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Contains fulltext : 165863.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (OA). In the current study, we investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and whether S100A8/A9 predicts osteophyte progression in early human OA. METHODS: OA was elicited in S100A9-/- mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN neoepitope was measured histologically. Chondrogenesis was induced in murine mesenchymal stem cells in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA participants of the Cohort Hip and Cohort Knee (CHECK) cohort study and osteophytes measured after 2 and 5 years. RESULTS: Osteophyte size was drastically reduced in S100A9-/- mice in ligaments and at medial femur and tibia on days 21 and 42 of collagenase-induced OA, in which synovial activation is high. In contrast, osteophyte size was not reduced in S100A9-/- mice during destabilised medial meniscus OA, in which synovial activation is scant. S100A8 increased expression and activation of matrix metalloproteinases during micromass chondrogenesis, thereby possibly increasing cartilage matrix remodelling allowing for larger osteophytes. Interestingly, early symptomatic OA participants of the CHECK study with osteophyte progression after 2 and 5 years had elevated S100A8/A9 plasma levels at baseline, while C-reactive protein, erythrocyte sedimentation rate and cartilage oligomeric matrix protein were not elevated at baseline. CONCLUSIONS: S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte progression in early symptomatic human OA.

Background:The pattern of changes in bone microarchitecture and mineralisation are distinctly different in osteoarthritis (OA) and osteoporosis (OP). However, the pathogenesis of OA is closely related with OP, making subchondral bone a promising target for OA treatment [1]. A detailed comparison of subchondral bone in OA and OP may help understand the relationship of the two diseases.Objectives:To carry out a comprehensive analysis of regional and compartmental variations in subchondral bone architecture and mineralisation in OA and OP.Methods:Femoral heads were collected from patients undergoing hip arthroplasty surgeries for hip OA (N=16) or osteoporotic fracture (N=7). For OP group, osteochondral plugs were collected from fixed sites: anterior, posterior and superior. For OA group, an optimised sampling procedure, based on a new macroscopic grading method and modified OARSI microscopic grading system, was used to collect plugs from regions with varying severity of cartilage degradation. Plugs were scanned by micro CT (voxel size 4.88µm). Regions of interest for cortical plate (Ct) and trabecular bone (Tb) were segmented from reconstructed images using semi-automatic approach. Densitometric (tissue and bone mineral density: TMD and BMD) and architectural parameters (cortical plate thickness (Ct.Th), trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), etc.) were measured using commercially available software. Unmatched inter-group regional comparisons were made between OA microscopic grades (1 to 4) and OP. Matched intra-sample regional analysis was made between ‘mild’ (Grade 1 and/or 2) and ‘severe’ (Grade 3 and/or 4) OA. TMD was also subjected to paired comparison between cortical (Ct.TMD) and trabecular (Tb.TMD) compartments. Correlations between densitometric and architectural parameters were also explored.Results:Regional analysis showed that Tb.TMD in OA Grade 3 and 4 was significantly lower than in OP and Grade 1 and 2, while Tb.TMD in OP was not significantly different from OA Grade 1 and 2 (Fig 1A, F). Ct.TMD in OA Grade 4 was significantly lower than in OP, but no difference was found in other comparisons (Fig 1B, G). For BMD of trabecular bone (Tb.BMD) and architectural parameters including BV/TV and Ct.Th, values for OA Grade 3 and/or 4 were significantly higher than OP and Grade 1 and/or 2, but the difference between OP and Grade 1 and 2 was not significant (Fig 1C-E, H-J). Compartmental analysis showed that Ct.TMD was significantly lower than Tb.TMD in all groups (Table I). Tb.TMD was inversely correlated with Tb.BMD and BV/TV in both OA and OP; Ct.TMD and Tb.TMD were inversely associated with Ct.Th and Tb.Th respectively in OA (Table II).Conclusion:In both OA and OP, material density (TMD) of cortical plate was lower than trabecular bone. In hip OA, densitometric and architectural changes of subchondral cortical and trabecular bone were related to severity of cartilage degradation. In OA trabecular bone, the decrease in material density was compensated by increased bone volume, leading to higher apparent density (BMD); while in OP, loss of bone volume was correlated with, but not compensated by increased mineralisation, leading to lower apparent density.References:[1]Castaneda, S., et al. Biochem. Pharmacol., 2012Table 1.Compartmental comparison of TMDOPOA Grade1234Ct.TMD1.19±0.071.18±0.091.15±0.061.16±0.061.12±0.07Tb.TMD1.34±0.06****1.37±0.10****1.33±0.07****1.22±0.06*1.19±0.09*Table 2.Correlation analysisOPOAPR2SlopePR2SlopeTb.TMD - BV/TV< 0.00010.69-0.01< 0.00010.84-0.01Tb.BMD - BV/TV< 0.00010.950.02< 0.00010.960.01Tb.BMD - Tb.TMD< 0.00010.60-1.25< 0.00010.81-1.37Ct.TMD - Ct.Thns0.00090.19-0.33Tb.TMD - Tb.Thns< 0.00010.55-1.97Acknowledgments:China Scholarship CouncilDisclosure of Interests:None declared

