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The matricellular protein Wnt-induced secreted protein 1 (WISP1) is the fourth member of the CCN family of proteins, which has been shown to affect tissues of the musculoskeletal system. In the context of the musculoskeletal disorder osteoarthritis, our lab studied the function of CCN4/WISP1 in joint tissues, including synovium and cartilage, using both gain- and loss-of-function approaches. In mice, this was done by genetic engineering and recombination to generate mice deficient in CCN4/WISP1 protein. Various experimental models of osteoarthritis with different characteristics were induced in these mice. Moreover, CCN4/WISP1 levels in joints were experimentally increased by adenoviral transfections. Osteoarthritis pathology was determined using histology, and the effect of different CCN4/WISP1 levels on gene expression was evaluated in individual tissues. Effects of high levels of CCN4/WISP1 on chondrocytes were studied with an in vitro chondrocyte pellet model. In this chapter, we describe the procedures to conduct these experiments.

Background Excessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1β are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn−/− mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn−/− mice with mice that have an additional deficiency for S100a9 (Il1rn−/−XS100a9−/−). Methods Il1rn−/−XS100a9−/− on a BALB/c background were obtained by crossing S100a9−/− mice and Il1rn−/− mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice. Results Macroscopically scored arthritis severity was comparable between Il1rn−/− and Il1rn−/−XS100a9−/− mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn−/−, but not significantly different between Il1rn−/−XS100a9−/− and Il1rn−/−. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn−/− strains, but the additional absence of S100a9 did not further affect tissue pathology. Conclusion S100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1β signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

OA is a complex and highly prevalent degenerative disease affecting the whole joint, in which factors like genetic predisposition, gender, age, obesity and traumas contribute to joint destruction. ∼50–80% of OA patients develop synovitis. OA-associated risk factors contribute to joint instability and the release of cartilage matrix fragments, activating the synovium to release pro-inflammatory factors and catabolic enzymes in turn damaging the cartilage and creating a vicious circle. Currently, no cure is available for OA. Mesenchymal stromal cells (MSCs) have been tested in OA for their chondrogenic and anti-inflammatory properties. Interestingly, MSCs are most effective when administered during synovitis. This review focusses on the interplay between joint inflammation and the immunomodulation by MSCs in OA. We discuss the potential of MSCs to break the vicious circle of inflammation and describe current perspectives and challenges for clinical application of MSCs in treatment and prevention of OA, focussing on preventing post-traumatic OA.

Contains fulltext : 287600.pdf (Publisher’s version ) (Open Access) Synovial macrophages are key mediators of OA pathology, and skewing of macrophage phenotype in favour of an M1-like phenotype is thought to underlie the chronicity of synovial inflammation in OA. Components of the metabolic syndrome (MetS), such as dyslipidaemia, can affect macrophage phenotype and function, which could explain the link between MetS and OA development. Recently published studies have provided novel insights into the different origins and heterogeneity of synovial macrophages. Considering these findings, we propose an important role for monocyte-derived macrophages in particular, as opposed to yolk-sac derived residential macrophages, in causing a pro-inflammatory phenotype shift. We will further explain how this can start even prior to synovial infiltration; in the circulation, monocytes can be trained by metabolic factors such as low-density lipoprotein to become extra responsive to chemokines and damage-associated molecular patterns. The concept of innate immune training has been widely studied and implicated in atherosclerosis pathology, but its involvement in OA remains uncharted territory. Finally, we evaluate the implications of these insights for targeted therapy directed to macrophages and metabolic factors.

Due to the complexity and heterogeneity of osteoarthritis (OA) pathophysiology, studying the interaction between intrinsic molecular changes in chondrocytes after hyper-physiological mechanical stress (MS) and aberrant signalling of OA risk genes remains a challenge. In this study we set out to set up anHuman primary chondrocyte neo-cartilage pellets were exposed for 2 days to 2 ​× ​10 ​min of hyper-physiological dynamic MS attained by a 20% strain and a frequency of 5 ​Hz. In order to assess cartilage damage, sulphated glycosaminoglycan (sGAG) content in the neo-cartilage was quantified using Alcian blue staining and a dimethyl methylene blue (DMMB) assay, while cleavage of aggrecan was visualized by immunohistochemical staining of aggrecan neo-epitope NITEGE. In addition, changes in expression levels of catabolic, anabolic and hypertrophic genes, and of three OA risk genes;Hyper-physiological MS induced cartilage damage, as reflected by decreased sGAG content. mRNA levels of aggrecanaseWe established a human

