M. van den Bosch

Alarmins S100A8/S100A9 aggravate osteophyte formation in experimental osteoarthritis and predict osteophyte progression in early human symptomatic osteoarthritis

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 5u2005years. 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 5u2005years 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.

Prophylactic treatment with S100A9 inhibitor paquinimod reduces pathology in experimental collagenase-induced osteoarthritis

Objectives Alarmins 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 methods Two 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. Results Paquinimod 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). Conclusions Prophylactic 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.

FRI0049 Fc gamma receptor iv enhances bone erosion in experimental arthritis by promoting influx of pmns

Background FcγRs are involved in regulation of synovial activation and bone destruction during immune complex (IC)-mediated arthritis. The balance between activating FcγRs (FcγRI,III and IV) and inhibiting FcγRII determines synovial activation. Here we investigated the particular role of activating FcγRIV in bone erosion in IC-mediated antigen induced arthritis (AIA) by comparing FcγRI,II,III,IV-/- mice, FcγRI,II,III-/- mice and wild type controls (WT). Objectives To investigate the role of FcγRIV in bone erosion during experimental arthritis. Methods AIA was induced by injection of mBSA into knee joints of mice previously immunized with mBSA/CFA. Joint inflammation, bone destruction, number of TRAP+ osteoclasts and S100A8/A9 positive cells was determined using histology and immunohistochemistry. In vitro osteoclastogenesis was assessed using TRAP staining. Results Seven days after induction of AIA, we observed decreased inflammation and bone erosion in the knee joints of FcγRI,II,III,IV-/- mice compared to WT. The ability of bone marrow cells of FcγRI,II,III,IV-/- mice to differentiate into osteoclasts in vitro was comparable to the one of WT controls. Moreover, we observed comparable numbers of TRAP+ osteoclasts on the bone surface of FcγRI/II/III/IV-/- and WT arthritic mice, suggesting that the observed decrease in bone erosion is mainly caused by a reduced osteoclast activity, rather than decreased osteoclast number. However, in contrast to FcγRI/II/III/IV-/-, AIA induction in knee joints of FcγRI/II/III -/- resulted in increased bone erosion and inflammation compared to WT, highlighting the possible crucial role of FcγRIV in the pathology. Interestingly, the number of PMNs infiltrated in the knee joint of FcγRI/II/III-/- resulted increased, whereas it was decreased in the knee joints of FcγRI/II/III/IV-/- compared to their WT controls. This observation suggests that particularly FcγRIV is involved in regulating influx of PMNs. PMNs are potent producers of alarmins S100A8/A9 which are described to promote osteoclast activity. In line the number of S100A8/A9 positive cells in synovium was increased in FcγRI/II/III-/- while decreased in FcγRI/II/III/IV-/-, compared to their WT control. Conclusions FcγRIV promotes bone erosion in AIA by enhancing influx of PMNs within the synovium. PMNs are potent producers of S100A8/A9 which has been described to induce osteoclast activity. Disclosure of Interest None declared

High LDL levels lead to increased synovial inflammation and accelerated ectopic bone formation during experimental osteoarthritis

OBJECTIVEnA relation between osteoarthritis (OA) and increased cholesterol levels is apparent. In the present study we investigate OA pathology in apolipoprotein E (ApoE)(-)(/-) mice with and without a cholesterol-rich diet, a model for high systemic low density lipoprotein (LDL) cholesterol levels independent of weight.nnnMETHODnWild type (WT), Apoe(-)(/-), S100a9(-/-) and Apoe(-)(/-)S100a9(-/-) mice (C57BL/6 background) received a standard or cholesterol-rich diet. Experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 10 and day 36.nnnRESULTSnAlthough minimal differences in cartilage damage were found between the WT and ApoE(-)(/-) mice, increased synovial thickening was found in the latter. Thirty-six days after OA-induction, ApoE(-)(/-) mice on a standard diet showed increased ectopic bone formation, particularly at the medial collateral ligament, compared with OA in WT mice. Furthermore, a significant increase in synovial gene expression of both S100a8 and S100a9 and S100A8/S100A9 protein levels was found in ApoE(-)(/-) mice, suggesting an activated inflammatory status of synovial cells. In both ApoE(-)(/-) and WT mice, addition of a cholesterol-rich diet resulted in excessive bone formation in the medial collateral ligament at late-time-point OA. Interestingly, at the early time point, proteoglycan deposition was already significantly increased in ApoE(-)(/-) mice compared with WT mice. Mice deficient for both ApoE and S100a9 also showed increased ectopic bone formation, but not synovial activation, suggesting a role for S100-proteins in cholesterol-mediated synovial activation.nnnCONCLUSIONSnIncreased cholesterol levels strongly elevate synovial activation and ectopic bone formation in early-stage collagenase-induced OA.

