Intensive cholesterol-lowering treatment reduces synovial inflammation during early collagenase-induced osteoarthritis, but not pathology at end-stage disease in female dyslipidemic E3L.CETP mice.

INTRODUCTION: The association between metabolic syndrome (MetS) and osteoarthritis (OA) development has become increasingly recognized. In this context, the exact role of cholesterol and cholesterol-lowering therapies in OA development has remained elusive. Recently, we did not observe beneficial effects of intensive cholesterol-lowering treatments on spontaneous OA development in E3L.CETP mice. We postulated that in the presence of local inflammation caused by a joint lesion, cholesterol-lowering therapies may ameliorate OA pathology. MATERIALS AND METHODS: Female ApoE3∗Leiden.CETP mice were fed a cholesterol-supplemented Western type diet. After 3 weeks, half of the mice received intensive cholesterol-lowering treatment consisting of atorvastatin and the anti-PCSK9 antibody alirocumab. Three weeks after the start of the treatment, OA was induced via intra-articular injections of collagenase. Serum levels of cholesterol and triglycerides were monitored throughout the study. Knee joints were analyzed for synovial inflammation, cartilage degeneration, subchondral bone sclerosis and ectopic bone formation using histology. Inflammatory cytokines were determined in serum and synovial washouts. RESULTS: Cholesterol-lowering treatment strongly reduced serum cholesterol and triglyceride levels. Mice receiving cholesterol-lowering treatment showed a significant reduction in synovial inflammation (P = 0.008, WTD: 95% CI: 1.4- 2.3; WTD + AA: 95% CI: 0.8- 1.5) and synovial lining thickness (WTD: 95% CI: 3.0-4.6, WTD + AA: 95% CI: 2.1-3.2) during early-stage collagenase-induced OA. Serum levels of S100A8/A9, MCP-1 and KC were significantly reduced after cholesterol-lowering treatment (P = 0.0005, 95% CI: -46.0 to -12.0; P = 2.8 × 10-10, 95% CI: -398.3 to -152.1; P = 2.1 × 10-9, -66.8 to -30.4, respectively). However, this reduction did not reduce OA pathology, determined by ectopic bone formation, subchondral bone sclerosis and cartilage damage at end-stage disease. CONCLUSION: This study shows that intensive cholesterol-lowering treatment reduces joint inflammation after induction of collagenase-induced OA, but this did not reduce end stage pathology in female mice.

IL-1ß inhibition combined with cholesterol-lowering therapies decreases synovial lining thickness and spontaneous cartilage degeneration in a humanized dyslipidemia mouse model.

INTRODUCTION: Both systemic inflammation and dyslipidemia contribute to osteoarthritis (OA) development and have been suggested as a possible link between metabolic disease and OA development. Recently, the CANTOS trial showed a reduction in knee and hip replacements after inhibition of IL-1ß in patients with a history of cardiovascular disease and high inflammatory risk. In this light, we investigated whether inhibition of IL-1ß combined with cholesterol-lowering therapies can reduce OA development in dyslipidemic APOE∗3Leiden mice under pro-inflammatory dietary conditions. MATERIALS AND METHODS: Female ApoE3∗Leiden mice were fed a cholesterol-supplemented Western-Type diet (WTD) for 38 weeks. After 14 weeks, cholesterol-lowering and anti-inflammatory treatments were started. Treatments included atorvastatin alone or with an anti-IL1ß antibody, and atorvastatin combined with proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor alirocumab without or with the anti-IL1ß antibody. Knee joints were analyzed for cartilage degradation, synovial inflammation and ectopic bone formation using histology at end point. RESULTS: Cholesterol-lowering treatment successfully decreased systemic inflammation in dyslipidemic mice, which was not further affected by inhibition of IL-1ß. Synovial thickening and cartilage degeneration were significantly decreased in mice that received cholesterol-lowering treatment combined with inhibition of IL-1ß (P < 0.01, P < 0.05, respectively) compared to mice fed a WTD alone. Ectopic bone formation was comparable between all groups. CONCLUSION: These results indicate that inhibition of IL-1ß combined with cholesterol-lowering therapy diminishes synovial thickening and cartilage degeneration in mice and may imply that this combination therapy could be beneficial in patients with metabolic inflammation.