ObjectivesAlarmins S100A8/A9 regulate pathology in experimental osteoarthritis (OA). Paquinimod is an immunomodulatory compound preventing S100A9 binding to TLR-4. We investigated the effect of paquinimod on experimental OA and human OA synovium.Materials and methodsTwo OA mouse models differing in level of synovial activation were treated prophylactic with paquinimod. Synovial thickening, osteophyte size and cartilage damage were measured histologically, using an arbitrary score, adapted Pritzker OARSI score or imaging software, respectively. Human OA synovia were stimulated with S100A9, with or without paquinimod.ResultsPaquinimod treatment of collagenase-induced OA (CIOA) resulted in significantly reduced synovial thickening (57%), osteophyte size at the medial femur (66%) and cruciate ligaments (67%) and cartilage damage at the medial tibia (47%) and femur (75%; n=7, untreated n=6). In contrast, paquinimod did not reduce osteophyte size and reduced cartilage damage at one location only in destabilised medial meniscus, an OA model with considerably lower synovial activation compared with CIOA. In human OA synovium, paquinimod blocked proinflammatory (interleukin (IL)-6, IL-8, tumour necrosis factor-α) and catabolic (matrix metalloproteinases 1 and 3) factors induced by S100A9 (n=5).ConclusionsProphylactic treatment of paquinimod reduces synovial activation, osteophyte formation and cartilage damage in experimental OA with high synovial activation (CIOA) and ameliorates pathological effects of S100A9 in OA synovium ex vivo.

ObjectivesIn osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse and studied the effect of elevated BMP2 expression on healthy joints and joints with experimental OA.MethodsWe cloned a lentivirus with BMP2 controlled by a tet-responsive element and transfected embryos of mice containing a collagen type II driven cre-recombinase and floxed rtTA to gain a mouse expressing BMP2 solely in chondrocytes and only upon dox exposure (Col2-rtTA-TRE-BMP2). Mice were treated with dox to induce elevated BMP2 expression. In addition, experimental OA was induced (destabilisation of the medial meniscus model) with or without dox supplementation and knee joints were isolated for histology.ResultsDox treatment resulted in chondrocyte-specific upregulation of BMP2 and severely aggravated formation of osteophytes in experimental OA but not in control mice. Moreover, elevated BMP2 levels did not result in alterations in articular cartilage of young healthy mice, although BMP2-exposure did increase VDIPEN expression in the articular cartilage. Strikingly, despite apparent changes in knee joint morphology due to formation of large osteophytes there were no detectible differences in articular cartilage: none with regard to structural damage nor in Safranin O staining intensity when comparing destabilisation of the medial meniscus with or without dox exposure.ConclusionsOur data show that chondrocyte-specific elevation of BMP2 levels does not alter the course of cartilage damage in an OA model in young mice but results in severe aggravation of osteophyte formation.

ObjectiveTo develop recommendations on monitoring for adverse events (AEs) of low-dose glucocorticoid (GC) therapy (≤7.5 mg prednisone or equivalent daily) in clinical trials and daily practice.MethodsLiterature was searched for articles containing information on incidence and monitoring of GC-related AEs using PubMed, EMBASE and Cochrane databases. Second, the authors searched for broad accepted guidelines on the monitoring of certain AEs (eg, WHO guidelines on screening for diabetes). Available data were summarised and discussed among experts (rheumatologists and patients) of the EULAR Task Force to decide which potential AEs should be monitored, how and at which interval.ResultsData on monitoring proved to be scarce; most articles were focused on therapeutic effects of GCs, not on occurrence and monitoring of AEs. Most recommendations had to be based on consensus. Those for clinical trials aimed at getting insights into incidence, prevalence and clinical relevance of AEs to create a comprehensive and valid AE-profile of GC therapy. The set of AEs to monitor is therefore more extensive, and often consists of assessments at baseline and at end of trials. Recommendations for daily practice are meant to protect patients from real dangers, which can be prevented or treated. Standard care monitoring needs NOT be extended for patients on low-dose GC therapy, except for osteoporosis (follow national guidelines), and baseline assessments of ankle edema, fasting blood glucose and risk factors for glaucoma.ConclusionGiven the incompleteness of literature data, consensus-based recommendations on monitoring for GC-related AEs were created, separately for daily practice and clinical trials.

