W. de Munter

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 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.

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

OBJECTIVE A 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. METHOD Wild 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. RESULTS Although 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. CONCLUSIONS Increased cholesterol levels strongly elevate synovial activation and ectopic bone formation in early-stage collagenase-induced OA.

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

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.

A1.14 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

OP0210 Apolipoprotein E Aggravates Inflammation and Joint Destruction in Murine Antigen-Induced Arthritis

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by severe bone destruction which has been associated with altered lipid metabolism. Apolipoprotein E (Apo E) is a lipoprotein crucial in lipid metabolism, mainly produced by macrophages. Moreover ApoE has been recently described as an important anti-inflammatory mediator regulating innate immunity and bone turnover. Objectives In the present study we investigated the role of Apo E in inflammation and bone destruction during antigen-induced arthritis (AIA). Methods Experimental arthritis (AIA) was induced by injection of 60 μg mBSA into the right knee joint of ApoE–/– and wild type (WT) control mice previously immunized with mBSA/CFA. Joint swelling was measured by uptake of 99mTechnecium (99mTc) and expressed as a ratio of the uptake in right (injected) and the left (non injected) knee joint. Humoral immunity (mBSA antibody titer) was measured by ELISA. Joint inflammation and bone erosion were measured by histological analysis using an arbitrary scale from 0 to 3. TRAP+ cells were determined using immunohistochemistry. Results ApoE–/– mice showed significantly less joint swelling at day 1, 3 and 7 after AIA induction compared to WT controls (21%, 17%, 18% lower, respectively). Serum mBSA antibody levels (total IgG, IgG1, IgG2a and IgG2b) are comparable between the two immunized mouse strains. At day 21, histology of the knee joints showed less infiltration of inflammatory cells (25% lower) and decreased bone erosion in the ApoE–/– mice compared to WT controls (25% lower from 1.5±0.2 to 1.1 ±0.1). In line with that, ApoE–/– mice showed a reduction of the number of osteoclasts present at the area of resorption within the arthritic knee joints (36% lower from 20±4 osteoclasts/section in WT mice to 12±5 in ApoE–/– mice), as measured by image analysis of TRAP staining. Conclusions ApoE aggravates bone destruction within the knee joints during AIA by increasing influx of inflammatory cells within the synovium and elevating the number of resorbing osteoclasts on the bone surface. 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

A4.1 Synovial macrophages promote TGF-β signalling after intra-articular injections of oxidised LDL in naÏve murine knee joints, preventing production of pro-inflammatory factors S100A8/9, chemokines and aggrecanase-induced neo-epitopes

Background and objectives In previous studies we found that synovial macrophages regulate joint pathology during experimental inflammatory osteoarthritis (OA) and that high systemic levels of LDL aggravate OA joint pathology. LDL in inflamed synovium is oxidised and taken-up by macrophages, leading to an activated macrophage phenotype. In this study, we investigate whether direct injection of oxLDL into a murine knee joint induces pathology and elucidate the role of synovial macrophages in that process. Materials and methods Knee joints of C57BL/6 mice were injected five consecutive days with 1.2 mg/mL oxLDL, LDL, or an equal volume of vehicle (PBS). This same procedure was performed 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 RNA expression and protein production by synovium were determined using RT-PCR and luminex, respectively. TGF-β signalling was measured using a functional CAGA-luciferase assay. Data are depicted as mean ± standard deviation. Results LDL and oxLDL injection in naïve knee joints did not increase synovial thickening, or production of pro-inflammatory factors (IL-1β, IL-6 and S100A8/9) compared to vehicle injection. TGF-β signalling in synovial wash-outs was, however, significantly increased by 33% (from 84.7 ng/mL/g synovium ± 14.4 to 113.0 ng/mL/g synovium ± 33.3; p < 0.05). Immunohistochemistry of total knee joints showed that oxLDL injection decreased formation of aggrecanase-induced neo-epitopes (NITEGE) compared with vehicle injections, especially in areas along the bone margins that are prone to develop osteophytes (from arbitrary score 1.19 ± 0.57 to 0.33 ± 0.30; p < 0.05). Repeated injections of oxLDL in macrophage-depleted knee joints led to a 3.1 fold increase of synovial thickening, compared with injection of vehicle (p < 0.01), while LDL injections did not alter synovial thickening. 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 vehicle (fold increase 8.3; p < 0.01) injection. RNA levels of chemokines CCL2 (Mcp-1) and CCL3 (Mip-1α) were also significantly upregulated after oxLDL injections (6.7 fold and 4.6 fold, respectively; p < 0.01). No raise in TGF-β signalling was measured in macrophage-depleted joints. NITEGE expression was markedly increased (fold increase 1.92) in the synovium-cartilage contact areas after oxLDL injection (p < 0.05). Conclusions Synovial macrophages promote anabolic effects after oxLDL injections, supporting earlier studies which show increased ectopic bone formation during LDL-rich conditions in experimental osteoarthritis. In absence of synovial macrophages, however, oxLDL induces cell influx, production of pro-inflammatory mediators and aggrecanase activity.

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.