A single dose of anti-IL-1ß antibodies prevents Western diet-induced immune activation during early stage collagenase-induced osteoarthritis, but does not ameliorate end-stage pathology.

OBJECTIVE: Metabolic dysfunction can cause IL-1ß mediated activation of the innate immune system, which could have important implications for the therapeutic efficacy of IL-1ß neutralizing drugs as treatment for OA in the context of metabolic syndrome (MetS). In the present study, we investigated whether early treatment with a single dose of IL-1ß blocking antibodies could prevent Western diet (WD) induced changes to systemic monocyte populations and their cytokine secretion profile and herewith modulate collagenase induced osteoarthritis (CiOA) pathology. METHODS: CiOA was induced in female C57Bl/6 mice fed either a standard diet (SD) or WD and treated with a single dose of either polyclonal anti-IL-1ß antibodies or control. Monocyte subsets and granulocytes in bone marrow and blood were analyzed with flow cytometry, and cytokine expression by bone marrow cells was analyzed using qPCR. Synovial cellularity, cartilage damage and osteophyte formation were assessed on histology. RESULTS: WD feeding of C57Bl/6 mice led to increased serum levels of low-density lipoprotein (LDL) and innate immune activation in the form of an increased number of Ly6Chigh cells in bone marrow and blood and increased cytokine expression of IL-6 and TNF-α by bone marrow cells. The increase in monocyte number and activity was ameliorated by anti-IL-1ß treatment. However, anti-IL-1ß treatment did not significantly affect synovial lining thickness, cartilage damage and ectopic bone formation during WD feeding. CONCLUSIONS: Single-dose systemic anti-IL-1ß treatment prevented WD-induced innate immune activation during early stage CiOA in C57Bl/6 mice, but did not ameliorate joint pathology.

Novel high-intensive cholesterol-lowering therapies do not ameliorate knee OA development in humanized dyslipidemic mice.

OBJECTIVE: High systemic cholesterol levels have been associated with osteoarthritis (OA) development. Therefore, cholesterol lowering by statins has been suggested as a potential treatment for OA. We investigated whether therapeutic high-intensive cholesterol-lowering attenuated OA development in dyslipidemic APOE∗3Leiden.CETP mice. METHODS: Female mice (n = 13-16 per group) were fed a Western-type diet (WTD) for 38 weeks. After 13 weeks, mice were divided into a baseline group and five groups receiving WTD alone or with treatment: atorvastatin alone, combined with PCSK9 inhibitor alirocumab and/or ANGPTL3 inhibitor evinacumab. Knee joints were analysed for cartilage degradation, synovial inflammation and ectopic bone formation using histology. Aggrecanase activity in articular cartilage and synovial S100A8 expression were determined as markers of cartilage degradation/regeneration and inflammation. RESULTS: Cartilage degradation and active repair were significantly increased in WTD-fed mice, but cholesterol-lowering strategies did not ameliorate cartilage destruction. This was supported by comparable aggrecanase activity and S100A8 expression in all treatment groups. Ectopic bone formation was comparable between groups and independent of cholesterol levels. CONCLUSIONS: Intensive therapeutic cholesterol lowering per se did not attenuate progression of cartilage degradation in dyslipidemic APOE∗3Leiden.CETP mice, with minor joint inflammation. We propose that inflammation is a key feature in the disease and therapeutic cholesterol-lowering strategies may still be promising for OA patients presenting both dyslipidemia and inflammation.

Increased IL-6 receptor expression and signaling in ageing cartilage can be explained by loss of TGF-ß-mediated IL-6 receptor suppression.

