high resolution accurate mass (HRAM) capability. Results We demonstrate a specific reduction in the size of the perirenal fat pad in CD248−/− mice compared to CD248+/+, despite similar food intake. More strikingly, we identify significant, diet-dependent differences in the serum metabolic phenotypes of CD248 null compared to age and sex-matched wildtype control mice. Generalised protection from HFD-induced lipid accumulation was observed in CD248 null mice compared to wildtype, with particular reduction noted in the lysophosphatidylcholines, phosphatidylcholines, cholesterol and carnitine. Conclusions Overall these results show a clear and protective metabolic consequence of CD248 deletion in mice, implicating CD248 in lipid metabolism or trafficking and opening new avenues for further investigation using anti-CD248 targeting agents.

Adiponectin is the most abundant circulating adipokine and is primarily involved in glucose metabolism and insulin resistance. Within the bone, osteoblasts and osteoclasts express the adiponectin receptors, however, there are conflicting reports on the effects of adiponectin on bone formation and turnover. Many studies have shown a pro-osteogenic role for adiponectin in in vivo murine models and in vitro: with increased osteoblast differentiation and activity, alongside lower levels of osteoclastogenesis. However, human studies often demonstrate an inverse relationship between adiponectin concentration and bone activity. Moreover, the presence of multiple isoforms of adiponectin and multiple receptor subtypes has the potential to lead to more complex signalling and functional consequences. As such, we still do not fully understand the importance of the adiponectin signalling pathway in regulating bone homeostasis and repair in health, with age and in disease. In this review, we explore our current understanding of adiponectin bioactivity in the bone; the significance of its different isoforms; and how adiponectin biology is altered in disease. Ultimately, furthering our understanding of adiponectin regulation of bone biology is key to developing pharmacological and non-pharmacological (lifestyle) interventions that target adiponectin signalling to boost bone growth and repair in healthy ageing, following injury or in 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 factorkappa;B 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.

Skin exhibits a complex structure consisting of three predominant layers (epidermis, dermis, and hypodermis). Extensive trauma may result in the loss of these structures and poor repair, in the longer term, forming scarred tissue and associated reduction in function. Although a number of skin replacements exist, there have been no solutions that recapitulate the chemical, mechanical, and biological roles that exist within native skin. This study reports the use of suspended layer additive manufacturing to produce a continuous tri-layered implant, which closely resembles human skin. Through careful control of the bioink composition, gradients (chemical and cellular) were formed throughout the printed construct. Culture of the model demonstrated that over 21 days, the cellular components played a key role in remodeling the supporting matrix into architectures comparable with those of healthy skin. Indeed, it has been demonstrated that even at seven days post-implantation, the integration of the implant had occurred, with mobilization of the adipose tissue from the surrounding tissue into the construct itself. As such, it is believed that these implants can facilitate healing, commencing from the fascia, up toward the skin surface—a mechanism recently shown to be key within deep wounds.

Imaging complex vascular structures by X‐ray microcomputed tomography (μ‐CT) is becoming vital for research purposes in pathology of vascular diseases. Acrylic‐based polymerizable resins are widely adopted for the contrast agent to prepare pathological specimens for imaging of vascular structures. For imaging of vascular structures at higher resolution, it is promising to develop inorganic‐type contrast agents with higher X‐ray attenuation coefficient as well as low viscosity, homogeneity, minimum shrinkage, curable (gellable) for replication, and low cost. Herein, a novel inorganic sol–gel system based on concentrated colloidal dispersion of NiAl layered double hydroxide (LDH) nanoparticles is described, allowing imaging of vascular structures at high resolution. NiAl LDH acts as nanofiller and alkaline catalyst to form a silica/LDH monolithic material with homogeneity from the nanoscale. Moreover, NiAl LDH nanoparticles contribute to the strong enhancement of the X‐ray attenuation. As a proof‐of‐concept, X‐ray μ‐CT imaging of the developed contrast agent in glass capillaries and of blood vessels of a human placenta and murine liver is demonstrated.

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.

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.