The alarmin S100A8/A9 is implicated in sterile inflammation-induced bone resorption and has been shown to increase the bone-resorptive capacity of mature osteoclasts. Here, we investigated the effects of S100A9 on osteoclast differentiation from human CD14

Recently, it was shown that interleukin-1&beta, (IL-1&beta, ) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn&minus, /&minus, ) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C&minus, ), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31&minus, Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn&minus, mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn&minus, mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of Nf&kappa, B ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn&minus, cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

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.

Item does not contain fulltext OBJECTIVES: Previously, we have shown the involvement of Wnt-activated protein Wnt-1-induced signaling protein 1 (WISP1) in the development of OA in mice. Here, we aimed to characterize the relation between WISP1 expression and human OA and its regulatory epigenetic determinants. METHODS: Preserved and lesioned articular cartilage from end-stage OA patients and non-OA-diagnosed individuals was collected. WISP1 expression was determined using immunohistochemistry and damage was classified using Mankin scoring. RNA expression and DNA methylation were assessed in silico from genome-wide datasets (microarray analysis and RNA sequencing, and 450 k-methylationarrays, respectively). Effects of WISP1 were tested in pellet cultures of primary human chondrocytes. RESULTS: WISP1 expression in cartilage of OA patients was increased compared with non-OA-diagnosed controls and, within OA patients, WISP1 was even higher in lesioned compared with preserved regions, with expression strongly correlating with Mankin score. In early symptomatic OA patients with disease progression, higher synovial WISP1 expression was observed as compared with non-progressors. Notably, increased WISP1 expression was inversely correlated with methylation levels of a positional CpG-dinucleotide (cg10191240), with lesioned areas showing strong hypomethylation for this CpG as compared with preserved cartilage. Additionally, we observed that methylation levels were allele-dependent for an intronic single-nucleotide polymorphism nearby cg10191240. Finally, addition of recombinant WISP1 to pellets of primary chondrocytes strongly inhibited deposition of extracellular matrix as reflected by decreased pellet circumference, proteoglycan content and decreased expression of matrix components. CONCLUSION: Increased WISP1 expression is found in lesioned human articular cartilage, and appears epigenetically regulated via DNA methylation. In vitro assays suggest that increased WISP1 is detrimental for cartilage integrity.

Background High systemic cholesterol levels have been associated with osteoarthritis (OA) development and lowering cholesterol using statins has been suggested as a potential treatment for OA1. Recently, novel therapeutic high-intensive cholesterol-lowering treatment has been shown to regress Western-type diet (WTD)-induced atherosclerosis in dyslipidemic APOE*3Leiden.CETP mice, a translational model with a human-like lipoprotein metabolism2. Objectives Here, we investigated the potency of these therapeutic cholesterol-lowering strategies to attenuate OA development in dyslipidemic APOE*3Leiden.CETP mice. Methods Female mice (n=13-16 per group) were fed a cholesterol-supplemented WTD for 38 weeks. After 13 weeks, mice were matched into a baseline group (sacrificed at t=13w) and 5 groups that received WTD alone or with treatment for 25 weeks: atorvastatin alone (A), in combination with PCSK9 inhibitor alirocumab (AA) or ANGPTL3 inhibitor evinacumab (AE) or triple treatment (AAE). Plasma lipids were monitored to assess metabolic dysfunction. Knee joints were analysed for cartilage degradation, synovial inflammation and osteophyte formation. Aggrecanase activity and S100A8 expression were determined using immunohistochemistry. Results Systemic administration of cholesterol-lowering interventions resulted in a significant reduction of body weight (A: -16%; AA: -8%; AE: -12%; AAE: -18% at end point) without changes in food intake. WTD-feeding manifested itself in dyslipidemia, with high cholesterol levels that were significantly reduced after administration of therapeutic cholesterol-lowering interventions (A: -46%; AA: -74%; AE: -74%; -AAE: 86% at end point). Using detailed OARSI scoring, dyslipidemic mice showed a mild but significant increase in cartilage degeneration at end point (7.8 ± 1.9) compared to baseline controls (3.8 ± 1.8, 2.1-fold increase, p=0.001), and a strong 52-fold increase in NITEGE staining, representing proteolytic activity by aggrecanases3. However, therapeutic cholesterol lowering did not ameliorate further progression of cartilage destruction. This was supported by comparable NITEGE staining in all treatment groups at end point. Dyslipidemic APOE*3Leiden.CETP mice developed minor synovial inflammation which was not affected by cholesterol-lowering interventions. Alarmin S100A8, a marker for macrophage activation, showed minor expression in the synovial lining of all treatment groups, supporting the absence of joint inflammation in this model4. Finally, we determined whether high plasma cholesterol levels promoted osteophyte formation. The total number of osteophytes and osteophyte maturation stage was comparable between all groups despite reduction of systemic cholesterol levels. Conclusion Therapeutic cholesterol-lowering interventions did not slow the progression of cartilage degradation and osteophyte formation in dyslipidemic APOE*3Leiden.CETP mice with minor joint inflammation. These findings suggest that therapeutic cholesterol-lowering interventions in the absence of local inflammation within the knee joint cannot prevent progression of OA. References [1] Gierman, LM, Ann Rheum Dis, 2014; 73, 921-927. [2] Dewey, F.E., New Engl J Med2017; 377, 211-221. [3] van Meurs JB, Arthritis Rheum42, 1999; 42, 1128-1139. [4] van Lent PL, Arthritis & Rheumatism2012; 64, 1466-1476. Disclosure of Interests None declared