OBJECTIVE: Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-ß (TGF-ß) signaling in cartilage. Recently, we showed that TGF-ß suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-ß signaling results in increased IL-6R expression and signaling in ageing cartilage. DESIGN: Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-ß1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR. RESULTS: Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-ß to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-ß-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed. CONCLUSIONS: Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-ß signaling, resulting in loss of TGF-ß-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.

Identifying effector molecules, cells, and cytokines of innate immunity in OA.

Inflammatory changes are observed in affected joints of osteoarthritis (OA) patients and are thought to be involved in the pathology that develops along OA progression. This narrative review provides an overview of the various cell types that are present in the joint during OA and which alarmins, cytokines, chemokines, growth factors, and other mediators they produce. Moreover, the involvement of more systemic processes like inflammaging and its associated cellular senescence in the context of OA are discussed.

High LDL levels lessen bone destruction during antigen-induced arthritis by inhibiting osteoclast formation and function.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by severe joint inflammation and bone destruction as the result of increased numbers and activity of osteoclasts. RA is often associated with metabolic syndrome, whereby elevated levels of LDL are oxidized into oxLDL, which might affect osteoclastogenesis. In this study, we induced antigen-induced arthritis (AIA) in Apoe-/- mice, which spontaneously develop high LDL levels, to investigate the effects of high LDL/oxLDL levels on osteoclast differentiation and bone destruction. Whereas basal levels of bone resorption were comparable between naive WT and Apoe-/- mice, induction of AIA resulted in a significant reduction of bone destruction in Apoe-/- mice as compared to WT controls. In line with that, the TRAP+ area on the cortical bone was significantly decreased. The absence of Apoe did affect neither the numbers of CD11b+Ly6Chigh and CD11b-/Ly6Chigh osteoclast precursors (OCPs) in the BM of naïve mice nor their in vitro osteoclastogenic potential as indicated by comparable mRNA expression of osteoclast markers. Addition of oxLDL, but not LDL, to pre-osteoclasts from day 3 and mature osteoclasts from day 6 of osteoclastogenesis strongly reduced the number of TRAP+ osteoclasts and their resorptive capacity. This coincided with a decreased expression of various osteoclast markers. Interestingly, oxLDL significantly lowered the expression of osteoclast-associated receptor (Oscar) and the DNAX adaptor protein-12 encoding gene Tyrobp, which regulate the immunoreceptor tyrosine-based activation motif (ITAM) co-stimulation pathway that is strongly involved in osteoclastogenesis. Collectively, our findings suggest that under inflammatory conditions in the joint, high LDL levels lessen bone destruction during AIA, probably by formation of oxLDL that inhibits osteoclast formation and activity through modulation of the ITAM-signaling.

IL-37 diminishes proteoglycan loss in human OA cartilage: donor-specific link between IL-37 and MMP-3.

OBJECTIVE: A hallmark of osteoarthritis (OA) is degradation of articular cartilage proteoglycans. In isolated human OA chondrocytes, the anti-inflammatory cytokine Interleukin-37 (IL-37) lowers the expression of the proteolytic MMP and ADAMTS enzymes, which mediate this degradation. Therefore, we investigated if IL-37 protects against proteoglycan loss in freshly obtained human OA explants. MATERIAL AND METHODS: Human OA cartilage explants were incubated with IL-37. Release of sulphated proteoglycans (sGAGs) was measured with the dimethylmethylene-blue assay. Production and degradation of newly synthesized proteoglycans was measured using 35S-sulphate. Proteoglycan and proteolytic enzyme expression were analyzed by qPCR and Western Blot. Proteolytic activity was determined by measuring MMP- and ADAMTS-generated aggrecan neo-epitopes with ELISA and by using MMP-3-, MMP-13- or ADAMTS-5-inhibitors. RESULTS: Over time, a linear release of sGAGs from OA cartilage was measured. IL-37 reduced this release by 87 µg/ml (24%) 95%CI [21.04-141.4]. IL-37 did not affect 35S-sulphate incorporation or proteoglycan gene expression. In contrast, IL-37 reduced loss of 35S-sulphate labeled GAGs and reduced MMP-3 protein expression, indicating that IL-37 inhibits proteoglycan degradation. Remarkably, we observed two groups of patients; one group in which MMP-3-inhibition lowered sGAG release, and one group in which ADAMTS5-inhibition had this effect. Remarkably, IL-37 was only functional in the group of patients that responded to MMP-3-inhibition. CONCLUSION: We identified a relationship between IL-37 and reduced sGAG loss in OA cartilage. Most likely, this effect is mediated by inhibition of MMP-3 expression. These results suggest that IL-37 could be applied as therapy in a subgroup of OA patients, in which cartilage degradation is mediated by MMP-3.