This review discusses the important role fibroblasts play in the process of inflammation and the evidence that these cells may drive the persistence of inflammation. Fibroblasts are key components of the stroma normally involved in maintenance of extracellular matrix and tissue function; however, the term 'fibroblast' is used to describe a heterogeneous population of cells that vary in phenotype both between and within anatomical sites. Fibroblasts possess Toll-like receptors allowing them to respond to pathogen and damage-related signals by producing proinflammatory mediators such as IL-6, PGE

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

Background Inflammation, local joint destruction and systemic bone loss are common complications in patients with rheumatoid arthritis (RA). We have identified that localised pre-receptor activation of glucocorticoids (GC) by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is increased within sites of inflammation and surrounding tissues, such as synovium and bone. Whilst this greatly increases local bioavailability of cortisol, which supports resolution of inflammation, in chronic disease, GCs drive may drive catabolic pathways that contribute to joint destruction and systemic bone loss. Objectives To determine the contribution of 11β-HSD1 activated glucocorticoids to joint destruction and inflammatory bone loss, we crossed an 11β-HSD1 null mouse onto a transgenic murine model of chronic polyarthritis (TNF-Tg) to generate TNF-tg 11bKO mice. Methods Clinical measures of joint inflammation, mobility and behaviour were collected between 4 and 9 weeks of age. Paw swelling was determined using calliper measurements. Histology was assessed in formalin fixed sections following staining with haematoxylin and eosin, safranin O or TRAP staining. Juxta articular and systemic bone losses were measured by micro-Ct. synovitis was determined by Image J analysis of histology sections. Results 11b-HSD1 was completely knocked out within sites of inflammation in the TNF-tg 11βKO mouse. At 9 weeks, both clinical and inflammation scores were markedly exacerbated in TNF-tg 11bKO animals relative to TNF-tg counterparts (inflammation score; TNF-tg, 4.3±2.26 versus TNF-tg 11βKO , 11.08±0.86; p 11βKO , 9.0±0.66; p 11bKO mouse (synovitis size, TNFtg, 26 763 (AU) ±3200 versus TNF-tg 11βKO , 530276±3225; p 11bKO mouse (TNF-tg, BV/TV 5.7±0.75, TT 73.5±6.4, TN 0.00077±0.00004 versus TNF-tg 11βKO BV/TV 1.8±0.36, TT 7359.77±3.7, TN 0.0003±0.00005; p Conclusions This study demonstrates that rather than contributing to catabolic pathways of tissue destruction, local GC activation by 11b-HSD1 is critical in mediating the suppression inflammation, joint destruction, synovitis and inflammatory bone loss in this murine model of chronic polyarthritis. Acknowledgements We would like to thank Professor George Kollias (Hellenic Pasteur Institute, Athens, Greece) for providing the hTNFtg mice. This research was supported by the Arthritis Research UK grants (Reference: 19859 and 20843). 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 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

This review discusses the important role fibroblasts play in the process of inflammation and the evidence that these cells may drive the persistence of inflammation. Fibroblasts are key components of the stroma normally involved in maintenance of extracellular matrix and tissue function; however, the term ‘fibroblast’ is used to describe a heterogeneous population of cells that vary in phenotype both between and within anatomical sites. Fibroblasts possess Toll-like receptors allowing them to respond to pathogen and damage-related signals by producing proinflammatory mediators such as IL-6, PGE2, and GM-CSF and can produce a range of chemokines such as CXCL12, CXCL13, and CXCL8 which attract B and T lymphocytes, monocytes, and neutrophils to sites of inflammation. Interactions between leukocytes and fibroblasts can facilitate increased survival of the leukocytes and modulate phenotypes leading to differential gene expression in the presence of mediators involved in inflammation. Fibroblasts also contribute to collateral tissue damage during inflammation through the production of members of the metalloproteinase family and cathepsins and also through induction of osteoclastogenesis leading to increased bone resorption rates. In persistent diseases, fibroblasts obtain an imprinted, aggressive phenotype leading to the production of higher basal levels of proinflammatory cytokines and the ability to damage tissue in the absence of continual stimuli. This aggressive phenotype offers an attractive new target for therapeutics that could help alleviate the burden of persistent inflammation.