Objectives:To assess the human complement inhibitor C4b-binding protein (C4BP) for treatment of arthritis.Methods:We have used two mouse models of rheumatoid arthritis (RA) to assess the therapeutic effect of C4BP on different phases of arthritis, the collagen antibody-induced arthritis (CAIA), an acute antibody-induced disease and the collagen-induced arthritis (CIA), which carries the full complexity of arthritis.Results:Purified human C4BP injected intraperitoneally alleviated CAIA significantly in a manner similar to cobra venom factor that depletes complement due to massive activation. Furthermore, C4BP was injected before and after the disease development into CIA mice. In the former case, the disease onset was delayed and in the latter, the severity of the disease was reduced in animals treated with C4BP. However, C4BP did not affect the anti-CII antibody synthesis. C4BP present in mouse sera decreased activity of the classical but not the alternative pathway of the complement system when these were assessed in a fluid phase. However, C4BP was efficiently inhibiting the alternative pathway when present on the activating surface. Taken together, the disease ameliorating effect of C4BP appears to be related to inhibition of both pathways of complement.Conclusions:Although human C4BP was cleared relatively fast from the circulation and was only moderately affecting complement activity, its effect on the disease severity was substantial, suggesting that minor alterations in complement activity can have significant therapeutic value in RA.

Item does not contain fulltext BACKGROUND: Recently, it has been found that collagen type II arthritis susceptible mouse strains are hyperreactive to immune complexes (ICs), locally deposited into their knee joints. OBJECTIVE: To investigate whether this strain specific knee joint hyperreactivity is related to a disturbed regulation of activatory and inhibitory FcgammaR on their macrophages before and after stimulation with ICs. METHODS: Macrophages from collagen induced arthritis susceptible strains (DBA/1 and B10.RIII) and non-susceptible strains (C57BL/6 and BALB/c) were compared. FcgammaR levels on macrophages were detected at protein level by flow cytometric analysis and at mRNA level by reverse transcriptase-polymerase chain reaction. Macrophages were stimulated with ICs, and production of cytokines and enzymes was measured at different times. RESULTS: On synovial and peritoneal macrophages of DBA/1 mice a higher basal FcgammaRII and III expression was found, which was skewed towards the activating FcgammaRIII. In B10.RIII macrophages, however, FcgammaRIII levels were much lower. Regulation of FcgammaR mRNA levels in macrophages was tested after stimulation with ICs for one and three days. DBA/1 and B10.RIII macrophages showed a prolonged up regulation of activating FcgammaRI and III, whereas the inhibiting FcgammaRII was significantly down regulated compared with non-susceptible strains. In line with this, DBA/1 and B10.RIII macrophages showed a higher interleukin 1 (IL1) and matrix metalloproteinase (MMP) production after IC exposure, whereas IL6 production was significantly reduced. CONCLUSIONS: This study indicates that macrophages derived from collagen type II arthritis susceptible mice show a disregulated FcgammaR expression before, and even more clearly, after activation by ICs involved in inflammation and cartilage degradation, resulting in prolonged expression of activatory FcgammaRI and III, down regulation of inhibitory FcgammaRII and increased release of IL1 and MMP.

Background Many osteoarthritis (OA) patients show synovial activation, which is suggested to be involved in joint destruction. Previously, we found strong upregulation of Wnt2b and Wnt16 in the synovium of two experimental OA models. In addition, we found strongly increased Wisp1 expression, a protein induced by canonical Wnt signaling. Objectives To determine whether there is a relation between WISP1 expression and progression of OA in a cohort study of patients with early symptomatic OA. Furthermore, we will elucidate whether WISP1 plays a role during experimental OA by inducing OA models in wild type (WT) and Wisp1 –/– mice. Methods Microarray analysis was performed on synovium from patients enrolled in the CHECK study, initiated to follow the progression of early symptomatic OA patients. Expression data were correlated with progression of OA (defined as a decrease in joint space width of ≥1mm and progression of osteophyte formation of ≥4x in size) between baseline and the five-year follow-up measurement. Human end-stage OA synovium was stimulated with WISP1. Joint pathology in WT or Wisp1 –/– mice was assessed by histology after induction of collagenase-induced OA (CIOA), destabilization of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT) experimental models of OA. The aggrecan neoepitope NITEGE was visualized using immunohistochemistry. Gene expression in the synovial tissue was evaluated using qRT-PCR. Results Microarray analysis of synovial tissue from patients in the CHECK cohort showed significantly increased WISP1 expression at baseline in OA progressors versus non-progressors. To determine the mechanism of how WISP1 might be involved in OA pathology, we stimulated human OA synovium with WISP1. This increased the expression of MMP2/3/9/13 and ADAMTS4/5 . Next, we determined the in vivo role of WISP1. First, we found that spontaneous cartilage damage was not different between WT and Wisp1 –/– mice at 3, 6 and 12 months of age. Next, we assessed joint pathology 42 days after induction of CIOA in WT and Wisp1 –/– mice. Cartilage damage was significantly decreased in the tibio-femoral joints of the Wisp1 –/– mice as compared with the WT controls. In line with the CIOA, we found significantly decreased cartilage degeneration in the Wisp1 –/– mice in the DMM and ACLT at day 56 after induction. In addition we found decreased expression of Mmp3 / 9 and the aggrecanases Adamts4/5 in the synovium, 7 days after induction of CIOA in Wisp1 –/– mice, in line with the increased expression of these factors after stimulation of human OA synovium with WISP1. Furthermore, the expression of the protease inhibitor Timp1 was decreased in the Wisp1 –/– mice, whereas the expression of Timp3 , an important inhibitor of ADAMTS-4/5, was unaffected. Finally, the protease activity in the cartilage, as assessed by the staining of the neoepitope NITEGE, was decreased in the Wisp1 –/– mice. Conclusions Increased WISP1 expression may play an important role in OA pathology via increased synovial MMP/ADAMTS expression. Furthermore, because of the tight regulation and complexity of Wnt signaling and its role in many physiological processes, targeting WISP1 may more specifically target the OA-related pathological events, while minimizing interference with physiological processes. Disclosure of Interest None declared