The role of NOX2-derived reactive oxygen species in collagenase-induced osteoarthritis.

OBJECTIVE: Synovitis in collagenase-induced osteoarthritis (CiOA) is driven by locally released S100A8/A9 proteins and enhances joint destruction. S100A8/A9 can induce reactive oxygen species (ROS) release by phagocytes in OA synovium via neutrophil cytosolic factor-1 (Ncf1)-regulated NOX2 activation. In the present study we investigated whether NOX2-derived ROS affect joint pathology during CiOA. METHODS: CiOA was induced in knee joints of wild type (WT) and Ncf1-deficient (Ncf1**) mice. Synovial gene expression of NOX2-subunits was measured with quantitative real-time polymerase chain reaction (qRT-PCR). Joint pathology was assessed using histology and immunohistochemistry for aggrecan neo-epitope VDIPEN. Levels of inflammatory proteins were measured with Luminex or ELISA. Phagocytes in synovium, blood, bone marrow (BM) and spleen were analyzed with flow cytometry. ROS release by phagocytes was measured with a ROS detection kit. RESULTS: CiOA induction in knee joints of WT mice caused significantly increased synovial gene expression of NOX2 subunits. On day 7 of CiOA, cartilage damage and MMP activity, as measured by VDIPEN, were comparable between WT and Ncf1** mice. Synovial thickening, synovial S100A8/A9 levels and percentages of synovial macrophages, polymorphonuclear cells (PMNs), and monocytes were not different, as were levels of inflammatory mediators in serum and phagocyte percentages in blood, BM and spleen. On day 42 of CiOA, synovitis, cartilage damage, and osteophyte formation in Ncf1** mice were unaltered when compared to WT mice. ROS detection confirmed that Ncf1** PMNs lack functional NOX2, but in vitro macrophages showed ROS production, suggesting activation of compensatory mechanisms. CONCLUSIONS: Absence of Ncf1-mediated ROS production does not alter joint pathology in CiOA.

WISP1/CCN4 aggravates cartilage degeneration in experimental osteoarthritis.

OBJECTIVE: Increased Wisp1 expression was previously reported in experimental and human osteoarthritis (OA). Moreover, adenoviral overexpression of Wisp1 in naïve mouse knee joints resulted in early OA-like cartilage lesions. Here, we determined how the matricellular protein WISP1 is involved in the pathology that occurs in the complex osteoarthritic environment with aging and experimental OA in wild type (WT) and Wisp1-/- mice. METHODS: WT and Wisp1-/- mice were aged or experimental OA was induced with intraarticular collagenase injection, destabilization of the medial meniscus (DMM) or anterior cruciate ligament transection (ACLT). Joint pathology was assessed using histology and microCT. Protease expression was evaluated with qRT-PCR and activity was determined by immunohistochemical staining of the aggrecan neoepitope NITEGE. Protease expression in human end-stage OA synovial tissue was determined with qRT-PCR after stimulation with WISP1. RESULTS: With aging, spontaneous cartilage degeneration in Wisp1-/- was not decreased compared to their WT controls. However, we observed significantly decreased cartilage degeneration in Wisp1-/- mice after induction of three independent experimental OA models. While the degree of osteophyte formation was comparable between WT and Wisp1-/- mice, increased cortical thickness and reduced trabecular spacing was observed in Wisp1-/- mice. In addition, we observed decreased MMP3/9 and ADAMTS4/5 expression in Wisp1-/- mice, which was accompanied by decreased levels of NITEGE. In line with this, stimulation of human OA synovium with WISP1 increased the expression of various proteases. CONCLUSIONS: WISP1 plays an aggravating role in the development of post-traumatic experimental OA.

