S. van Dalen

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

OBJECTIVE Interleukin-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). METHODS CiOA 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). RESULTS Synovial 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. CONCLUSIONS IL-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.

SAT0067 Role of nox2-derived reactive oxygen species in s100a8/a9-driven inflammatory osteoarthritis

Background Synovitis in inflammatory osteoarthritis (OA) is driven by locally released DAMPs like S100A8/A9 proteins that have been shown to enhance joint destruction. S100A8/A9 induce ROS (reactive oxygen species) release by phagocytes in OA synovium via the NADPH-oxidase 2 (NOX2)-complex. Assembly of this complex is dependent on the neutrophils cytosolic factor (Ncf1). In this complex, the NOX2 protein is responsible for ROS production. Objectives In the present study we investigated whether NOX2-derived ROS are involved in joint pathology during collagenase-induced OA (CiOA). Methods CiOA was induced in knee joints of wild type (WT) and Ncf1-deficient (Ncf1*/*) mice. Synovial gene expression of NOX2-subunits was measured with qRT-PCR. Joint pathology was assessed using histology and antibodies against aggrecan neo-epitope VDIPEN. Levels of inflammatory proteins were measured with Luminex or ELISA. Phagocytes present in synovium, blood, bone marrow and spleen were analysed with flow cytometry. Extracellular ROS production by bone marrow-derived phagocytes was measured using an Amplex Red ROS detection assay. Results CiOA induction in knee joints of WT mice caused significantly increased synovial gene expression of NOX2 subunits. On day 7 of CiOA, synovial thickening, synovial S100A8/A9 levels and synovial percentages of inflammatory macrophages, PMNs, and monocytes were comparable between WT and Ncf1*/* mice. Cartilage damage and MMP activity, as measured by VDIPEN staining, were comparable, as well as levels of inflammatory mediators in serum and phagocyte percentages in blood, bone marrow and spleen. On day 42 of CiOA, synovitis, cartilage damage, and osteophyte formation in Ncf1*/* mice were unaltered when compared to WT mice. ROS production by Ncf1*/* PMNs was completely absent but Ncf1*/* macrophages, the more predominant phagocyte involved in development of pathology during CiOA, produced ROS in similar amounts as WT macrophages. Ncf1 deficiency thus seems to exclusively affect PMNs, this surprising finding might explain the lack of differences observed between CiOA development in WT and Ncf1*/* mice. ROS production by WT and Ncf1*/* macrophages was strongly upregulated by S100A8 and almost completely inhibited by the pan-NOX inhibitor diphenyleneiodonium chloride (DPI). In order to determine whether NOX1 complexes caused the compensation responsible for Ncf1 independent ROS production, we co-stimulated Ncf1*/* macrophages with NOX1-specific inhibitor ML171. However, no ML171-induced inhibition of ROS production was observed. Conclusions Absence of PMN-derived ROS does not alter synovitis and joint pathology in S100A8/A9-driven experimental inflammatory OA. The mechanism that enables Ncf1-independent ROS production by macrophages should be further investigated. Disclosure of Interest None declared

SAT0004 Intra-articular injection of adipose-derived mesenchymal stromal cells reduce experimental oa pathology via il-1Β-mediated reallocation and enhanced phagocytosis of polymorphonuclear leucocytes

Background Injection of adipose-derived mesenchymal stromal cells (ASCs) into knee joints after induction of experimental inflammatory osteoarthritis (CiOA) reduces development of joint pathology.1 This protection is only achieved when ASCs are applied in early CiOA, which is characterised by synovitis and high levels of S100A8/A9 and IL-1β, suggesting that inflammation boosts the protective effect of ASCs.2 Objectives To examine the role of synovitis in ASC-mediated amelioration of CiOA pathology. Methods CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with haematoxylin/eosin and immunolocalization of polymorphonuclear leucocytes (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1β or S100A8/A9 was assessed with qPCR and Luminex. Migration of PMNs through transwell membranes towards ASC-conditioned medium (CM) was examined using flow cytometry. ASC-PMN co-cultures were analysed microscopically and with Luminex. Phagocytic capacity of PMNs was measured with labelled zymosan particles. Results Intra-articular injection of saline in knee joints of day 7 CiOA induced a flare already after 6 hours, characterised by particularly PMNs scattered throughout the synovium. Although ASC injection resulted in comparable numbers of PMNs, these cells however, were clustered around ASCs. IL-1β-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines KC, CXCL5, and CXCL7, whereas S100A8/A9 did not. Migration of PMNs towards CM of IL-1β-stimulated ASCs (IL-1β-CM) was significantly enhanced (2.9-fold increase) when compared to CM of non-stimulated ASCs (NS-CM). After 6 hours co-culturing PMNs with IL-1β-stimulated ASCs, the number of clustered PMNs per ASC was significantly increased. Interestingly, association of PMNs with ASCs significantly diminished the release of KC protein by ASCs (69% lower after 24 hour), and also strongly reduced the release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with CM of IL-1β-stimulated ASCs. Conclusions Local application of ASCs in inflamed CiOA knee joints results in attraction and clustering of PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of chemokine release by ASCs. This results in lowered S100A8/A9 levels and enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis and promote tissue repair. References [1] ter Huurne M, et al. Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis. Arthritis Rheum2012. Nov;64(11):3604–13. [2] Schelbergen RF, et al. Treatment efficacy of adipose-derived stem cells in experimental osteoarthritis is driven by high synovial activation and reflected by S100A8/A9 serum levels. Osteoarthritis Cartilage 2014 Aug;22(8):1158–66. Disclosure of Interest None declared

A1.12 Local experimental osteoarthritis induces systemic changes in monocyte populations regulated by S100A8/A9

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 (CCR2high) and patrolling Ly6C-low monocytes (CCR2low). 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)lowCD11bhigh(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 naïve 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.