Background High systemic levels of low density lipoprotein (LDL) is a shared metabolic risk factor for osteoarthritis (OA) and cardiovascular disease (CVD), which may point to common biochemical pathways. One of the key events in atherosclerosis is formation and uptake of oxidized LDL (oxLDL) as a consequence of macrophage-mediated inflammation. Studies performed in our lab have previously shown that in mice, high LDL levels enhance synovial activation and ectopic bone formation during development of collagenase-induced OA. Furthermore, injection of oxLDL in naive macrophage depleted joints led to infiltration of myeloid progenitor cells. Although these results strongly indicate towards (ox)LDL-mediated events that enhance OA severity, these processes have not yet been shown to occur in human osteoarthritic synovium. Objectives In the present study, we investigated whether the inflammatory status of OA synovium is associated to systemic and local levels of (ox)LDL. Methods Blood was collected from patients planned to undergo total knee replacement and LDL serum levels were determined (n=16). After surgery, synovial explants from the same patients (2-8 per patient) were collected and incubated at 37°C for 24 hours in presence of LDL (50 µg ApoB/mL), oxLDL (50 µg ApoB/mL) or control medium. Levels of IL-1β, TNF-α, IL-10 and MCP-1 secreted in the conditioned medium were measured using Luminex. Levels of S100A8/A9 were measured using ELISA. Concentrations of secreted factors were normalized for tissue sample weight. Synovial explants were further processed for histological analysis. Immunohistochemical staining of ApoB was performed to assess presence and uptake of (ox)LDL in the synovium. Hematoxylin & eosin-stained sections were used to arbitrarily score cellular infiltration and synovial hyperplasia. Results Around 50% of the patients in our cohort showed synovial activation exemplified by high secretion levels of IL-1β, TNF-α and IL-10. Cellular infiltration and synovial hyperplasia was observed in the majority of synovial tissue samples, their grades did however not correlate with cytokine secretion. Secretion levels of IL-1β positively correlated with serum levels of LDL and total cholesterol and did not correlate with BMI, suggesting a link that runs via systemic metabolic processes rather than increased weight-mediated joint loading. Considering that around 50% of OA patients suffers from synovitis characterized by increased macrophage presence and activity, high LDL in these patients may trigger oxLDL formation and uptake in the synovium. Staining of LDL-associated protein ApoB in histological sections of synovia showed distinct speck-like staining within macrophages and endothelial cells especially, suggesting oxLDL accumulation in these cells. Unexpectedly, presence of high concentrations of LDL or oxLDL during incubation of synovial explants did not significantly affect secretion of IL-1β, TNF-α, IL-10 and MCP-1, whereas presence of LDL but not oxLDL negatively affected secretion of S100A8/A9. Conclusion IL-1β secretion by end stage OA synovium is associated to systemic levels of LDL. Intervention with high concentrations of LDL or oxLDL for 24-hours after synovium extraction did however not alter cytokine secretion, suggesting that LDL and synovitis are linked through more long-term local interactions, or alternatively via systemic inflammatory factors. Disclosure of Interests: None declared