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

OBJECTIVE: Interleukin-1 (IL-1) is an alleged important cytokine in osteoarthritis (OA), although the exact contribution of IL-1 to joint destruction remains unclear. Here we investigated the involvement of IL-1α and IL-1ß in joint pathology during collagenase-induced OA (CiOA). METHODS: CiOA was induced in wild type (WT) and IL-1αß-/- mice. Additionally, IL-1 signaling was inhibited in WT mice with CiOA using osmotic pumps containing IL-1RA. Joint pathology was assessed using histology. Activity of cartilage-degrading enzymes was determined using antibodies against aggrecan neo-epitopes VDIPEN and NITEGE. Synovial gene expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Serum protein levels were measured with Luminex or enzyme-linked immunosorbent assay (ELISA). RESULTS: Synovial IL-1ß expression was strongly elevated 7 days after induction of CiOA in WT mice but decreased afterwards, whereas S100A8/A9, previously described to aggravate OA, remained elevated for 21 days. Remarkably, synovial inflammation was comparable between WT and IL-1αß-/- mice on day 7 of CiOA. In line, synovial mRNA expression of genes involved in IL-1 signaling and inflammatory mediators was comparable between WT and IL-1αß-/- mice, and serum levels for Keratinocyte Chemoattractant (KC)/IL-6/S100A8/S100A9/IL-10 were equal. Synovial matrix metalloproteinase (MMP)/aggrecanase expression and activity in cartilage was not different in WT and IL-1αß-/- mice on day 7 of CiOA. Cartilage destruction on day 42 was not different between WT and IL-1αß-/- mice, which was supported by our finding that IL-1RA treatment in WT mice with CiOA did not alter joint destruction. CONCLUSIONS: IL-1α and IL-1ß are not involved in synovial inflammation and cartilage destruction during CiOA, implicating that other mediators are responsible for the joint damage.

Synovial macrophages promote TGF-ß signaling and protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidized low-density lipoproteins.

OBJECTIVE: Low-density lipoproteins (LDL) in inflamed synovium is oxidized and taken-up by synoviocytes. In this study, we investigate whether direct injection of oxidized LDL (oxLDL) into a normal murine knee joint induces joint pathology and whether synovial macrophages are involved in that process. DESIGN: Synovium was obtained from end-stage osteoarthritis (OA) patients in order to analyze LDL-uptake. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (phosphate buffered saline (PBS)). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes 7 days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. RESULTS: Synovial tissue of OA patients showed extensive accumulation of apolipoprotein B. Multiple injections of oxLDL in murine knee joints significantly increased TGF-ß activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to increased synovial thickening in combination with significantly upregulated protein and RNA levels of CCL2 and CCL3. 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 TGF-ß concentrations was measured in macrophage-depleted joints. CONCLUSIONS: OxLDL can affect joint pathology, since synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity combined with increased influx of monocytes and neutrophils.

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.

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.

Prophylactic treatment with S100A9 inhibitor paquinimod reduces pathology in experimental collagenase-induced osteoarthritis.