Background: Tissue fibrosis and microvascular rarefaction are hallmarks of progressive renal disease. CD248 is a transmembrane glycoprotein expressed by key effector cells within the stroma of fibrotic kidneys including pericytes, myofibroblasts and stromal fibroblasts. In human disease, increased expression of CD248 by stromal cells predicts progression to end-stage renal failure. We therefore, hypothesized that the genetic deletion of the CD248 gene would protect against fibrosis following kidney injury. Methods: Using the unilateral ureteral obstruction (UUO) model of renal fibrosis, we investigated the effect of genetic deletion of CD248 on post obstructive kidney fibrosis. Results: CD248 null mice were protected from fibrosis and microvascular rarefaction following UUO. Although the precise mechanism is not known, this may to be due to a stabilizing effect of pericytes with less migration and differentiation of pericytes toward a myofibroblast phenotype in CD248-/- mice. CD248-/- fibroblasts also proliferated less and deposited less collagen in vitro. Conclusion: These studies suggest that CD248 stromal cells have a pathogenic role in renal fibrosis and that targeting CD248 is effective at inhibiting both microvascular rarefaction and renal fibrosis through modulation of pericyte and stromal cell function.

Rheumatoid arthritis (RA) is an autoimmune disease in which chronic inflammation of the synovial joints can lead to destruction of cartilage and bone. Pre-clinical studies attempt to uncover the underlying causes by emulating the disease in genetically different mouse strains and characterising the nature and severity of bone shape changes as indicators of pathology. This paper presents a fully automated method for obtaining quantitative measurements of bone destruction from volumetric micro-CT images of a mouse hind paw. A statistical model of normal bone morphology derived from a training set of healthy examples serves as a template against which a given pathological sample is compared. Abnormalities in bone shapes are identified as deviations from the model statistics, characterised in terms of type (erosion / formation) and quantified in terms of severity (percentage affected bone area). The colour-coded magnitudes of the deviations superimposed on a three-dimensional rendering of the paw show at a glance the severity of malformations for the individual bones and joints. With quantitative data it is possible to derive population statistics characterising differences in bone malformations for different mouse strains and in different anatomical regions. The method was applied to data acquired from three different mouse strains. The derived quantitative indicators of bone destruction have shown agreement both with the subjective visual scores and with the previous biological findings. This suggests that pathological bone shape changes can be usefully and objectively identified as deviations from the model statistics.

Spectra computed from multispectral images of murine models of Rheumatoid Arthritis show a characteristic decrease in reflectance within the 600-800nm region which is indicative of the reduction in blood oxygenation and is consistent with hypoxia.

Transgenic tumour necrosis factor alpha (TNFα)-driven models of polyarthritis such as the TNFΔARE mouse have proven to be invaluable in delineating aspects of inflammatory disease pathophysiology in humans. Unfortunately, the onset of joint destruction and inflammation in these models represents a significant detriment to breeding management. We examined whether TNFα depleting therapy ‘infliximab’ might represent a significant refinement in routine breeding. Clinical scores of joint inflammation were assessed in TNFΔARE males receiving either infliximab (10 mg/kg) or saline by twice-weekly intraperitoneal injection. Joint histology and bone morphology were assessed by histological analysis and micro-computed tomography (CT), respectively. Analysis of breeding was examined retrospectively in TNFΔARE males prior to, and following, regular introduction of infliximab. Clinical scores of inflammation were significantly reduced in TNFΔARE males receiving infliximab (control 6.6 arbitrary units [AU] ± 0.88 versus infliximab 4.4 AU ± 1.4; P