Background In previous studies we found that synovial macrophages regulate joint pathology during experimental osteoarthritis (OA) and, more recently, we found that high systemic levels of low-density lipoproteins (LDL) aggravate joint pathology during experimental OA with synovitis. LDL in inflamed synovium is oxidized and taken-up by macrophages, leading to an activated macrophage phenotype. Objectives In this study, we investigate whether injection of oxidized LDL directly into a murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Methods Synovium was obtained from end-stage OA patients and stained for apolipoprotein B (APOB). Murine knee joints were injected five consecutive days with oxLDL, LDL, or an equal volume of vehicle (PBS). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry and flow cytometry (FCM) analysis, and RNA expression and protein production by synovium were determined using RT-PCR and luminex, respectively. Aggrecanase activity was measured using NITEGE-staining and active TGF-β was measured using a functional CAGA-luciferase assay. Data are depicted as mean ± standard deviation. Results Synovial macrophages and fibroblasts of end-stage OA patients showed extensive accumulation of APOB, the main protein present in LDL and oxLDL. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs by 28% [from 16562 ± 2326 relative light units (RLU) to 21151 ± 3823 RLU; P P P P P P P Conclusions Synovial macrophages promote anabolic processes after intra-articular oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity, in combination with increased influx of monocytes and neutrophils. Disclosure of Interest None declared

Background The cause for chronic pain in OA is largely unknown. Over 50% of osteoarthritis (OA) patients show synovial inflammation, even at early stages of the disease. However, if and how this synovial activation contributes to joint pathology and pain, is not known. Objectives To identify pathways that predict progression of cartilage damage and osteophyte (OP) formation in OA. In addition, we identified association between gene expression and pain in a cross-sectional approach. Methods From 25 patients with knee OA that entered the CHECK Cohort study (Cohort Hip and Cohort Knee) synovial biopsies were collected at baseline. CHECK is a prospective 10-year follow-up study on patients with early osteoarthritis-related complaints initiated by the Dutch Arthritis Foundation. Progression over 5 years was determined radiographically based on change of joint space width (JSW) and OP size in these radiographs. Pain was assessed at baseline by the WOMAC pain questionnaire. Synovial samples from baseline were studied using histology and microarray (affymetrix U133-plus-2.0), and Functional Annotation Clustering (FAC). Results We identified patients that were marked progressors or non-progressors, either based on JSW (respectively n=13 vs n=8) or OP size (respectively n=10 vs n=11) at these time points. Among the genes that were differentially expressed by OP progressors were MMP1, 2, 3, 9 and -14, whereas in JSW-progressors only MMP1 was differentially expressed. In the group of JSW-progressors, macrophage markers like CD14, S100A8, S100A9, MHC class II genes, and CXCR2 were positively associated with progression. This indicates that expression of these factors predict progression of cartilage damage in OA patients. The OP-progressors showed an increase of most of these markers, but to a far lesser extent. Using FAC we identified inflammatory response, macrophage differentiation, blood vessel formation, ossification and cell migration to be enriched in JSW-progressors. Blood vessel formation, wound healing, ossification and cell proliferation were enriched in OP-progressors. Histologically, in the JSW-progressors the lining layer was thicker and the cellularity was higher in the sublining compared to non-progressors, and compared to OP progressors. Both JSW-progressors and osteophyte-progressors showed increased vascularisation compared to non-progressors. At baseline, FAC analysis pointed out that dendrite formation (p=0.036) response to heatshock/stress (p=0.015) and voltage gated ion transport (p=0.013) were annotation clusters that were related to pain. Surprisingly, no positive relation was found between inflammatory mediators and pain. In line with this, no relation between histological inflammation or lining thickness and pain was found. Conclusions We found evidence for a difference in underlying processes in the synovium regarding progression of cartilage damage and progression of osteophyte formation. The presence of macrophages, especially in the lining layer, is associated with progression of cartilage damage, whereas synovial expression of MMPs correlated to progression of osteophyte formation. Pain was associated with several nerve fiber related processes. Disclosure of Interest None declared