Background: Inflammatory mediators released during synovitis, including S100A8 and -9, have been implicated in the regulation of pain. S100A8 and -9 may regulate pain either via direct stimulation of TLR4 on the nerve endings in the synovium or via stimulation at the site of the dorsal root ganglia (DRG), thereby inducing local inflammation which may cause sensitization. Objectives: We aimed to elucidate the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, comparing S100A9-/- mice and their WT controls. Methods: Synovial biopsies of 25 patients with early symptomatic OA were studied by micro-array analysis and subsequent functional annotation clustering (FAC). Patients with pain at t=5 years after biopsy isolation were compared to those without pain. Acute synovitis was induced by a single i.a. injection of SCW in the knee joint of C57Bl6 (WT) mice and S100A9-/- mice, control mice received an i.a. saline injection. Serum S100A8/A9 levels were investigated by ELISA and expression of S100A8 and S100A9 in synovium and DRG by immunohistochemistry (IHC). Joint swelling and cell influx was assessed by 99mTc accumulation and histology, respectively. Pain response were investigated Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey’s filaments (mechanical allodynia). Gene expression of inflammatory mediators and neuron activation markers in DRG were determined by q-PCR. Monocyte influx and protein expression of NAV1.7 and GAP43 was monitored by IHC. Results: In synovial biopsies of early symptomatic OA patients, approximately 750 genes were upregulated in patients that had pain 5 years after inclusion compared to patients without pain. FAC showed that inflammatory genes were highly significantly enriched (p=6,4*10-12). Among the upregulated genes were the alarmins S100A8 and S100A9. In mice, a single i.a. injection of SCW resulted in increased synovial expression of S100A8 and S100A9 and subsequent increased serum S100A8/A9 levels (2.6-fold, P Conclusion: These findings show that S100A9, which is released from the synovium upon inflammation, is an important mediator of inflammatory nociceptive pain response in the knee, rather than by being involved in peripheral sensitization. In acute inflammation S100A8/A9 is likely regulated via direct activation of TLR4 on nerve endings in the synovium. Disclosure of Interests: None declared

Background High levels of the damage-associated molecular pattern (DAMP) S100A8/A9 are produced in the inflamed synovium during experimental and human rheumatoid arthritis (RA), which have been implicated in sterile inflammation-induced bone resorption. We and others have previously shown that stimulation of mature osteoclasts with S100A8/A9 increases their bone-resorptive capacity. In agreement, reduced bone destruction was observed after induction of experimental RA models in S100a9-/- mice. However, its effects on the differentiation of osteoclasts from their precursors remains elusive. Objectives Here, we investigated the effects of S100A9 on osteoclast differentiation from CD14+ circulating precursors. Methods CD14+ monocytes were isolated from buffy coats of healthy donors and differentiated towards osteoclasts with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B (RANK) ligand in the presence or absence of S100A9. Differentiation state of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP) staining and resorption capacity was quantified using hydroxyapatite-like-coated plates. RNA expression was analyzed with RNA sequencing and qPCR. RANK expression was assessed using FACS. Underlying epigenetic programming was studied using chromatin immunoprecipitation. Secretion of pro-/anti-inflammatory mediators was analyzed with Luminex analysis. Results S100A9 stimulation during monocyte-to-osteoclast differentiation resulted in a strong decrease in the numbers of multinucleated osteoclasts, underlined by a decreased resorptive capacity. The thus differentiated cells showed a high production of pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNFα) after 16h of stimulation. In contrast, at day 4, the cells showed a decreased expression of the osteoclast-promoting factor TNFα. Interestingly, we showed that S100A9 stimulation only during the first 16h of culture was sufficient to reduce osteoclastogenesis. To determine the mechanism of how this short S100A9 stimulation might reduce the osteoclast differentiation, we determined the protein expression of RANK. We observed that within this 16h time frame, S100A9 inhibited the M-CSF-mediated induction of RANK, which we found to be associated with changes in various histone marks at the epigenetic level. This S100A9-induced reduction in RANK could be partially reversed by blocking TNFα using etanercept, but not by blocking interleukin-1 (IL-1) with the IL-1 receptor antagonist. Conclusion Whereas S100A8/A9 was previously shown to stimulate the resorptive capacity of mature osteoclasts, we here show that early S100A9 stimulation impedes monocyte-to-osteoclast differentiation via reduction of RANK expression that is partially TNFα-mediated. This suggests that the timing of exposure to S100A8/A9 is an important determinant for monocyte-to-osteoclast differentiation. Disclosure of Interests Martijn van den Bosch: None declared, Irene Di Ceglie: None declared, Arjen Blom: None declared, Robab Davar: None declared, Colin Logie: None declared, Ehsan Habibi: None declared, Johannes Roth: None declared, Thomas Vogl: None declared, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie, Peter van der Kraan: None declared, Peter van Lent: None declared