Background During synovial inflammation, platelets and their associated microparticles escape from the synovial microvasculature and provide pro-inflammatory factors leading to the activation of synovial fibroblasts (SF) that actively contribute to joint damage.1 In patients with rheumatoid arthritis (RA), SF up-express the surface protein Podoplanin (PDPN), a ligand for CLEC-2.2,3 Although the function of PDPN is still poorly understood, recent data suggest that the PDPN/CLEC-2 axis can modulate cellular responses. Within the RA synovium, platelets are considered the sole source of CLEC-2.3 Despite these observations, clear experimental approaches that explore the role of PDPN/CLEC-2 interactions in RA pathogenesis are lacking. Materials and methods PDPN expression by freshly isolated mouse synoviocytes was measured by flow cytometry (FACS) during joint inflammation and after resolution. PDPN expressing cells were characterised by FACS and quantitative PCR. Arthritis severity in the absence of CLEC-2 was assessed in the global tamoxifen-inducible Clec1b deleting mice (ie, Rosa26-Ert2Cre x Clec1bFlox/Flox) as well as in the platelet-specific Clec1b deleting mice (ie, Pf4-Cre x Clec1bFlox/Flox). Wild-type mice with auto-immune arthritis were injected with anti-PDPN antibody. The disease was monitored by body weight, clinical scores, ankle and paw thicknesses. The synovium cellular contents from arthritic animals were analysed by FACS and cartilage damage quantified by histology while bone erosion and remodelling were monitored by MicroCT. 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. In the absence of CLEC-2, arthritis was more severe and led to more pronounced bone erosion and remodelling. Mice treated with an agonist anti-PDPN antibody were partially protected from induced auto-immune arthritis as demonstrated by their clinical features and reduced leucocyte infiltrations into the joints. Conclusions We provide the first in vivo evidence that the PDPN/CLEC-2 interaction restrains arthritis as ablation of platelet-derived CLEC-2 leads to worse arthritis, bone erosion and remodelling. Accordingly, mimicking PDPN/CLEC-2 interactions with an agonist anti-PDPN restrains auto-immune arthritis in mice. These observations suggest that platelets, known for promoting joint inflammation, also contribute to the suppression of arthritis in a CLEC-2 dependent manner. The mechanisms underlying this anti-inflammatory process are currently under investigation. References 1. Boilard E. et al. Nature Review Rheumatol. 2012;8:534–42. 2. Ekwall A.K. et al. Arthritis Res Ther. 2011;13:R40. 3. Del Rey M.J. et al. PLoS One.2014;9:e0099607.

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

Summary Inflammation is an unstable state; it either resolves or persists. Inflammatory reactions often have a propensity for specific anatomical sites. Why inflammation persists with specific tissue tropism remains obscure. Increasing evidence suggests that stromal cells which define tissue architecture are the key cells involved, and therefore make attractive therapeutic targets. Research on stromal cells in general and fibroblasts in particular has so far been hampered by a lack of fibroblast-specific cell markers. This review highlights our increasing understanding of the role of fibroblasts in inflammation, and suggests that these cells provide the cellular basis for site specific chronic inflammation.

Background We investigated two distinct synovial fibroblast populations that were located preferentially in the lining or sub-lining layers and defined by their expression of either podoplanin (PDPN) or CD248, and explored their ability to undergo self-assembly and transmigration in vivo. Methods Synovial fibroblasts (SF) were cultured in vitro and phenotypic changes following stimulation with interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 were examined. To examine the phenotype of SF in vivo, a severe combined immunodeficiency (SCID) human-mouse model of cartilage destruction was utilised. Results SF in the lining layer in rheumatoid arthritis (RA) expressed high levels of PDPN compared to the normal synovium, whereas CD248 expression was restricted to sub-lining layer cells. TNF-α or IL1 stimulation in vitro resulted in an increased expression of PDPN. In contrast, stimulation with TGF-β1 induced CD248 expression. In the SCID human-mouse model, rheumatoid SF recapitulated the expression of PDPN and CD248. Fibroblasts adjacent to cartilage expressed PDPN, and attached to, invaded, and degraded cartilage. PDPN+ CD248– SF preceded the appearance of PDPN– CD248+ cells in contralateral implants. Conclusions We have identified two distinct SF populations identified by expression of either PDPN or CD248 which are located within different anatomical compartments of the inflamed synovial membrane. These markers discriminate between SF subsets with distinct biological properties. As PDPN-expressing cells are associated with early fibroblast migration and cartilage erosion in vivo, we propose that PDPN-expressing cells may be an attractive therapeutic target in RA.

Monte Carlo modelling of light propagation through mouse paw tissues reveals that hypoxia and erythema occurring in arthritis characteristically alter the shape of reflectance spectra. Measurements from normal and arthritic mice show similar trends.

CD248 (endosialin) is a transmembrane glycoprotein that is dynamically expressed on pericytes and fibroblasts during tissue development, tumour neovascularization and inflammation. Its role in tissue remodelling is associated with increased stromal cell proliferation and migration. We show that CD248 is also uniquely expressed by human, but not mouse (C57BL/6), CD8(+) naive T cells. CD248 is found only on CD8(+) CCR7(+) CD11a(low) naive T cells and on CD8 single-positive T cells in the thymus. Transfection of the CD248 negative T-cell line MOLT-4 with CD248 cDNA surprisingly reduced cell proliferation. Knock-down of CD248 on naive CD8 T cells increased cell proliferation. These data demonstrate opposing functions for CD248 on haematopoietic (CD8(+)) versus stromal cells and suggests that CD248 helps to maintain naive CD8(+) human T cells in a quiescent state.