OP0214 S100A8/A9 Produced Locally during Experimental Osteoarthritis Induces Local and Systemic Changes in Monocyte Populations

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 (CCR2low). 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)lowCD11bhigh(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 naïve 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

FRI0035 Joint Inflammation and Cartilage Destruction in Experimental Osteoarthritis Is Not Mediated by Interleukin-1

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 naïve 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

A1.30 Locally induced experimental osteoarthritis results in a system bone marrow shift towards a pro-inflammatory monocyte subpopulation

Background and objectives A significant role for inflammation during osteoarthritis (OA) is increasingly recognised, which involves the recruitment of immune cells, including monocytes, towards the inflamed synovium. In mice two functionally distinct monocyte populations are described; Ly6C-high monocytes, which express CCR2 and are considered pro-inflammatory and Ly6C-low monocytes, which express CX3CR1 and are suggested to be involved in repair processes. These monocytes arise from the bone marrow (BM) where monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives the monocyte efflux. As second tissue from which monocytes may originate is the spleen. The aim of this study is to investigate systemic effects of locally induced OA on BM and splenic monocyte subpopulations and the recruitment of these monocytes to the OA joint synovium in collagenase induced osteoarthritis (CiOA). Materials and methods CiOA was locally induced in C57Bl/6 mice by injection of collagenase in the right knee joint. Seven and 42 days after induction, mice (n = 6) were sacrificed, together with age-matched naive C57Bl/6 mice. Cells from BM, spleen, blood and knee synovial tissue were isolated and analysed by FACS. Ly6C-high monocytes were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)lowLy6Chigh and Ly6C-low monocytes as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)lowLy6Clow. BM expression of MCP-1, CCR2 and CX3CR1 mRNA was determined at day 7 and 42 by q-PCR. Results In naive synovium few monocytes were present (1012 ± 925 Ly6C-high and 621 ± 488 Ly6C-low monocytes), which is likely an artefact of residual blood. At day 7 after CiOA induction, the number of Ly6C-high and -low monocytes in the OA synovium was 420% and 300% increased, respectively, compared to naive synovium. In blood, monocyte subpopulations were not changed, but in BM, the number of Ly6C-high monocytes was 160% increased, while Ly6C-low monocytes were decreased by 170%. Furthermore, expression of MCP-1 and CCR2 was increased by 3.2 and 2.8 times, while CX3CR1 expression remained unchanged. Even at day 42 increased levels of both monocyte subpopulations were observed in the OA synovium (Ly6C-high; 280% and Ly6C-low; 220%). In the BM, no change in both monocyte subpopulations was observed anymore as well as no change in expression of MCP-1, CCR2 and CX3CR. In spleen no changes in Ly6C-high or -low monocytes were observed throughout the course of CiOA, indicating no role for splenic monocytes in the recruitment of monocytes towards the OA synovium. Conclusions These data indicate that compared to naïve synovium, increased numbers of both Ly6C-high and -low monocytes are present in the OA synovium throughout the course of CiOA, but that a systemic effect on the BM monocyte subpopulations and their efflux is only observed in the early phase of OA. In the BM a clear skew towards a pro-inflammatory monocyte subset is visible, indicating that locally induced OA may also be systemically regulated.

OP0146 Locally Administered Adipose Derived Mesenchymal Stem Cells Augment their Anti-Inflammatory Efficacy Through IL-1β Mediated Influx of Neutrophils into Knee Joints with Experimental Osteoarthritis