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1β levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (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. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1β-stimulated ASCs (ASCIL-1β-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1β-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1β-stimulated ASCs. Also migration of PMNs toward ASCIL-1β-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1β-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

Contains fulltext : 208632.pdf (Publisher’s version ) (Open Access) Bone erosion is one of the central hallmarks of RA and is caused by excessive differentiation and activation of osteoclasts. Presence of autoantibodies in seropositive arthritis is associated with radiographic disease progression. ICs, formed by autoantibodies and their antigens, activate Fcgamma-receptor signalling in immune cells, and as such stimulate inflammation-mediated bone erosion. Interestingly, ICs can also directly activate osteoclasts by binding to FcgammaRs on their surface. Next to autoantibodies, high levels of alarmins, among which is S100A8/A9, are typical for RA and they can further activate the immune system but also directly promote osteoclast function. Therefore, IC-activated FcgammaRs and S100A8/A9 might act as partners in crime to stimulate inflammation and osteoclasts differentiation and function, thereby stimulating bone erosion. This review discusses the separate roles of ICs, FcgammaRs and alarmins in bone erosion and sheds new light on the possible interplay between them, which could fuel bone erosion.

In this study, we used hypercholesterolaemic apolipoprotein E-deficient (Apoe-/-) mice to investigate LDL/oxLDL effect on synovial inflammation and cartilage destruction during antigen-induced arthritis (AIA). Further, as macrophage FcγRs are crucial to immune complex-mediated AIA, we investigated in vitro the effects of high cholesterol levels on the expression of FcγRs on macrophages.AIA was induced by intra-articular injection of mBSA into knee joints of immunised Apoe-/- and wild type (WT) control mice. Joint swelling was measured by uptake of 99mTc pertechnetate (99mTc). Joint inflammation and cartilage destruction were assessed by histology. Anti-mBSA IgGs were measured by ELISA and specific T-cell response by lymphocyte stimulation test. Upon oxLDL stimulation of WT macrophages, protein levels of FcγRs were measured by flow cytometry.Local induction of AIA resulted in less joint swelling, synovial infiltrate and exudate in the joint cavity in Apoe-/- mice compared to WT controls, even though both their humoral and adaptive immune response were comparable. Whereas Apoe deficiency alone did not affect macrophage expression of FcγRs, oxLDL sharply reduced the protein levels of activating FcγRs, crucial in mediating cartilage damage. In agreement with the reduced inflammation in Apoe-/- mice, we observed decreased MMP activity and destruction in the articular cartilage.Taken together, our findings suggest that high levels of LDL/oxLDL during inflammation, dampen the initiation and chronicity of joint inflammation and cartilage destruction in AIA by regulating macrophage FcγR expression.