We describe an automated method for building a statistical model of the mouse hind limb from micro-CT data, based on articulated registration. The model was initialised by hand-labelling the constituent bones and joints of a single sample. A coarse alignment of the entire model mesh to a sample mesh was followed by consecutive registration of individual bones and their descendants down a hierarchy. Transformation parameters for subsequent bones were constrained to a subset of vertices within a frustum projecting from a terminal joint of an already registered parent bone. Samples were segmented and transformed into a common coordinate frame, and a statistical shape model was constructed. The results of ten registered samples are presented, with a mean registration error of less than 40 μm (~ 3 voxels) for all samples. The shape variation amongst the samples was extracted by PCA to create a statistical shape model. Registration of the model to three unseen normal samples gives rise to a mean registration error of 5.84 μm, in contrast to 27.18 μm for three unseen arthritic samples. This may suggest that pathological bone shape changes in models of RA are detectable as departures from the model statistics.

Background Aberrant activation of MAPK p38 signalling contributes to pathogenesis of RA 1 . MAPK p38 promotes expression of inflammatory mediators via phosphorylation of the mRNA destabilising protein Tristetraprolin (TTP) at two serine residues. These phosphorylations a) protect TTP protein from destruction by the proteasome, and b) inactivate it, resulting in stabilisation of target mRNAs such as TNF 2,3 . Dephosphorylation and activation of TTP is mediated by the phosphatase PP2A. Disruption of the murine TTP gene ( Zfp36 ) causes a severe, pervasive inflammatory syndrome, including erosive arthritis. The phenotype is largely due to increased stability of TNF mRNA and overexpression of TNF protein 4 . TTP protein is reported to be strongly expressed in the RA synovium 5 . However, the cells expressing TTP were not identified, and the relationship with inflammation was not explored. Objectives To test the hypothesis that synovial inflammation requires phosphorylation and inactivation of TTP, and that, conversely, synovial inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. Methods The expression of TTP and its relationship with p38 activation in normal and RA synovial tissue were examined by confocal microscopy. A knock-in mouse strain ( Zfp36aa/aa ) was generated, in which sites of p38-dependent phosphorylation were subsituted by non-phosphorylatable alanine residues 3 . Experimental arthritis was induced by K/BxN serum transfer. In vitro and in vivo experiments were performed to test anti-inflammatory effects of a compound that activates the phosphatase PP2A. Results TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some synovial fibroblasts, and co-localised with MAPK p38 activation. The Zfp36aa/aa mouse, in which TTP is non-phosphorylatable and constitutively active, were resistant to induction of experimental arthritis. Protection against arthritis was dependent on constitutive TTP function in both haematopoietic and non-haematopoietic compartments. In vitro , a PP2A activating compound impaired the expression of TNF in a manner dependent on the modulation of TTP function. In vivo , it reduced inflammation and prevented bone erosion in the K/BxN serum transfer model of RA. Conclusions The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammmatory treatments. References Clark, A.R. & Dean, J.L. The p38 MAPK Pathway in Rheumatoid Arthritis: A Sideways Look. Open Rheumatol J 6, 209–219 (2012). Sandler, H. & Stoecklin, G. Control of mRNA decay by phosphorylation of tristetraprolin. Biochem Soc Trans 36, 491–496 (2008). Ross, E.A., et al. Dominant Suppression of Inflammation via Targeted Mutation of the mRNA Destabilizing Protein Tristetraprolin. J Immunol 195, 265–276 (2015). Brooks, S.A. & Blackshear, P.J. Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action. Biochim Biophys Acta 1829, 666–679 (2013). Brooks, S.A., et al. Analysis of the function, expression, and subcellular distribution of human tristetraprolin. Arthritis Rheum 46, 1362–1370 (2002). Acknowledgement This work was supported by programme grant 19614 from ARUK Disclosure of Interest None declared