Background Previous studies have reported medication adherence ranging from 49.5% to 98.5% in patients with rheumatoid arthritis (RA) 1. Adherence to TNFi has been estimated to be 59%2. Non-adherence has been associated with poorer health outcomes, such as physical disability and increased need for health services. Objectives The aim of the study was to analyse adherence to self-injectable biologic disease-modifying antirheumatic drugs (bDMARDs), conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and glucocorticoids in Finnish RA patients using their first bDMARDs. Methods RA patients starting their first bDMARD in 2007–2011 were identified from the National Register for Biologic Treatment in Finland (ROB-FIN). Restriction to the patients with available baseline data and at least 360 days follow-up with their first bDMARD was made. Data on prescribed medication was recorded in ROB-FIN while medication claims were retrieved from the register of the Social Insurance Institution of Finland. The proportion of days covered (PDC) during a 360-day period was calculated to define adherence. Patient was considered adherent if PDC was ≥80%. Results Fifty-two percent (n=424) of the 823 patients remained on the first bDMARD and were followed for at least 360 days. Forty-three percent of them (205) were adherent to all prescribed anti-rheumatic medication. CsDMARDs were prescribed to 372 patients of which 53% (196) were adherent while 5% (20) claimed no cdDMARD at all. Methotrexate was the most common csDMARD and was prescribed to 281 patients. The proportions of patients adherent to methotrexate and patients who claimed no methotrexate at all were 60% (170) and 9% (26), respectively. Self-injectable bDMARDs were prescribed to 319 of which 59% (187) were adherent and 8% (25) did not claim any of the prescribed bDMARD. Mean PDC for csDMARDs, methotrexate and self-injectable bDMARDs were 73%, 74% and 74%, respectively. Conclusions Less than half of the RA patients using bDMARDs were adherent to their antirheumatic medication. However, the current data measures only the medication claims instead of actual use. Moreover, the high proportion of patients lost to follow-up may cause bias to the results. References Pasma A, van9t Spijker A, Hazes JMW, et al. Factors associated with adherence to pharmaceutical treatment for rheumatoid arthritis patients: a systematic review. Semin Arthritis Rheum. United States; 2013 Aug;43(1):18–28. Fidder HH, Singendonk MMJ, van der Have M, et al. Low rates of adherence for tumor necrosis factor-alpha inhibitors in Crohn9s disease and rheumatoid arthritis: results of a systematic review. World J Gastroenterol. China; 2013 Jul;19(27):4344–50. Acknowledgement ROB-FIN has received restricted financial support from Abbvie, Roche, MSD and Pfizer. Disclosure of Interest None declared

Background Osteoarthritis (OA) is a degenerative disease of the joint characterized by severe cartilage destruction, with a putative role for synovial macrophages. Up to 50% of the patients also show low grade joint inflammation reflected by a thickened synovial lining and elevated release of macrophage-derived mediators like interleukin-1 (IL-1) and S100A8/A9. The deteriorating role of S100A8/A9 in OA has been studied extensively (1), but the contribution of IL-1 to OA pathology is still unclear. IL-1 mediates cartilage destruction by degrading existing proteoglycans through stimulating degradative enzyme production, and by inhibiting new formation of proteoglycans. However, treatment of OA patients with IL-1 inhibitors has so far been disappointing. Objectives To investigate the role of IL-1α/β in synovitis and cartilage destruction during experimental collagenase-induced OA (CiOA). Methods Experimental OA was induced by intra-articular injection of collagenase into WT and IL-1αβ –/– mice. At day 7 and 42 after CiOA induction, total knee joints were stained with haematoxylin/eosin (H&E) to score synovial activation (arbitrary score from 0–3). Cartilage destruction was determined in knee sections stained with safranin O/fast green using a modified Pritzker score. Gene expression in inflamed synovium was analyzed using qPCR with primers for several cytokines. Serum protein levels of inflammatory mediators were measured with Luminex. Results At early stage (day 7) of CiOA, gene expression of IL-1 within inflamed synovium of knee joints was elevated 65 times compared to synovium of naive control knees. However in later stages (day 21 and 42), IL-1 expression levels were reduced to basal levels. This is in contrast to pro-inflammatory mediators like S100A8 and S100A9 which remained elevated at day 21 and 42 (30–35-fold and 66–69-fold, respectively). Histological examination of knee joints showed a moderate synovitis at day 7 which waned thereafter but was still significant at day 21 and 42. Remarkably, synovial inflammation at day 7 in IL-1αβ –/– mice was not different from WT controls (2.89±0.15 vs. 2.71±0.44), suggesting that IL-1 does not aggravate synovitis. Absence of IL-1α/β had no effect on the synovial gene expression levels of pro-inflammatory factors KC, S100A8 and S100A9. IL-6 mRNA levels, however, were significantly decreased in the synovium of IL-1αβ –/– mice (3-fold reduction). The lack of IL-1α/β also did not affect gene expression of anti-inflammatory cytokines IL-10, TGFβ and iNOS. Moreover, serum protein levels of KC, IL-6, MCP-1, IL-10 and S100A8/A9 at day 7 of CiOA in IL-1αβ –/– mice were not different compared to WT mice. Cartilage destruction in CiOA clearly aggravated over time, but we found no significant differences between IL-1αβ –/– mice and WT controls. No difference in cartilage damage was measured at day 42 of CiOA when compared to WT (medial femur (95.3±42.0 vs. 65.5±54.5), medial tibia (84.5±36.6 vs. 55.1±38.9), lateral femur (69.6±43.9 vs. 60.8±35.8), and lateral tibia (49.8±26.3 vs. 55.4±17.1)). Conclusions IL-1 does not affect synovial inflammation and cartilage destruction during collagenase-induced osteoarthritis, implicating that other macrophage-derived mediators such as S100A8/A9 are responsible for the joint damage. References van Lent et al. 2012 Acknowledgement This research was supported by the Dutch Arthritis Association. Disclosure of Interest None declared

