R.F. Schelbergen

Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis.

OBJECTIVEnIn experimental collagenase-induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose-derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA.nnnMETHODSnASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint.nnnRESULTSnASCs were injected into the knee joints of mice 7 days after the induction of collagenase-induced OA. On day 1, green fluorescent protein-labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42.nnnCONCLUSIONnThese findings indicate that a single injection of ASCs into the knee joints of mice with early-stage collagenase-induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.

Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4.

OBJECTIVEnS100A8 and S100A9 are two Ca(2+) binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators.nnnMETHODSnUsing immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metalloproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcription-polymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific inhibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carboxylated glycans were used.nnnRESULTSnIn cartilage from OA patients, the expression of S100A8 and S100A9 protein close to chondrocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimulation of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favoring cartilage breakdown. Blocking TLR-4, but not carboxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pronounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression.nnnCONCLUSIONnS100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chondrocytes to S100 stimulation.

Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis

OBJECTIVEnTo investigate whether alarmins S100A8 and S100A9 are involved in mediating cartilage destruction during murine and human osteoarthritis (OA).nnnMETHODSnTwo different murine models of OA that differed in terms of synovial activation were compared. Cartilage destruction was measured histologically. Synovial biopsy and serum samples from OA patients were derived from the Cohort Hip and Cohort Knee (CHECK) patients with symptomatic early OA. Expression of mediators in the synovium was measured by reverse transcription-polymerase chain reaction analysis and immunolocalization.nnnRESULTSnIn collagenase-induced OA, which showed marked synovial activation, interleukin-1β was expressed at significant levels only during the early stages of disease, whereas S100A8 and S100A9 expression remained high for a prolonged period of time (up to day 21 after induction). In S100A9-knockout mice, we found a major impact of S100A8 and S100A9 on synovial activation (62% inhibition) and OA cartilage destruction (45-73% inhibition) as compared to wild-type controls. In contrast, in the surgically induced destabilized medial meniscus model, in which synovial involvement is scant, we found no role of S100A8 and S100A9 in the focal OA cartilage destruction. Examination of arthroscopic synovial biopsy samples from patients in the early symptomatic OA CHECK cohort revealed substantial levels of S100A8 and S100A9 messenger RNA and protein, which correlated significantly with synovial lining thickness, cellularity in the subintima, and joint destruction. Levels of S100A8/A9 serum protein were significantly enhanced (19%) at baseline in patients who had pronounced progression of joint destruction after 2 years.nnnCONCLUSIONnOur data suggest that the S100A8 and S100A9 proteins are crucially involved in synovial activation and cartilage destruction during OA and that high levels may predict joint destruction in humans with OA.

S100A8 causes a shift toward expression of activatory Fcγ receptors on macrophages via toll‐like receptor 4 and regulates Fcγ receptor expression in synovium during chronic experimental arthritis

OBJECTIVEnThe levels of both Fcγ receptor (FcγR) and the alarmins S100A8 and S100A9 are correlated with the development and progression of cartilage destruction during antigen-induced arthritis (AIA). This study was undertaken to study the active involvement of S100A8, S100A9, and S100A8/S100A9 in FcγR regulation in murine macrophages and synovium during AIA.nnnMETHODSnRecombinant murine S100A8 (rS100A8) was injected into normal mouse knee joints, and the synovium was isolated for analysis of FcγR messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR). Macrophages, including bone marrow macrophages derived from Toll-like receptor 4-deficient (TLR-4(-/-)) mice, and polymorphonuclear cells (PMNs) were stimulated with S100 proteins, and levels of FcγR mRNA and protein were measured using RT-PCR and fluorescence-activated cell sorting analyses. AIA was induced in the knee joints of S100A9-deficient (S100A9(-/-)) mice, compared with wild-type (WT) controls, and the extent of cartilage destruction was determined using immunohistochemical analysis.nnnRESULTSnIntraarticular injection of rS100A8 into the knee joints of normal mice caused a strong up-regulation of mRNA levels of activating FcγRI (64-fold increase) and FcγRIV (256-fold increase) in the synovium. Stimulation of macrophages with rS100A8 led to significant up-regulation of mRNA and protein levels of FcγRI and FcγRIV, but not FcγRIII, while the effects of S100A9 or S100A8/S100A9 complexes were less potent. Stimulation of PMNs (32Dcl3 cell line) with S100 proteins had no effect on FcγR expression. Up-regulation of FcγRI and FcγRIV was abrogated in rS100A8-stimulated macrophages from TLR-4(-/-) mice, indicating that the induction of FcγR expression by S100A8 is mediated by TLR-4. FcγR expression in the inflamed synovium of S100A9(-/-) mice was significantly lower on day 14 after arthritis induction when compared with WT controls, and these findings correlated with reduced severity of matrix metalloproteinase-mediated cartilage destruction.nnnCONCLUSIONnS100A8 is a strong promoter of activating FcγRI and FcγRIV in macrophages through the activation of TLR-4, and acts as a regulator of FcγR expression in inflamed synovium in chronic experimental arthritis.