Background and objectives During synovial inflammation, platelets and their microparticles escape from the vasculature to fuel the synovial membrane with pro-inflammatory factors leading to the activation of synovial fibroblasts (SF) that actively contribute to joint damage. 1 Patients with rheumatoid arthritis (RA) show an up-regulation of surface protein Podoplanin (PDPN) on SF. 2,3 Although the function of PDPN is still poorly understood, recent data suggest that PDPN ligation to its ligand CLEC-2 can modulate cellular responses. Within the RA synovium, platelets are considered the sole source of CLEC-2. 3 Despite these observations, clear experimental approaches that explore the role of PDPN/CLEC-2 interactions in RA are lacking. Materials and methods PDPN expression by freshly isolated mouse synoviocytes was measured by flow cytometry during joint inflammation and after resolution. Tamoxifen-inducible Clec1b deletion mice (TIC mice) were used to assess the disease severity in absence of CLEC-2. CLEC-2 deletion was confirmed on circulating platelets by flow cytometry. Arthritis was induced by anti-collagen antibodies and LPS injections. The disease severity was monitored by body weight, clinical scores, ankle and paw thicknesses. Bone erosion and bone remodelling were studied by MicroCT scans and 3D reconstructions. Results Joint inflammation triggers PDPN up-regulation on SF and an accumulation of PDPN+ leucocytes in the synovium. These high levels of PDPN expression disappear when inflammation resolved. In absence of CLEC-2, arthritis is more severe, bone erosion and bone remodelling are more pronounced. Conclusions In this work, we provide the first in vivo evidence that PDPN/CLEC-2 interactions act to restrain arthritis by showing that ablation of CLEC-2 expression leads to worse arthritis, bone erosion and bone remodelling. These observations suggest that platelets, known for promoting joint inflammation, also contribute to the suppression of arthritis in a CLEC-2 dependent manner. The mechanisms underlying this anti-inflammatory process are currently under investigation. References Boilard E, et al . Nat Rev Rheumatol. 2012;8:534–542 Ekwall AK, et al . Arthritis Res Ther . 2011; 13 :R40 Del Rey MJ, et al . PLoS One. 2014; 9 :e0099607

Introduction: Inflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis.Methods: We examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array.Results: DKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1.Conclusions: These results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.

CD248 (also known as endosialin or tumor endothelial marker 1) is a single-pass transmembrane receptor whose ligands are reported to be extracellular matrix molecules (fibronectin and type I/IV collagen) (1). CD248 is widely expressed on mesenchymal cells in the developing embryo and is required for proliferation and migration of pericytes and fibroblasts (2). Expression of CD248 is dramatically reduced in adults, but can be up-regulated during fibrosis, inflammation, and malignancy (3-6). This distinctive temporal presentation, exclusive expression on stromal cells (2,5), structural similarity to other family members (including thrombomodulin and C1qRp), association with matrix metalloproteinase activity (1,6), and expression on mesenchymal stem/stromal cells (MSCs) (7) suggest a role for CD248 in tissue remodeling and repair. CD248 is required for the progression of certain tumors. Abdominal tumors implanted into CD248-knockout (CD248−/−) mice exhibit a reduction in growth, invasiveness, and metastasis (8). This defect is thought to be due to the role of CD248 in perivascular cells and tumor-associated fibroblasts, which support the vascularization of the tumor and facilitate malignant cell migration through the basement membrane. These findings have led to the development of anti-CD248 antibodies for use as therapeutic agents in cancer (9). In human kidney disease, the level of CD248 expression is predictive of fibrosis and disease outcome (4). CD248 is also highly expressed in the rheumatoid synovium. In particular, it has been shown to exacerbate inflammatory arthritis in mice, although the molecular basis of this phenotype has not been fully elucidated (6). CD248 has also been identified as an MSC marker (3). Given this finding, and considering that findings from in silico data-mining of an online gene expression database have shown that CD248 is very highly expressed on osteoblasts (10), we explored the function of CD248 in bone formation using mice deficient in CD248. We found that CD248 acts as a negative regulator of bone formation, since mice lacking CD248 demonstrated increased bone formation in vivo and in vitro. Moreover, we were able to identify the molecular mechanism for this bone phenotype, determined to be an impairment of platelet-derived growth factor (PDGF) signaling in osteoblasts lacking CD248.