Background The etiology of osteoarthritis (OA) is multi-factorial, and is mainly driven by an activated synovium wherein inflammation plays an important role. In response to pro-inflammatory cytokines, monocytes can be recruited from the bone marrow (BM) to the site of inflammation. In mice, two functionally distinct monocyte populations are described: pro-inflammatory Ly6C-high monocytes (C-C chemokine receptor type 2 (CCR2) high ) and patrolling Ly6C-low monocytes (CCR2 low) . In the BM, monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives monocyte efflux via binding with CCR2. Objectives The objectives of our study are to investigate the systemic effects of locally induced OA on BM monocyte populations and their recruitment to the OA joint in collagenase induced OA (CiOA), and the role of the alarmins S100A8/A9 in that. Methods CiOA was induced by unilateral-articular collagenase-injection in C57BL/6 mice. At day 7, 21 and 42, mice were sacrificed together with age-matched naive mice (n=6/group), and synovial mRNA expression of several pro-inflammatory cytokines were measured. During CiOA and control conditions, the absolute amount of cells in the BM per femur was measured and the mRNA expression of BM MCP-1 and CCR2 was determined. Cells from BM, blood and synovial tissue were isolated and analyzed by FACS. Monocyte subsets were identified as (B220/CD90/CD49b/NK1.1/Ly6G) low CD11b high (F4/80/MHCII/CD11c) low and further distinguished by their Ly6C expression. In addition, we investigated synovitis in S100A9 –/– mice during CiOA. Results Expression of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, S100A8 and S100A9 in the synovium were increased at day 7, but only expression of S100A8 and S100A9 remained high until day 21. Local induction of CiOA resulted in systemic effects within the BM as shown by the decrease in cell numbers at day 7 and 21 (15% and 14% respectively). Concurrently, the expression of MCP1 at day 7 in BM was increased (3.3-fold), suggesting an increased efflux of BM-cells. Relative number of BM-Ly6C-high monocytes was increased at day 7 (164%), being reflected by increased CCR2 expression (2.8-fold). This suggests a specific regulation of BM Ly6C-high monocytosis and recruitment during CiOA and emphasizes the systemic effects following CiOA. During the course of CiOA increased numbers of Ly6C-high monocytes were also observed in the synovium: 398% at day 7 and 510% at day 42, compared to naive mice. These effects may be caused by the sustained S100A8/A9 levels; we therefore investigated synovitis in S100A9 –/– mice during CiOA. The number of cell layers and cell influx in the synovium of S100A9 –/– mice was decreased compared to C57BL/6 mice. Conclusions Local induction of OA induces systemic release of BM-derived cells and increased Ly6C-high monocyte populations systemically and locally in the synovium, a process that may be regulated by the sustained release of S100A8/A9 from the synovium. Disclosure of Interest None declared