Background Osteoarthritis (OA) is characterized by cartilage destruction and ectopic bone formation in joints. Several studies have demonstrated that mild synovitis in early phases is conducive to joint damage. This inflammation is reflected by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). Recently we found that adipose derived mesenchymal stem cells (ASCs) exhibit immunosuppressive characteristics (1) and reduce joint pathology after local application into mouse knee joints with experimental OA (2). This anti-inflammatory effect is only perceived after intra-articular injection in early but not late stage OA, suggesting that the effect may be mediated by an inflammatory environment (1). Objectives To examine the effect of IL-1β on the immunosuppressive potency of ASCs in early experimental OA. Methods Experimental OA was induced by injection of collagenase into murine knee joints (CIOA). Total knee joints were stained with haematoxylin/eosin and the PMN specific antibody NIMPR14. ASCs were isolated from adipose tissue and stimulated for 24 hours with IL-1β or interferon-gamma (IFN-γ). Gene expression in synovium and stimulated cells were analyzed using qPCR. Protein levels of chemokines and cytokines were measured in the supernatant and washouts using Luminex. ASCs were co-cultured with MACS isolated bone marrow (BM-) PMNs and analyzed using histology, qPCR and Luminex. Results Injection of ASCs into day 7 CIOA knee joints (when synovitis and IL-1β levels are highest) caused a strong influx of immune cells into the joint cavity shortly after injection (6 hours), which had largely vanished after 24 hours. The attraction of particularly PMN-like cells was confirmed by immunohistochemistry. Synovial gene expression of neutrophil attracting chemokines KC, CXCL5, and CXCL7 were increased. In line with this, IL-1β stimulated ASCs injected in naive knee joints also resulted in a strong influx of PMN-like cells. IL-1β and IFN-γ (as a positive control) stimulation of ASCs in vitro strongly increased gene expression of KC, CXCL5, and CXCL7 as well as protein levels of KC. Finally, we co-cultured ASCs with BM-PMNs in the presence of IL-1β or IFN-γ. After 3 hours, a clear clustering of neutrophils around ASCs was observed which significantly diminished protein levels of KC (-69% after 24h; -76% after 48h). Conclusions Local injection of ASCs into a day 7 CIOA knee-joint expressing low levels of IL-1β causes attraction of PMN-like cells. In vitro, IL-1β stimulated ASCs show an increase in chemokine expression, leading to attraction of and clustering with neutrophils which ultimately results in significantly decreased levels of pro-inflammatory factors like KC. The anti-inflammatory effect of locally applied ASCs into OA joints showing synovitis may be triggered by IL-1β and attraction of PMN-like cells. References ter Huurne M et al. Arthritis Rheum. 64(11):3604-13, 2012. Schelbergen R et al. Osteoarthritis Cartilage. 22(8):1158-66, 2014. Acknowledgements This research was supported by the Dutch Arthritis Association (RF 12-2-405). Disclosure of Interest None declared

THU0026 A Systemic Shift Towards a Pro-Inflammatory Monocyte Subpopulation in Bone Marrow Upon Locally Induced Experimental Osteoarthritis

Background A significant role for inflammation during osteoarthritis (OA) is increasingly recognized, which involves the recruitment of immune cells, including monocytes, to the inflamed synovium. Monocytes have been shown to regulate joint destruction in OA, by the release of pro-inflammatory molecules, like S100-DAMPs. In mice two functionally distinct monocyte populations are described; Ly6C-high monocytes, which express high levels of CCR2 and are considered pro-inflammatory and Ly6C-low monocytes, which express low levels of CCR2 but high levels of CX3CR1 and are suggested to be involved in repair processes. Both monocytes arise from the bone marrow (BM) where monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives the monocyte efflux via binding with CCR2. Objectives The objective of this study is to investigate systemic effects of locally induced OA on BM monocyte subpopulations and the recruitment of these monocytes to the OA joint synovium in collagenase induced osteoarthritis (CiOA). Methods CiOA was induced in C57BL/6 mice by injection of collagenase in the right knee joint. Seven and 42 days after induction, mice (n=6) were sacrificed, together with age-matched naive C57BL/6 mice. Cells from BM, blood and knee synovial tissue were isolated and analyzed by FACS. Ly6C-high monocytes were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)lowLy6Chigh cells and Ly6C-low monocytes as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)lowLy6Clow cells. BM expression of MCP-1, CCR2 and CX3CR1 mRNA was determined at day 7 and 42 by q-PCR. Results In naive synovium few monocytes were present (645±146 Ly6C-high and 231±32 Ly6C-low monocytes per synovium). At day 7 after CiOA induction, the number of Ly6C-high and -low monocytes in the OA synovium was increased by 398% and 299%, respectively, compared to naive synovium. In blood, monocyte subpopulations were not changed, but in BM, a clear shift in the subpopulations was observed; the number of Ly6C-high monocytes was increased by 164%, while Ly6C-low monocytes were decreased 1.7 fold. Furthermore, expression of MCP-1 and CCR2 was increased 3.2 and 2.8 fold respectively, while CX3CR1 expression remained unchanged. When measured at day 42, levels of both monocyte subpopulations were still significantly increased in the OA synovium (Ly6C-high; 510% and Ly6C-low; 222%), while in the BM, no change in monocyte subpopulations and expression of MCP-1, CCR2 and CX3CR1 was observed anymore. Conclusions These data show that, compared to naive synovium, both Ly6C-high and -low monocytes are increased in the OA synovium throughout the course of CiOA, but that particularly the pro-inflammatory Ly6C-high monocytes are increased. This indicates an important role for Ly6C-high monocytes in late stage OA pathology, possibly by increased release of S100-DAMPs. Indeed serum S100A8/A9 levels are elevated for a longer period than other pro-inflammatory molecules during CiOA. A systemic effect of CiOA on the BM monocyte subpopulations is only observed in the early phase of OA, here a clear skew towards the pro-inflammatory monocyte subset is visible, indicating that locally induced OA may have systemic effects on BM monocytosis. Disclosure of Interest None declared