OBJECTIVES: Alarmins S100A8/A9 regulate pathology in experimental osteoarthritis (OA). Paquinimod is an immunomodulatory compound preventing S100A9 binding to TLR-4. We investigated the effect of paquinimod on experimental OA and human OA synovium. MATERIALS AND METHODS: Two OA mouse models differing in level of synovial activation were treated prophylactic with paquinimod. Synovial thickening, osteophyte size and cartilage damage were measured histologically, using an arbitrary score, adapted Pritzker OARSI score or imaging software, respectively. Human OA synovia were stimulated with S100A9, with or without paquinimod. RESULTS: Paquinimod treatment of collagenase-induced OA (CIOA) resulted in significantly reduced synovial thickening (57%), osteophyte size at the medial femur (66%) and cruciate ligaments (67%) and cartilage damage at the medial tibia (47%) and femur (75%; n=7, untreated n=6). In contrast, paquinimod did not reduce osteophyte size and reduced cartilage damage at one location only in destabilised medial meniscus, an OA model with considerably lower synovial activation compared with CIOA. In human OA synovium, paquinimod blocked proinflammatory (interleukin (IL)-6, IL-8, tumour necrosis factor-α) and catabolic (matrix metalloproteinases 1 and 3) factors induced by S100A9 (n=5). CONCLUSIONS: Prophylactic treatment of paquinimod reduces synovial activation, osteophyte formation and cartilage damage in experimental OA with high synovial activation (CIOA) and ameliorates pathological effects of S100A9 in OA synovium ex vivo.

Association between serum levels of the proinflammatory protein S100A8/A9 and clinical and structural characteristics of patients with established knee, hip, and hand osteoarthritis.

OBJECTIVES: To explore the association between S100A8/A9 serum levels with clinical and structural characteristics of patients with established knee, hip, or hand osteoarthritis (OA). METHOD: A cross-sectional exploratory study was conducted with 162 OA patients. Measures for pain, stiffness, and function included the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) questionnaire or the Australian Canadian Osteoarthritis Hand (AUSCAN) Index and for structural abnormalities, osteophytes and joint space narrowing grades. The association between S100A8/A9 and clinical or structural characteristics was analysed using linear regression or logistic regression where appropriate. RESULTS: The mean age of the OA patients was 56 years, 71% were female, and 61% had a Kellgren and Lawrence (K&L) score ≥ 2. The serum S100A8/A9 level did not differ between knee, hip, and hand OA patients and no association was found between serum S100A8/A9 and clinical characteristics. The serum S100A8/A9 level was negatively associated with the sum score of osteophytes after adjusting for sex and body mass index (BMI) [adjusted ß -0.015, 95% confidence interval (CI) -0.030 to 0.001, p = 0.062] and positively associated with erythrocyte sedimentation rate (ESR) > 12 mm/h (adjusted OR 1.002, 95% CI 1.000-1.004 p = 0.049) for each increase in S100A8/A9 of 1 ng/mL. For hand OA patients, a negative association of S100A8/A9 with sum score of joint space narrowing was found (adjusted ß -0.007, 95% CI -0.016 to 0.001, p = 0.099). CONCLUSIONS: The results from this cross-sectional exploratory study do not support an important role for serum S100A8/A9 levels as a biomarker for clinical and structural characteristics in established knee, hip, and hand OA patients. The inverse association with structural abnormalities and the positive association with ESR may reflect inflammatory synovial processes in patients with OA before structural abnormalities occur.

TGF-ß is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

OBJECTIVE: Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-ß (TGF-ß), which in turn stimulates chondrocytes to produce NGF. DESIGN: Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-ß1 and/or Interleukin-1 (IL-1)ß. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-ß pathway inducing NGF. RESULTS: NGF expression was consistently induced in higher levels by TGF-ß than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-ß-induced NGF whereas it fully blocked IL-1ß-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-ß-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-ß exposure led to a consistent high level of NGF induction. CONCLUSION: We show for the first time that TGF-ß induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage.

OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse and studied the effect of elevated BMP2 expression on healthy joints and joints with experimental OA. METHODS: We cloned a lentivirus with BMP2 controlled by a tet-responsive element and transfected embryos of mice containing a collagen type II driven cre-recombinase and floxed rtTA to gain a mouse expressing BMP2 solely in chondrocytes and only upon dox exposure (Col2-rtTA-TRE-BMP2). Mice were treated with dox to induce elevated BMP2 expression. In addition, experimental OA was induced (destabilisation of the medial meniscus model) with or without dox supplementation and knee joints were isolated for histology. RESULTS: Dox treatment resulted in chondrocyte-specific upregulation of BMP2 and severely aggravated formation of osteophytes in experimental OA but not in control mice. Moreover, elevated BMP2 levels did not result in alterations in articular cartilage of young healthy mice, although BMP2-exposure did increase VDIPEN expression in the articular cartilage. Strikingly, despite apparent changes in knee joint morphology due to formation of large osteophytes there were no detectible differences in articular cartilage: none with regard to structural damage nor in Safranin O staining intensity when comparing destabilisation of the medial meniscus with or without dox exposure. CONCLUSIONS: Our data show that chondrocyte-specific elevation of BMP2 levels does not alter the course of cartilage damage in an OA model in young mice but results in severe aggravation of osteophyte formation.