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.

Alarmins S100A8 and S100A9 stimulate production of pro-inflammatory cytokines in M2 macrophages without changing their M2 membrane phenotype

Background Synovial activation, which plays an important role in a subgroup of osteoarthritis (OA) patients, is regulated by M1/M2 macrophage subpopulations: pro-inflammatory M1-macrophages and regulatory M2-macrophages. Alarmins S100A8 and S100A9 are found in high concentrations in synovial fluid of rheumatoid arthritis (RA) and OA patients and are correlated to inflammation and cartilage destruction. In the current study, the authors investigated the production of S100A8 and/or S100A9 by M1- and M2-macrophages and whether S100A8 and/or S100A9 could stimulate M2-macrophages, giving them a more pro-inflammatory character. Methods Monocytes were isolated from blood of healthy volunteers by isolating peripheral blood mononuclear cells (PBMCs) using a Ficoll-gradient followed by isolating CD14+ monocytes using magnetic-activated cell sorting (MACS). CD14+ monocytes were differentiated into M1- or M2-macrophages by 6 day stimulation with either GM-CSF or M-CSF, respectively. mRNA levels of S100A8 and S100A9 in M1 and M2 macrophages were measured with RT-qPCR. Intracellular protein production was determined using FACS analysis and S100A8/A9 secretion was measured using an ELISA.M1 and M2 macrophages were stimulated with human recombinant S100A8 and S100A9. M1 and M2 markers (IL-1β, tumour necrosis factor α (TNFα), IL-6 and CD163, CD206 respectively) were measured using RT-qPCR, FACS and/or Luminex. Results S100A8 and S100A9 had a significantly higher mRNA expression (65- and 2.5-fold respectively, n=10) in human monocyte derived M1-macrophages compared to M2. Also, the levels of intracellular S100A8 and S100A9 were higher in M1 than in M2 macrophages (4.3-fold and 6.6-fold higher respectively, n=6). The secreted protein level of the S100A8/A9 heterodimer was elevated from 55.5 ng/ml in M2 to 102.8 ng/ml in M1 macrophages (n=10). Stimulation of human monocyte derived M2-macrophages for 24 h with S100A8 and S100A9 showed a marked upregulation of several pro-inflammatory markers: IL-1β expression was increased by S100A8 and S100A9 on mRNA level (33- and 16-fold, n=10). At the protein level IL-6 and TNFα were strongly upregulated by S100A8 71- and 146-fold and by S100A9 93- and 121-fold, respectively (n=6). Interestingly however, S100A8 or S100A9 stimulation did not alter the level of M2 membrane markers CD163 and CD206, both at mRNA as well as protein level. Conclusions Stimulation of M2 macrophages with S100A8 and S100A9, produced in high amounts by M1- macrophages, upregulates pro-inflammatory cytokines IL-1β, IL-6 and TNFα while M2 membrane markers CD163 and CD206 remain unchanged. S100A8 and S100A9 production during OA could therefore contribute to synovial activation by stimulating the M2 macrophage towards a more pro-inflammatory phenotype.