Background It is becoming clear that platelets play a more significant role in the immune system than previously recognized 1-3 . Furthermore they are known to play a role in the induction of inflammation but the mechanisms by which they do so remain unclear. Both CLEC-2, which is highly expressed by platelets, and its ligand PDPN (gp38) are found in the joints of patients with rheumatoid arthritis (RA) 4 . However little is known about the role of CLEC-2-PDPN axis in synovial inflammation. Objectives To determine whether platelet-derived CLEC-2 induces synovial inflammation by triggering PDPN-expressing fibroblasts to adopt a pro-inflammatory phenotype. Methods Using flow cytometry we analyzed the PDPN expression in biopsies from RA patients as well as in mouse joints using the TNFΔare/+ and the anti-collagen antibodies induced arthritis (CAIA) models. We also induced arthritis in tamoxifen-induced CLEC-2 deficient mice to monitor the disease onset and resolution. After resolution, bones erosion and remodelling was assessed by micro-CT. We generated mouse PDPN Pos. and PDPN Neg. synovial fibroblasts (SF) that we co-cultured with wild-type or CLEC-2 deficient platelets. After incubation pro- and anti-inflammatory makers were measured at the mRNA (qPCR) and the protein (Luminex) levels. Results In human and mouse synovium, high levels of PDPN expression are found on SF as well as on a subset of haematopoietic cells. PDPN expression on mouse SF and myeloid cells is dynamic and depends on the level of inflammation. In vivo studies in which CLEC-2 is selectively deleted in platelets show that arthritis is more severe in absence of platelet-derived CLEC-2 suggesting that platelet-derived CLEC-2 plays a protective role in physiological conditions. However, in vitro co-culture data indicate that platelet-derived CLEC-2 induce SF to adopt a pro-inflammatory phenotype. This discrepancy between our in vitro and in vivo data might be explained by the fact that platelet-derived CLEC-2 induces myeloid cells to adopt an anti-inflammatory phenotype. Conclusions Our observations suggest that CLEC-2-PDPN interactions play a dual role in arthritis depending on whether they occur between platelets and fibroblasts or platelets and macrophages. References Benezech C, et al. CLEC-2 is required for development and maintenance of lymph nodes. Blood. 2014;123:3200-3207. Acton SE, et al. Dendritic cells control fibroblastic reticular network tension and lymph node. Nature. 2014;514:498-502 LID - 410.1038/nature13814 [doi]. Astarita JL, et al. The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture. Nat Immunol. 2014:doi: 10.1038/ni.3035. Del Rey MJ, et al. Clinicopathological correlations of podoplanin (gp38) expression in rheumatoid synovium and its potential contribution to fibroblast platelet crosstalk. PLoS One. 2014;9:e99607. Disclosure of Interest None declared

Chronic inflammatory conditions such as Rheumatoid Arthritis are characterised by over expression of pro-inflammatory factors resulting in prolonged and destructive inflammation. Dysregulation of endogenous anti-inflammatory control mechanisms contribute to this aetiology. The mRNA destabilising protein Tristetraprolin (TTP) targets pro-inflammatory transcripts for destruction, limiting the production of key inflammatory factors. Expression and activity of TTP are controlled via phosphorylation of serines 52 and 178. MAPK p38-dependent phosphorylation of these residues stabilises and inactivates TTP, promoting expression of inflammatory mediators and driving the on-phase of an inflammatory response. As MAPK p38 activity declines, accumulated TTP is activated by PP2A mediated dephosphorylation, driving the off-phase of the inflammatory response. We investigated the role of the TTP phosphorylation switch in inflammatory arthritis. Immuno-staining of human RA synovial biopsy tissue revealed abnormally high expression of TTP protein compared to control tissues, with highest expression in synovial macrophages. We hypothesise that TTP protein accumulates in a phosphorylated, inactive form, contributing to sustained expression of inflammatory mediators. To assess the therapeutic potential of targeting TTP we generated TTP aa/aa mice in which the two key serine residues in endogenous TTP are substituted. These mice produced significantly lower amounts of pro-inflammatory factors after systemic LPS challenge, due to constitutive mRNA destabilising activity of the mutant form of TTP, yet were still able to generate a protective immune response to bacterial infection. Intriguingly TTP aa/aa mice were protected from K/BxN induced inflammatory arthrtitis with no adverse histological pathology or bone remodelling compared to WT mice. Heterozygote mice, in which 20% of endogenous TTP is mutant, also demonstrated significant protection from inflammatory arthritis. Therefore, therapeutically altering the balance of activation in the total TTP pool could lead to a significant anti-inflammatory effect. An experimental reagent that activates TTP decreased clinical score, joint inflammation and bone erosions in K/BxN induced arthritis. Taken together, these data suggest that targeting of the equilibrium between phosphorylated (inactive) and dephosphorylated (active) TTP may exert therapeutic effects in arthritis without compromising immune function.