OBJECTIVE: Both alarmins S100A8/A9 and canonical Wnt signaling have been found to play active roles in the development of experimental osteoarthritis (OA). However, what activates canonical Wnt signaling remains unknown. This study was undertaken to investigate whether S100A8 induces canonical Wnt signaling and whether S100 proteins exert their effects via activation of Wnt signaling. METHODS: Expression of the genes for S100A8/A9 and Wnt signaling pathway members was measured in an experimental OA model. Selected Wnt signaling pathway members were overexpressed, and levels of S100A8/A9 were measured. Activation of canonical Wnt signaling was determined after injection of S100A8 into naive joints and induction of collagenase-induced OA in S100A9-deficient mice. Expression of Wnt signaling pathway members was tested in macrophages and fibroblasts after S100A8 stimulation. Canonical Wnt signaling was inhibited in vivo to determine if the effects of S100A8 injections were dependent on Wnt signaling. RESULTS: The alarmins S100A8/A9 and members of the Wnt signaling pathway showed coinciding expression in synovial tissue in an experimental OA model. Synovial overexpression of selected Wnt signaling pathway members did not result in increased expression of S100 proteins. In contrast, intraarticular injection of S100A8 increased canonical Wnt signaling, whereas canonical Wnt signaling was decreased after induction of experimental OA in S100A9-deficient mice. S100A8 stimulation of macrophages, but not fibroblasts, resulted in increased expression of canonical Wnt signaling members. Overexpression of Dkk-1 to inhibit canonical Wnt signaling decreased the induction of matrix metalloproteinase 3, interleukin-6, and macrophage inflammatory protein 1alpha after injection of S100A8. CONCLUSION: Our findings indicate that the alarmin S100A8 induces canonical Wnt signaling in macrophages and murine knee joints. The effects of S100A8 are partially dependent on activation of canonical Wnt signaling.

Item does not contain fulltext The Wnt signalling pathway is gaining increasing attention in the field of joint pathologies, attributable to its role in the development and homeostasis of the tissues found in the joint, including bone and cartilage. Imbalance in this pathway has been implicated in the development and progression of OA, and interference with the pathway might therefore depict an effective treatment strategy. Though offering multiple opportunities, it is yet to be decided which starting point will bring forth the most promising results. The complexity of the pathway and its interaction with other pathways (such as the TGF-beta signalling pathway, which also has a central role in the maintenance of joint homeostasis) means that acting directly on proteins in this signalling cascade entails a high risk of undesired side effects. Therefore, interference with Wnt-induced proteins, such as WISP1, might be an overall more effective and safer therapeutic approach to inhibit the pathological events that take place during OA.

Background Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). Methods AIA was induced in knee joints of wild-type (WT), FcγRI,II,III−/−, and FcγRI,II,III,IV−/− mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. Results FcγRI,II,III,IV−/− mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV−/− mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV−/− and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV−/− mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV−/− mice, AIA induction in knee joints of FcγRI,II,III−/− mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. Conclusions FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint. Electronic supplementary material The online version of this article (10.1186/s13075-018-1584-1) contains supplementary material, which is available to authorized users.

Contains fulltext : 153161.pdf (Publisher’s version ) (Closed access) Proteins from the Wnt signaling pathway are very important for joint development. Curiously, osteoarthritis (OA) is thought to be a recapitulation of developmental processes. Various members of the Wnt signaling pathway are overexpressed in the synovium during experimental OA. Here, we investigated the potency of specific Wnt proteins, when expressed in the synovium, to induce OA pathology. We overexpressed Wnt5a, Wnt8a, Wnt16, and WISP1 in the synovium using adenoviral vectors. We determined whether overexpression resulted in OA pathology by histology, and we measured whether Wnt signaling led to increased protease activity in the joint. Synovial overexpression of Wnt8a and Wnt16 led to canonical Wnt signaling in the cartilage, whereas overexpression of Wnt5a did not. Canonical Wnt signaling increased protease activity and induced cartilage damage shortly after overexpression. Specific blocking of the canonical Wnt signaling pathway with Dickkopf-1 reduced the Wnt-signaling-induced cartilage damage. By contrast, the noncanonical signaling Wnt5a did not cause cartilage lesions. Overexpression of WISP1, a downstream protein of canonical Wnt signaling, resulted in increased cartilage damage. In conclusion, our data show that canonical Wnts and WISP1, which we found overexpressed in the synovium during experimental OA, may conduce to OA pathology.