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

A9.10 Synovial activation drives anti-inflammatory effects of adipose-derived stem cells after local administration in experimental oa which is reflected by s100a8/a9 levels in the serum

Backgound and Objectives Synovitis is evident in a substantial subpopulation of patients with early osteoarthritis (OA) and has been associated with development of pathophysiology. Recently we have shown that adipose-derived stem cells (ASC) inhibit joint destruction after local application to knee joints with experimental OA. In the current study we explored the effect of synovitis on the immunomodulatory capacity of ASCs after local administration in two experimental OA models differing in synovitis. Material and Methods ASCs were isolated from fat surrounding the popliteal lymph nodes. ASCs were injected into knee joints after induction of collagenase-induced OA (CiOA) characterised by synovitis and surgically induced DMM model in which synovitis is scant. Synovial activation, chondrogenesis in ligaments and osteophytes were measured using histology. Cytokines in synovial washouts and serum were determined using Luminex. Active TGFβ was measured using the CAGAluc assay. Results ASCs injected into knee joints at different time-points after induction of DMM (day 7, day 14 or day 7 and day 14) had no effect on development of ligament damage or osteophyte formation. In contrast, ASC treatment of collagenase-induced OA knee joints, rapidly inhibited synovitis and ligament damage when administered at day 7 after induction. Washouts of synovium taken at different time points after injection of ASCs (6 hrs, day 2, day 14 and day 42) showed significantly decreased levels of IL-1β and S100A8/A9 already at 48 hrs after ASC treatment. No effect was found on levels of active TGFβ. Serum levels of S100A8/A9 were significantly decreased (85% lower) at day 14 whereas IL-1 levels were not detectable at that time-point. Next, we explored the effect in CiOA in a condition with less synovial inflammation. Synovial thickness at day 42 was 62% lower when compared to the former study. Injection of the same dose of ASCs at day 7 after induction of CiOA, only marginally inhibited synovial thickening when measured at day 42. Serum levels of S100A8/A9 were low at day 14 (around 50 ng/ml compared to 800 ng/ml in the first experiment) and were not altered by the ASC treatment. Chondrogenesis in collateral and cruciate ligaments but also osteophyte formation at the bone margins was also not diminished. Conclusion Our study indicates that synovial activation rapidly drives anti-inflammatory effects of ASCs after local administration in murine OA knee joints with synovitis protecting against development of ligament damage and osteophyte formation.

AB0073 The s100a9 inhibitor paquinimod (abr-215757) ameliorates experimental osteoarthritis with high synovial involvement

Background There is increasing evidence that synovial activation contributes to osteoarthritis (OA) pathology and that it is present in a large subset of OA patients. Previously, we found that alarmins S100A8 and S100A9 are elevated in the synovium of OA patients and that high S100A8/A9 serum levels correlate with 2-year progression of the disease. Furthermore, these S100-proteins are involved in cartilage degradation and synovial activation in experimental OA. Paquinimod is a quinoline-3-carboxamide compound with immunomodulatory properties that is currently in clinical development for treatment of systemic sclerosis. It targets the S100A9 protein and disrupts the binding of S100A9 to RAGE and TLR-4. Objectives In the current study we investigated the effect of the S100A9-blocking compound paquinimod on experimental osteoarthritis with different degrees of synovial involvement. Methods Collagenase induced OA (CIOA) was induced by two times intra-articular injection of 1U collagenase and DMM was induced by transsection of the medial anterior meniscotibial ligament leading to destabilization of the medial meniscus (DMM), both in C57Bl6 mice. CIOA measurements were done at day 42, DMM at day 56. Paquinimod (71,5 μM) was administered in the drinking water 4 days before induction of OA in both CIOA and DMM and refreshed twice a week. Synovial thickening and cellularity was measured using an arbitrary score from 0-3. OA-like cartilage pathology was scored using a modified Pritzker OARSI score. Osteophyte size was assessed by a blinded observer using imaging software. Results We assessed the effect of paquinimod-treatment on two different experimental OA models. In CIOA, synovial activation is high throughout day 42 of the model, whereas during DMM synovial activation is marginal. We started with paquinimod-treatment of DMM mice and focused first on osteophytes. These are bony outgrowths that limit joint formation, originating from the periosteum or from ligaments. No differences were observed on osteophyte size between paquinimod-treated and non-treated animals at both medial tibia and medial femur. Furthermore, OA-like cartilage pathology was only significantly reduced by paquinimod-treatment at the medial femur (64%), not at other surfaces and not in the total joint score (16%). Next, we investigated the effect of paquinimod-treatment in an OA model with synovial activation. In CIOA, synovial activation was significantly reduced by paquinimod-treatment at the medial side of the patella-femur region (57%). Osteophyte size was significantly reduced at the medial femur (66%) and cruciate ligaments (67%). Finally, OA-like cartilage pathology was significantly reduced in CIOA after paquinimod treatment on the medial side of both tibia and femur (47% and 75% respectively) as well as in the total joint score (46%). Conclusions Blocking S100A9 with paquinimod reduces synovial activation, osteophyte formation and OA-like cartilage pathology in CIOA. In contrast, in an experimental OA model where synovial activation is nearly absent (DMM), the effect of paquinimod is marginal. Paquinimod could prove a very promising treatment for osteoarthritis patients with high synovial activation by blocking S100A9. Disclosure of Interest None Declared

