E.J. Geven

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.

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.

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.

A1.09 S100-damps in IL-1RA-/-mice, a serum biomarker andin vivoimaging tool to assess joint inflammation and destruction in experimental seronegative arthritis

Background and objectives Seronegative joint diseases are characterised by the lack of autoantibodies and rheumatoid factor, potent biomarkers for disease activity in seropositive arthritis. Promising alternative biomarkers in seronegative arthritis are the Damage Associated Molecular Patterns (DAMPs), S100A8 and S100A9, proteins specifically expressed by infiltrating phagocytes. In this study we explore the biomarker potential of S100A8/S100A9 to asses joint inflammation and damage in IL-1Ra-/- mice, a mouse model for seronegative arthritis in which serum autoantibodies are not correlated to disease activity. Materials and methods Serum of IL-1Ra-/- and BALB/c mice was collected every two weeks and swelling in the hind paws was macroscopically scored. Serum levels of IL-1β, IL-6 and TNFα were measured by Luminex technology and S100A8/A9 complex levels by ELISA. Hind paws were isolated and histologically scored for cell influx, cartilage damage and bone erosion, sections were also stained for S100A9 expression. Synovial S100A8 was imaged by injection of anti-S100A8-Cy7 antibody and 24 h p.i. fluorescent images were acquired in the IVIS Lumina optical imaging system. Results Starting at week 8 up to week 16, serum levels of S100A8/A9 in IL-1Ra-/- mice were significantly increased (1640 ± 1008 ng/ml) compared to BALB/c mice (429 ± 191 ng/ml, P = 0.005) and strongly correlated to joint swelling (r = 0.740, P < 0.0001) and microscopic parameters for joint pathology (r = 0.672–0.770, P < 0.0001), in contrast to levels of IL-1β, IL-6, and TNFα. Serum S100A8/A9 levels already correlated to joint swelling (r = 0.410, P = 0.047) as early as week 8 and high serum levels at week 10 were predictive for increased joint swelling at week 16 (r = 0.563, P = 0.004). Local expression of S100A9 within the synovium correlated to joint damage and serum S100A8/A9 levels and imaging of S100A8 using specific anti-S100A8-Cy7 antibody showed a specific targeting in inflamed joints of IL-1Ra-/- mice when compared to a control isotype (P = 0.044). Conclusions High levels of serum S100A8/A9 correlate to joint inflammation and destruction in experimental seronegative arthritis and local synovial expression of S100-DAMPs can be monitored in vivo by molecular imaging. These findings underline the potential of S100-DAMPs as a serum and imaging biomarker for disease severity in seronegative arthritis.

A1.14 Synovial macrophages promote TGF-β signalling but protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidised low-density lipoproteins

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.

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

SAT0022 S100a8/a9 Is A Potent Serum and Imaging Biomarker Tool for Assessing Joint Inflammation and Destruction in Seronegative Experimental Arthritis

Background Seronegative joint diseases, including psoriatic arthritis and juvenile idiopathic arthritis, are characterized by the lack of autoantibodies, which are relevant biomarkers for predicting disease activity in rheumatoid arthritis. Promising alternative biomarkers are the Damage Associated Molecular Patterns (DAMPs), S100A8, S100A9 and the heterodimer S100A8/A9. These proteins are specifically expressed and released by infiltrating phagocytes and may therefore serve as relevant biomarkers for joint inflammation and destruction in seronegative arthritis. Objectives In this study we determined the biomarker potential of serum S100A8/A9 and in vivo imaging of synovial S100A8 to asses joint inflammation and damage in the IL-1 receptor antagonist deficient (IL-1Ra–/–) mice, a mouse model for seronegative arthritis in which serum autoantibodies are not correlated to disease activity. Methods Serum levels of S100A8/A9 and various cytokines were monitored during arthritis development in IL-1Ra–/– mice using ELISA and Luminex and were correlated to macroscopic and microscopic parameters for joint inflammation and damage. Local S100A9 expression and matrix metalloproteinase (MMP) mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF-489-Cy7, a specific tracer for activated MMPs. Results Starting at week 8, serum levels of S100A8/A9 were significantly increased (1640 ± 1008 ng/ml at week 16) in IL-1Ra–/– mice compared to WT BALB/c control mice (429 ± 191 ng/ml, P =0.005) and strongly correlated to joint swelling (r =0.766, P<0.0001), while serum levels of IL-1β, IL-6, TNFα, IL-17, IL-4 or IFN-α did not. In addition, high serum S100A8/A9 levels at week 10 were predictive for increased joint swelling at week 16 (r =0.576, P =0.0002). Next to macroscopic swelling, increased serum S100A8/A9 also correlated to microscopic cell influx (r =0.794, P <0.0001) and was reflected by local expression of S100A9 within the synovium, indicating the activated synovial lining as the source of increased serum S100A8/A9. Local expression of S100-DAMPs could also be monitored non-invasively by in vivo optical imaging using anti-S100A8-Cy7. Next to a biomarker for inflammation, S100-DAMPs may also be used for assessing joint damage. Indeed, arthritic IL-1Ra–/– mice showed increased cartilage damage (r =0.687, P <0.0001) which coincided with MMP-mediated neoepitope (VDIPEN) expression and in vivo imaging of activated MMPs. Conclusions These findings underline the potential of S100-DAMPs as a systemic and local biomarker in seronegative arthritis, not only for assessing inflammation but also joint damage. Disclosure of Interest None declared

FRI0039 Synovial Macrophages Promote TGF-Beta Signaling but Protect against Influx of S100a8/s100a9-Producing Cells Following Intra-Articular Injections of Oxidized Low-Density Lipoproteins

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<0.05], but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to a 3.1 fold increase of synovial thickening, compared with injection of PBS (P<0.01), while LDL injections did not alter synovial thickening. Protein and RNA levels of chemokines CCL2 and CCL3 were significantly upregulated in macrophage-depleted joints after oxLDL injections (6.7 fold and 4.6 fold, respectively; P<0.01). Furthermore, FCM-analysis revealed increased presence of monocytes (from 1422 ± 1105 to 3029 ± 1644 cells) and neutrophils (from 4014 ± 3511 to 12708 ± 7829 cells) in the synovium of macrophage-depleted joints after injection of oxLDL (P<0.05), which was confirmed by immunohistochemical staining. Also protein levels of S100A8/A9, markers for inflammation, were significantly increased in synovial wash-outs of oxLDL-injected joints compared with LDL (fold increase 5.6; P<0.05) or PBS (fold increase 8.3; P<0.01) injection, as was NITEGE expression (fold increase 1.92; P<0.05). Interestingly, no raise in active TGF-β was measured in these macrophage-depleted joints. 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

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.

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