Alarmin S100A9 Induces Proinflammatory and Catabolic Effects Predominantly in the M1 Macrophages of Human Osteoarthritic Synovium

Objective. The alarmins S100A8 and S100A9 have been shown to regulate synovial activation, cartilage damage, and osteophyte formation in osteoarthritis (OA). Here we investigated the effect of S100A9 on the production of proinflammatory cytokines and matrix metalloprotease (MMP) in OA synovium, granulocyte macrophage colony-stimulating factor (GM-CSF)-differentiated/macrophage colony-stimulating factor (M-CSF)-differentiated macrophages, and OA fibroblasts. Methods. We determined which cell types in the synovium produced S100A8 and S100A9. Further, the production of proinflammatory cytokines and MMP, and the activation of canonical Wnt signaling, was determined in human OA synovium, OA fibroblasts, and monocyte-derived macrophages following stimulation with S100A9. Results. We observed that S100A8 and S100A9 were mainly produced by GM-CSF–differentiated macrophages present in the synovium, and to a lesser extent by M-CSF–differentiated macrophages, but not by fibroblasts. S100A9 stimulation of OA synovial tissue increased the production of the proinflammatory cytokines interleukin (IL) 1β, IL-6, IL-8, and tumor necrosis factor-α. Additionally, various MMP were upregulated after S100A9 stimulation. Experiments to determine which cell type was responsible for these effects revealed that mainly stimulation of GM-CSF–differentiated macrophages and to a lesser extent M-CSF-differentiated macrophages with S100A9 increased the expression of these proinflammatory cytokines and MMP. In contrast, stimulation of fibroblasts with S100A9 did not affect their expression. Finally, stimulation of GM-CSF–differentiated, but not M-CSF–differentiated macrophages with S100A9 activated canonical Wnt signaling, whereas incubation of OA synovium with the S100A9 inhibitor paquinimod reduced the activation of canonical Wnt signaling. Conclusion. Predominantly mediated by M1-like macrophages, the alarmin S100A9 stimulates the production of proinflammatory and catabolic mediators and activates canonical Wnt signaling in OA synovium.

Interleukin-1 is not involved in synovial inflammation and cartilage destruction in collagenase-induced osteoarthritis

OBJECTIVEnInterleukin-1 (IL-1) is an alleged important cytokine in osteoarthritis (OA), although the exact contribution of IL-1 to joint destruction remains unclear. Here we investigated the involvement of IL-1α and IL-1β in joint pathology during collagenase-induced OA (CiOA).nnnMETHODSnCiOA was induced in wild type (WT) and IL-1αβ-/- mice. Additionally, IL-1 signaling was inhibited in WT mice with CiOA using osmotic pumps containing IL-1RA. Joint pathology was assessed using histology. Activity of cartilage-degrading enzymes was determined using antibodies against aggrecan neo-epitopes VDIPEN and NITEGE. Synovial gene expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Serum protein levels were measured with Luminex or enzyme-linked immunosorbent assay (ELISA).nnnRESULTSnSynovial IL-1β expression was strongly elevated 7 days after induction of CiOA in WT mice but decreased afterwards, whereas S100A8/A9, previously described to aggravate OA, remained elevated for 21 days. Remarkably, synovial inflammation was comparable between WT and IL-1αβ-/- mice on day 7 of CiOA. In line, synovial mRNA expression of genes involved in IL-1 signaling and inflammatory mediators was comparable between WT and IL-1αβ-/- mice, and serum levels for Keratinocyte Chemoattractant (KC)/IL-6/S100A8/S100A9/IL-10 were equal. Synovial matrix metalloproteinase (MMP)/aggrecanase expression and activity in cartilage was not different in WT and IL-1αβ-/- mice on day 7 of CiOA. Cartilage destruction on day 42 was not different between WT and IL-1αβ-/- mice, which was supported by our finding that IL-1RA treatment in WT mice with CiOA did not alter joint destruction.nnnCONCLUSIONSnIL-1α and IL-1β are not involved in synovial inflammation and cartilage destruction during CiOA, implicating that other mediators are responsible for the joint damage.

OP0146 Alarmins S100a8/S100a9 Aggravate Osteophyte Formation in Experimental Osteoarthritis and PREDICT Osteophyte Progression in Early Human Osteoarthritis in the Dutch CHECK Cohort