Background Efficacy of Tumor Necrosis Factor (TNF)-inhibitors in treatment of rheumatoid arthritis (RA) measured in randomized controlled trials (RCTs) may not be fully generalizable to routine care owing to the stringent inclusion criteria [1]. Objectives The objective of this study was to observe the effectiveness of TNF-inhibitors in Finland and to assess the real-world patients9 potential eligibility for the RCTs. Methods RA patients starting a TNF-inhibitor as their first, second or third biologic treatment between 2004 and 2014 were identified from the National Register for Biologic Treatment in Finland (ROB-FIN), which is an observational cohort study. Effectiveness was measured using the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) response criteria and as the proportion of patients reaching Disease Activity Score 28 (DAS28) remission. Logistic regression was employed to identify any predictors of achieving EULAR response. The patients9 baseline characteristics were compared against the inclusion criteria of 28 RCTs identified from two systematic reviews [2,3]. Treatment responses measured in the RCTs were compared to those achieved by routine care patients either eligible or not eligible for the same RCTs. Results Of the 4067 TNF-inhibitor treatment periods, 2259 periods from 1899 patients with valid baseline visit were included in this study. The included patients were predominantly female (73%) with a median age of 55. Adalimumab, etanercept infliximab, golimumab and certolizumab pegol accounted for 41%, 40%, 9.7%, 5.1% and 4.6% of the medication periods, respectively. Moderate and good EULAR responses at 6 months were achieved by 69% and 40% of the users of the first biologic drug, respectively. ACR20, ACR50 and ACR70 responses were reached by 47%, 25% and 13%, respectively. DAS28 remission was reached by 45%. Concomitant use of methotrexate (MTX) was associated with higher odds of achieving at least moderate EULAR response (odds ratio 1.8, 95% confidence interval 1.1– 3.0) as compared to TNF-inhibitor monotherapy. Golimumab and certolizumab pegol were possibly more effective than infliximab, depending on whether the discontinuers were included in the analysis or not. Only 3.8% to 50% of the patients would have been eligible for the included RCTs. The eligible patients had generally better ACR50 responses as compared to the non-eligible ones. Conclusions Different TNF-inhibitors were mostly equipotent in routine clinical practice, but the use of MTX co-therapy had a major influence on treatment response. The efficacy of TNF-inhibitors measured in RCTs cannot be directly generalized into Finnish routine healthcare. References Zink A et al. Arthritis Rheum 2006;54:3399–407. Nam JL et al. Ann Rheum Dis 2010;69:976–86. Nam JL et al. Ann Rheum Dis 2014;73:516–28. Acknowledgement ROB-FIN has received financial support from the following pharmaceutical companies: Abbvie, MSD, Pfizer and Roche Disclosure of Interest None declared

Inflammation is increasingly recognised to be involved in osteoarthritis (OA) pathology. In response to pro-inflammatory cytokines, monocytes can be recruited from the bone marrow (BM) where monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives monocyte efflux via binding with the C-C chemokine receptor type 2 (CCR2). In mice, two functionally distinct monocyte populations are described: pro-inflammatory Ly6C-high monocytes (CCR2 high) and patrolling Ly6C-low monocytes (CCR2 low) . The objectives of our study are to investigate the systemic effects of locally induced OA on BM monocyte populations and their recruitment to the OA joint in collagenase induced OA (CiOA), and the role of the alarmins S100A8/A9 in that. CiOA was induced in C57BL/6 mice by unilateral-articular collagenase-injection and were sacrificed at day 7, 21 and 42, together with age-matched naive mice (n = 6/group), and synovial mRNA expression of several pro-inflammatory cytokines were measured. During CiOA, the absolute amount of cells in the BM per femur was measured and the mRNA expression of BM MCP-1 and CCR2 was determined. Cells from BM, blood and synovial tissue were isolated and analysed by FACS. Monocyte subsets were identified as (B220/CD90/CD49b/NK1.1/Ly6G) low CD11b high (F4/80/MHCII/CD11c) low and further distinguished by their Ly6C expression. In addition, we investigated synovitis in S100A9 -/- mice during CiOA. Synovial expression of IL-1β, IL-6, TNF-α, S100A8 and S100A9 was increased at day 7, but only expression of S100A8 and S100A9 remained high until day 21. Local induction of CiOA resulted in systemic effects within the BM showing decreased cell numbers at day 7 and 21 (15% and 14% respectively). Concurrently, the expression of MCP1 at day7 in BM was increased 3.3-fold, suggesting increased BM-efflux. Relative number of BM-Ly6C-high monocytes was increased at day 7 (164%), being reflected by increased CCR2 expression (2.8-fold). This suggests a specific regulation of BM Ly6C-high monocytosis and recruitment during CiOA. During the course of CiOA increased number of Ly6C-high monocytes were observed in the synovium: 398% at day 7 and 510% at day 42, compared to naive mice. These effects may be caused by the sustained S100A8/A9 levels, we therefore investigated synovitis in S100A9 -/- mice during CiOA. The number of cell layers and cell influx in the synovium of S100A9 -/- mice was decreased compared to C57BL/6 mice Local induction of OA induces systemic release of BM-derived cells and increased Ly6C-high monocyte populations systemically and locally in the synovium, a process that may be regulated by the sustained release of S100A8/A9 from the synovium. Disclosure of Interest None declared.