Background It is increasingly recognized that part of the pathology in osteoarthritis (OA) is driven by a low-grade inflammation in the synovium. Pro-inflammatory cytokines released locally during OA, such as S100A8/A9 which are expressed for prolonged periods when compared to IL-1β, IL-6, and TNF-α, may recruit monocytes from the bone marrow (BM) to the joint. In mice, two functionally distinct monocyte populations are described: (i) pro-inflammatory Ly6Chigh monocytes; and (ii) patrolling Ly6Clow monocytes. Objectives The objective of our study is to investigate the role of S100A8/A9 in the recruitment of the different monocyte populations during early collagenase-induced OA (CiOA). Methods S100A8 was intra-articularly injected into the knee joint of naive wild type C57BL/6 (WT) mice to investigate their direct role in recruitment of monocytes. CiOA was induced by unilateral intra-articular collagenase injection in WT, and S100A9KO mice to investigate the role of S100A8/A9 in more detail. At CiOA day 7, WT and S100A9KO mice were sacrificed together with age-matched saline-injected control mice (n=6/group), and expression of several monocyte cell markers, chemokines, and adhesion molecules were measured in the synovium and BM, in which the cells were also analyzed by FACS. Monocytes were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)low, which were further divided into subsets based on their Ly6C expression. Results Injection of S100A8 into the knee joints of naive mice led to a significantly elevated expression of monocyte-related markers (Ly6C, CCR2, and CX3CR1) and monocyte attracting chemokines (MCP-1, CX3CL1, MIP1α, and KC) within the synovium after 1 and 3 days, suggesting that S100A8/A9 is directly involved in the attraction of monocytes. At CiOA day 7 in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium as compared to saline-injected control joints. In contrast, S100A9KO mice showed a significant increase in Ly6Clow monocytes (2-fold), whereas the number of Ly6Chigh monocytes remained unaffected. Concurrently, a strong upregulation of several chemokines (MCP1, CX3CL1, KC, and MIP1α) was observed locally in the synovium, of which only the Ly6Clow mobilization marker CX3CL1 was significantly higher in S100A9KO mice, corresponding with the increased Ly6Clow monocytes in the synovium of S100A9KO mice. This could however not explain the local increased number of Ly6Chigh monocytes at CiOA day 7 in WT mice, and therefore we next investigated the main source of the monocytes, which is the BM. We observed a decrease of 14% of myeloid cells (consisting partly of Ly6Chigh monocytes) in the BM of WT mice at CiOA day 7, whereas there were no changes in the BM of S100A9KO mice, suggesting that S100A8/A9 affects the release of myeloid populations from the BM. In line with that, expression of adhesion molecules (LFA-1, VCAM, VE-cadherin, PECAM1, and L-selectin) was lower at CiOA day 7 in the BM of S100A9KO mice when compared to WT mice. Conclusions Local induction of OA leads to S100A8/A9 production, and subsequently to the mobilization of Ly6Chigh monocytes into the joint, driving OA pathology. Disclosure of Interest None declared

Background Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. Methods Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)–/– mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 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 AF489-Cy5.5, a specific tracer for activated MMPs. Results Serum levels of S100A8/A9 were significantly increased in IL-1Ra–/– mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra–/– mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. Conclusions Expression of S100A8 and S100A9 in IL-1Ra–/– mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1121-z) contains supplementary material, which is available to authorized users.

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.

Item does not contain fulltext OBJECTIVE: S100A8 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. METHODS: Using 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. RESULTS: In 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. CONCLUSION: S100A8 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. 01 mei 2012

OBJECTIVE: Increased Wnt signaling in chondrocytes is associated with development of osteoarthritis (OA). However, OA is considered a disease of the entire joint, where the synovium has been attributed an important role in disease pathogenesis and progression. This study was undertaken to determine whether Wnt signaling in synovial tissue could contribute to pathologic development of OA through the production of matrix metalloproteinases (MMPs), and to assess the relationship of synovial expression of Frizzled (FZD) receptors and the Wnt inhibitor FRZB to MMP expression and disease progression in patients with early OA in the Dutch Cohort Hip and Cohort Knee (CHECK) study cohort. METHODS: In mouse knee joints, human WNT8A and mouse Wnt16 were overexpressed using adenoviral vectors, and expression of messenger RNA (mRNA) for MMPs in the synovium was determined by reverse transcription-polymerase chain reaction or Luminex assay. In human synovial tissue from a subgroup of patients with early OA with knee pain enrolled in the CHECK cohort, levels of Wnt family members were assessed for linkage to MMP expression and disease progression. In addition, MMP production in human synovium from patients with end-stage OA was determined after stimulation of Wnt signaling with WNT3A or inhibition with FRZB or DKK1 in the synovium. RESULTS: Overexpression of WNT8A and Wnt16 in mouse knee joints induced MMP expression in vivo. Expression of MMPs relevant to human OA in the synovium from CHECK study participants significantly correlated with expression of FZD1, FZD10, and FRZB mRNA. Moreover, increased FZD1 mRNA expression and decreased FRZB mRNA expression were observed in CHECK study patients who experienced disease progression compared to those who were nonprogressors. Stimulation of human OA synovium with WNT3A induced the production of various MMPs, whereas inhibition of Wnt signaling with FRZB or DKK1 reduced the production of MMPs. CONCLUSION: Wnt signaling in the synovium may potently induce progression of OA via increased production of MMPs.