FRI0044 Cholesterol accumulation by low density lipoprotein receptor deficiency or a cholesterol-rich diet enhances osteophyte formation during experimental osteoarthritis by activating transforming growth factor-beta

Background Synovial macrophages have previously shown to play a significant role in the etiopathology of experimental collagenase-induced osteoarthritis (OA)1. In addition to production of the pro-inflammatory protein S100A8/9 in early OA2, synovial lining macrophages have also shown to play a crucial role in promotion of transforming growth factor-beta (TGFβ) mediated osteophyte formation3. In an inflammatory milieu such as OA, accumulated low density lipoprotein (LDL) is oxidized, resulting in high intra-articular levels of oxidized LDL (oxLDL). OxLDL is taken up by macrophages via scavenger receptors, resulting in an more aggressive phenotype4. Objectives To investigate whether LDL accumulation by either LDL-receptor (LDLr) deficiency or a cholesterol-rich diet leads to increased oxLDL uptake by synovial macrophages and affects synovial activation and osteophyte formation. Methods LDLr deficient mice and their wild type (WT) controls received either a high cholesterol or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase on day 84 and 86. Paraffin sections of OA knee joints were analyzed for cartilage destruction and osteophyte formation using the Pritzker score and image analysis, respectively. ApoB and S100A8 were detected using immunohistochemistry. Murine bone marrow derived macrophages were stimulated with 50 μg/mL oxLDL, after which supernatant was functionally tested for active TGFβ presence using a TGFβ reporter gene assay. Results Mice receiving a cholesterol-rich diet not only developed increased serum LDL cholesterol levels, but also showed enhanced ApoB expression in synovial lining macrophages. In line with that, LDLr deficient mice, which already had systemically high basal levels of LDL, showed a much higher accumulation of ApoB in the synovial lining after receiving a cholesterol-rich diet. Although increased LDL levels did not enhance thickening of the synovium, S100A8 expression within macrophages was markedly increased, reflecting an elevated activation status. Even though no effect of LDL accumulation on cartilage destruction was found, both a cholesterol-rich diet and LDLr deficiency stongly increased bone formation in ligaments with a fold change of 6.7 and 6.1, respectively. Moreover, an increase in osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr deficient mice compared to WT mice). To elucidate the mechanism, we finally studied the presence of active TGFβ, which is crucial in driving osteophyte formation, in culture supernatant of oxLDL stimulated macrophages. Using a TGFβ reporter assay, stimulation of macrophages with oxLDL showed an increased presence of functional TGFβ in supernatant compared to that of unstimulated macrophages (fold change of 2.9). Conclusions LDL cholesterol accumulation by either LDLr deficiency or a cholesterol-rich diet leads to increased synovial activation and osteophyte formation in experimental OA. Uptake of oxLDL by synovial macrophages may activate TGFβ. References Bondeson J et al. Arthritis Rheum 2010; 62: 647-57. van Lent et al. Arthritis Rheum 2012; 64: 1466-76. van Lent PL et al. Arthritis Rheum 2004; 50: 103-11. van Tits LJ et al. Atherosclerosis 2011; 214: 345-9. Disclosure of Interest None Declared