Background The main pathological feature of osteoarthritis (OA) is degradation of the articular cartilage. Other important hallmarks include subclinical inflammation of the synovium and ectopic formation of new bone and cartilage at the ligaments or joint margins, termed osteophytes. Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (1) (OA). Objectives In the current study we investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and if S100A8/A9 predicts osteophyte progression in early human OA. Methods OA was elicited in S100A9 -/- and wild-type C57Bl/6 mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN expression was measured on histology. Chondrogenesis was induced in murine mesenchymal stem cells (MSCs) in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA patients of the CHECK cohort study and osteophyte size measured at baseline and after 2 and 5 years. Results S100A8 and S100A9 protein levels in the synovial lining and serum coincide with osteophyte development in collagenase-induced OA (CIOA), in which synovial activation is high. Osteophyte size was drastically reduced in S100A9 -/- mice on day 21 and 42 of CIOA, in the medial collateral ligaments (58% and 93% reduction) and at medial femur and tibia (62% and 67% reduction). In contrast, osteophyte size was not reduced in S100A9 -/- mice during destabilized medial meniscus OA, in which synovial activation is scant. One explanation for the reduced osteophyte size in S100A9-/- mice may be a direct effect of S100-proteins on chondrogenesis. During in vitro chondrogenesis using murine MSCs, S100A8 caused a marked increase in MMP-3 mRNA and VDIPEN expression (as measure for MMP activity) as well as a strongly altered morphology, indicating increased remodeling allowing for larger osteophytes. Interestingly, early symptomatic OA patients of the CHECK study with osteophyte progression after two and five years had significantly elevated S100A8/A9 plasma levels at baseline, while CRP, COMP and ESR were not higher. Conclusions S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte formation in early human OA. References van Lent PL, Blom AB, Schelbergen RF, Sloetjes A, Lafeber FP, Lems WF, et al. Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis. Arthritis and rheumatism. 2012 May; 64(5):1466-1476 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3178

Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo

Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated by CRISPR/Cas9-mediated gene editing. We analyzed the migration of wildtype and knockout leukocytes in vivo by optical and nuclear imaging in mice with irritant contact dermatitis, cutaneous granuloma, experimental arthritis and myocardial infarction. Results: Transient immortalization, gene editing and in vivo imaging can be combined to analyze migratory mechanisms of murine leukocytes, even for gene deletions resulting in lethal phenotypes in mice. We reliably confirmed known migratory defects of leukocytes deficient for the adhesion molecules CD18 or VLA4α. Also, using our new method we identified a new role of the most abundant calcium-binding proteins in phagocytes and major alarmins in many inflammatory diseases, MRP8 and MRP14, for transmigration in vivo. Conclusion: We provide a combinatorial approach to rapidly characterize molecular mechanisms of leukocyte recruitment in vivo, with the potential to aid in identification of diagnostic and therapeutic targets in inflammatory pathologies.

OP0068 Wisp1 Induces Pathology in Experimental Osteoarthritis and Predicts Disease Progression in Early Osteoarthritis Patients

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

THU0455 Transcriptomics to Identify Synovial Genes and Pathways Associated with Disease Progression in A Cohort of Early Osteoarthritis Patients (CHECK)

Background If and how the synovial activation that is observed in over 50% of osteoarthritis (OA) patients contributes to irreversible joint pathology, is not known. Objectives To identify pathways that may determine progression of cartilage damage in this disease. Methods From 25 patients with knee OA that entered the CHECK Cohort study (Cohort Hip and Cohort Knee) and 6 controls, synovial biopsies were collected at baseline. CHECK is a prospective 10-year follow-up study on participants with early osteoarthritis-related complaints initiated by the Dutch Arthritis Association. Progression was determined based on change of joint space width (JSW) and osteophyte formation in radiographs, as analyzed using the KIDA (Knee Image Digital Analysis) system. Synovial samples from baseline were studied using histology and affymetrix U133-plus-2.0 chips, which were analyzed using Partek Genomics Suite software and DAVID. Results Histologically, lining thickness and synovitis were enhanced in the CHECK biopsies compared to control synovia. Next we compared synovial tissue of CHECK-patients with radiological damage with CHECK-patients without joint damage at baseline. Among the genes that were strongest associated with cartilage damage were MMP1 (18-fold), MMP3 (10-fold), and S100A8 (6-fold). Immunohistochemical staining revealed that expression of MMP-1 and MMP-3 was highest in the synovial lining layer. Enrichment analysis showed that chemotaxis, innate immune response and MMPs were significantly associated with joint damage at baseline. To determine whether any of the regulated genes and pathways were predictive for progression of joint damage between baseline and t=5 yrs, we identified 13 patients that were marked progressors and 8 non-progressors, based on JSW and osteophyte size. At baseline, neither minimum JSW nor osteophyte size differed between the groups. Approximately 200 genes were expressed more than 2-fold higher in synovium of progressors, versus non-progressors. Among these genes were genes from the wnt-signaling pathway: WISP1, FZD1, FZD8 and FZD10, whereas FRZB was downregulated. In addition, pro-inflammatory factors like IL1, IL6, S100A9 and MMP1 were increased. Macrophage markers like CD14, MHC class II genes, scavenger receptor A3 and CXCR2 were positively associated with progression. This indicates that expression of these factors may predict, or even be involved in, progression of joint damage in OA patients. Using DAVID we identified inflammatory response, macrophage differentiation, blood vessel formation, ossification and cell migration to be enriched in patients that show progression of damage 5 yrs later. Histologically, the progressors showed a higher thickness of the lining layer at baseline compared to non-progressors, 2.0 vs 1.2 respectively on an arbitrary scale from 0-3. Conclusions These data reveal an active role for the synovium in OA pathology, and identify pathways that may be involved. From histology and the expression data, it appears that presence of macrophages is associated with progression of joint damage in OA. In addition, synovial expression of wnt-signaling genes seems important in progression of damage. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2125