Background Rheumatoid arthritis synovial fibroblasts (RASF) migrate to distant tissue sites and damage articular cartilage. Using novel markers of RASF subsets to identify lining and sublining layer RASF, we investigated the ability of RASF to undergo self-assembly, transmigration, and cartilage degradation in vivo. Methods Healthy human cartilage was co-implanted subcutaneously into the flank of SCID mice together with RASF. On the contralateral flank, cartilage was implanted without cells. After 60 days, implants and blood were analysed. Human cells were detected with immunohistochemistry for species-specific antibodies. For in-vitro studies, RASF were isolated from patients with established rheumatoid arthritis and from healthy controls, and the expression of cellular markers was defined. Findings RASF at the ipsilateral implant differentiated into distinct fibroblast subsets in the presence of cartilage. Cells proximal to cartilage expressed markers of a lining layer phenotype (GP38, FAP, VCAM-1, and Cadherin-11). These cells attached to, invaded, and degraded cartilage. Cells more distal to cartilage expressed sub-lining layer phenotype markers including CD248 and CD90. Cells expressing CD248 and CD90 were never observed in the lining layer (proximal to cartilage) and never invaded cartilage. The development of this stromal architecture mirrored that observed in vivo in the inflamed synovial membrane. This stromal pattern of distinct differentiation of lining layer and sub-lining layer was repeated in the contralateral implant that contained only cartilage. In addition, we showed that RASF in vitro could be directed towards either a lining layer (GP38, FAP, VCAM-1, and Cadherin-11) or sublining layer phenotype (CD248 and CD90) after cytokine treatment. The lining layer, but not sub-lining cell phenotype, was associated with increased cartilage degradation in vitro. Interpretation RASF have an activated cell phenotype ex vivo. In vitro and in vivo they display plasticity with the capacity to differentiate into distinct cell subpopulations that morphologically distinguish between the lining and sublining layer of the human joint. In-vivo cell subpopulation differentiation occurs locally at the site of engraftment and is the same as the lining anatomy observed at the site of origin. This occurrence is dependent on the release of tissue factors present in the joint microenvironment after cartilage damage. This inflammatory microenvironment is required for cartilage destruction by RASF. Cellular therapies targeting RASF specific subsets are an unexplored but important therapeutic target to modulate inflammation and might provide a way to minimise joint damage in patients with rheumatoid arthritis. Funding None.

Introduction Monocytic cells play a central role in the aetiology of rheumatoid arthritis, and manipulation of the activation of these cells is an approach currently under investigation to discover new therapies for this and associated diseases. CD148 is a transmembrane tyrosine phosphatase that is highly expressed in monocytes and macrophages and, since this family of molecules plays an important role in the regulation of cell activity, CD148 is a potential target for the manipulation of macrophage activation. For any molecule to be considered a therapeutic target, it is important for it to be increased in activity or expression during disease. Methods We have investigated the expression of CD148 in two murine models of arthritis and in joints from rheumatoid arthritis (RA) patients using real-time PCR, immunohistochemistry, and studied the effects of proinflammatory stimuli on CD148 activity using biochemical assays. Results We report that CD148 mRNA is upregulated in diseased joints of mice with collagen-induced arthritis. Furthermore, we report that in mice CD148 protein is highly expressed in infiltrating monocytes of diseased joints, with a small fraction of T cells also expressing CD148. In human arthritic joints both T cells and monocytes expressed high levels of CD148, however, we show differential expression of CD148 in T cells and monocytes from normal human peripheral blood compared to peripheral blood from RA and both normal and RA synovial fluid. Finally, we show that synovial fluid from rheumatoid arthritis patients suppresses CD148 phosphatase activity. Conclusions CD148 is upregulated in macrophages and T cells in human RA samples, and its activity is enhanced by treatment with tumour necrosis factor alpha (TNFα), and reduced by synovial fluid or oxidising conditions. A greater understanding of the role of CD148 in chronic inflammation may lead to alternative therapeutic approaches to these diseases.