Background An important role for canonical Wnt signalling in osteoarthritis pathology has been established. However, the role of Wnt1-induced secreted protein-1 (WISP1), which we found increased in experimental and human OA, remains to be elucidated. Here, we determined the relation between WISP1 expression and OA-progression in early OA patients. Additionally, we determined how WISP1 might be involved in OA pathology using experimental OA models. Methods WISP1 expression levels, determined with microarray analysis on synovial biopsies obtained from patients enrolled in the CHECK study of early OA patients, were coupled to OA-progression. Progression was defined as ≥1mm decrease in joint space width and ≥4x increased osteophyte size after 5 years. Human OA synovium was stimulated with WISP1. Joint pathology was assessed after induction of experimental OA models (DMM, ACLT and CIOA) in WT or WISP1-deficient mice. Immunohistochemical staining was used to visualise NITEGE aggrecan neoepitopes resulting from protease activity. Gene expression was evaluated using qRT-PCR. Results WISP1 expression at baseline was significantly higher in CHECK patients that showed OA-progression between baseline and the five-year follow-up measurement compared to non-progressors. Stimulation of human OA synovium with WISP1 increased the expression of the proteases MMP-2/-3/-9/-13 and ADAMTS-4/-5. To further pinpoint the role of WISP1 during OA, we induced three experimental OA models with different characteristics in WT and WISP1-deficient mice. We observed significantly decreased cartilage damage in the tibio-femoral joints of WISP1-deficient mice, compared with the WT controls in all models. Accordingly, we found decreased expression of MMP-3/-9 and the aggrecanases ADAMTS-4 and -5 in synovial tissue from WISP1-deficient mice. Moreover, we observed decreased expression of IL-1 and IL-6, which have the potential to induce protease expression, but not of the anti-inflammatory cytokine IL-10 in the WISP1-deficient mice. Finally, the protease activity in the cartilage, as assessed by the staining of the neoepitope NITEGE, was decreased in the WISP1-deficient mice. Conclusions Increased WISP1 expression in OA joints may play an important role in OA pathology via increased expression of MMPs and ADAMTSs in the synovium. Specific downregulation of WISP1 may avoid undesired side-effects that are likely to occur after targeting upstream Wnt signalling.

Background and objectives Osteoarthritis (OA) is a degenerative joint disease characterised by severe cartilage destruction, with a putative role for synovial macrophages. Recently it has become clear that over 50% of the patients also show low grade joint inflammation reflected by a thickened synovial lining and elevated production of cytokines like interleukin-1 (IL-1)by different cell types in the synovium. IL-1 mediates cartilage destruction by degrading existing proteoglycans and inhibiting new formation ofproteoglycans by stimulating degradative enzyme production. However, treatment of OA patients with IL-1 inhibitors has so far been disappointing. In this study we investigated the role of IL-1α/β in synovitis and cartilage destruction during experimental collagenase-induced OA (CiOA). Materials and methods Experimental OA was induced by intra-articular injection of collagenase into WT and IL-1αβ -/- mice. At day 7 after CiOA induction, total knee joints were stained with haematoxylin/eosin (H&E) to score synovial activation (arbitrary score from 0–3). Cartilage destruction at day 7 and 42 after induction of CiOA was determined in knee sections stained with safranin O/fast green using a modified Pritzker score. Gene expression in inflamed synovium was analysed using qPCR with primers for several pro- and anti-inflammatory cytokines. Results Synovitis in IL-1αβ -/- mice at day 7 of CiOAwas not different from WT controls (2.9 ± 0.2 and 2.7 ± 0.4 respectively). Absence of IL-1α/β had no effect on synovial mRNA levels of pro-inflammatory mediators KC, S100A8 and S100A9. IL-6 mRNA levels, however, were significantly down-regulated in the synovium of IL-1αβ -/- mice (3-fold reduction). The lack of IL-1α/β also did not affect gene expression of anti-inflammatory cytokines IL-10, TGFβ and iNOS. Cartilage destruction in CiOA clearly aggravated over time. Cartilage lesions in IL-1αβ -/- mice were not significantly different from WT controls at day 7. Also no difference in cartilage damage was measured at day 42 of CiOA when compared to WT (at the medial femur (95.3 ± 42.0 vs. 65.5 ± 54.5), medial tibia (84.5 ± 36.6 vs. 55.1 ± 38.9), lateral femur (69.6 ± 43.9 vs. 60.8 ± 35.8), and lateral tibia (49.8 ± 26.3 vs. 55.4 ± 17.1)). Conclusions IL-1β does not affect synovial inflammation nor cartilage destruction during collagenase-induced osteoarthritis, implicating that other macrophage-derived mediators are responsible for the joint damage.

Background and objectives LDL in inflamed synovium is oxidised and taken-up by macrophages, leading to an activated macrophage phenotype. In this study, we investigate whether injection of oxLDL directly into a murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Materials and Methods Synovium was obtained from end-stage OA patients. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (PBS). This procedure was repeated in mice depleted of synovial lining macrophages by intraarticular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. Results Synovial macrophages and fibroblast of OA patients showed extensive accumulation apolipoprotein B, the main protein present in LDL and oxLDL. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of specifically oxLDL in macrophage-depleted knee joints led to increased synovial thickening. Furthermore, protein and RNA levels of CCL2 and CCL3 were significantly upregulated in macrophage-depleted joints after oxLDL injections and FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in active TGF-β was measured in macrophage-depleted joints. Conclusions Synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity in combination with increased influx of monocytes and neutrophils.