Item does not contain fulltext BACKGROUND: Both Wnt signaling and TGF-beta signaling have been implicated in the regulation of the phenotype of many cell types including chondrocytes, the only cell type present in the articular cartilage. A changed chondrocyte phenotype, resulting in chondrocyte hypertrophy, is one of the main hallmarks of osteoarthritis. TGF-beta signaling via activin-like kinase (ALK)5, resulting in Smad 2/3 phosphorylation, inhibits chondrocyte hypertrophy. In contrast, TGF-beta signaling via ALK1, leading to Smad 1/5/8 phosphorylation, has been shown to induce chondrocyte hypertrophy. In this study, we investigated the capability of Wnt3a and WISP1, a protein downstream in canonical Wnt signaling, to skew TGF-beta signaling in chondrocytes from the protective Smad 2/3 towards the Smad 1/5/8 pathway. RESULTS: Stimulation with Wnt3a, either alone or in combination with its downstream protein WISP1, decreased TGF-beta-induced C-terminal phosphorylation of Smad 2/3. In addition, both Wnt3a and WISP1 increased Smad 1/5/8 phosphorylation at the C-terminal domain in both murine and human chondrocytes. DKK-1, a selective inhibitor of canonical Wnt signaling, abolished these effects. TGF-beta signaling via Smad 2/3, measured by the functional CAGA12-Luc reporter construct activity, was decreased by stimulation with Wnt3a in accordance with the decrease in Smad 2/3 phosphorylation found on Western blot. Furthermore, in vivo overexpression of the canonical Wnt8a decreased Smad 2/3 phosphorylation and increased Smad 1/5/8 phosphorylation. CONCLUSIONS: Our data show that canonical Wnt signaling is able to skew TGF-beta signaling towards dominant signaling via the ALK1/Smad 1/5/8 pathway, which reportedly leads to chondrocyte hypertrophy. In this way canonical Wnts and WISP1, which we found to be increased during experimental osteoarthritis, may contribute to osteoarthritis pathology.

Introduction In a previous study, we showed that LDL accumulation by LDL receptor deficient mice resulted in increased ectopic bone formation during experimental osteoarthritis (OA). Furthermore, we found that S100A8/A9 proteins are crucial in mediating joint pathology during experimental OA. In the present study we investigate OA pathology and its correlation with S100A8/9 in ApoE-/- mice, which is a different model for studying effects of systemically high LDL cholesterol levels. Material and Methods Wild type (WT) and ApoE deficient (ApoE -/- ) mice received a normal or cholesterol-rich diet for 54 days. At day 18, experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 28 and 54. Results ApoE -/- mice on a normal diet showed remarkably higher LDL levels than WT mice (8.90 mmol/L and 0.40 mmol/L, respectively; p -/- mice showed no increase in synovial thickening and ectopic bone formation, but a significant increase of cartilage damage was found in ApoE -/- mice compared to WT mice at the lateral side of the femoral chondyle (OARSI score 13.7 and 6.8, respectively; p -/- mice compared to WT mice (fold increase 1.8 and 1.4, respectively; p -/- mice at day 28 (fold increase 5.8; p In addition, we investigated whether a cholesterol-rich diet could increase joint pathology after induction of OA. This diet increased differences in LDL levels even more (18.4 mmol/L in ApoE -/- mice versus 1.2 mmol/L in WT mice; p -/- mice compared to WT mice (p -/- mice, both on protein and gene expression level and significantly correlated with ectopic bone formation. Conclusion LDL cholesterol accumulation by ApoE deficiency results in increased S100A8 and S100A9 production by synovial cells. A cholesterol-rich diet further increases this production which correlates with increased synovial thickening and ectopic bone formation in experimental OA. This suggests a important role for LDL cholesterol in developing OA joint pathology.