Treatment efficacy of adipose-derived stem cells in experimental osteoarthritis is driven by high synovial activation and reflected by S100A8/A9 serum levels.

OBJECTIVE: Synovitis is evident in a substantial subpopulation of patients with osteoarthritis (OA) and is associated with development of pathophysiology. Recently we have shown that adipose-derived stem cells (ASC) inhibit joint destruction in collagenase-induced experimental OA (CIOA). In the current study we explored the role of synovitis and alarmins S100A8/A9 in the immunomodulatory capacity of ASCs in experimental OA. METHOD: CIOA, characterized by synovitis, and surgical DMM (destabilization of medial meniscus) OA were treated locally with ASCs. Synovial activation, cartilage damage and osteophyte size were measured on histological sections. Cytokines in synovial washouts and serum were determined using Luminex or enzyme-linked immunosorbent assay (S100A8/A9), mRNA levels with reverse-transcriptase (RT)-qPCR. RESULTS: Local administration of ASCs at various time-points (days 7 or 14) after DMM induction had no effect on OA pathology. At day 7 of CIOA, already 6 h after ASC injection mRNA expression of pro-inflammatory mediators S100A8/A9, interleukin-1beta (IL-1ß) and KC was down-regulated in the synovium. IL-1ß protein, although low, was down-regulated by ASC-treatment of CIOA. S100A8/A9 protein levels were very high at 6 and 48 h and were decreased by ASC-treatment. The protective action of ASC treatment in CIOA was only found when high synovial inflammation was present at the time of deposition which was reflected by high serum S100A8/A9 levels. Finally, successful treatment resulted in significantly lower levels of serum S100A8/A9. CONCLUSION: Our study indicates that synovial activation rapidly drives anti-inflammatory and protective effects of intra-articularly deposited ASCs in experimental OA which is reflected by decreased S100A8/A9 levels.

Liposomal targeting of glucocorticoids to the inflamed synovium inhibits cartilage matrix destruction during murine antigen-induced arthritis.

UNLABELLED: Encapsulation of glucocorticoids into long-circulating liposomes provides targeting of these drugs to the inflamed synovium in experimental arthritis and thereby strongly improves their therapeutic index. The goal of this study was to evaluate the effect and mechanisms of intravenous liposomal delivery of prednisolone phosphate (Lip-PLP) on protease mediated cartilage destruction during murine antigen-induced arthritis (AIA). Mice treated with a single injection of Lip-PLP showed a pronounced suppression of synovial immune cell infiltration compared to control, PBS-treated mice. Liposomal PLP also significantly suppressed interleukin 1ß (3.6 fold) in the synovium, but not in the blood serum. Furthermore, expression of the proteases MMP-3, -9, -13 and -14 and ADAMTS-4 and -5 was suppressed by Lip-PLP in the synovium, but not within the articular cartilage of the femur and tibia, demonstrating the specific action of Lip-PLP on the synovium. Lip-PLP is phagocytosed by macrophages in vitro and suppresses their expression of IL-1ß and MMPs after LPS activation. Moreover, Lip-PLP suppresses destruction of the cartilage matrix during AIA mediated by active MMPs and ADAMTS, as assessed by immunostaining of their respective neoepitopes VDIPEN and NITEGE in various cartilage layers of the knee joint. CONCLUSION: liposomal delivery of glucocorticoids protects against cartilage matrix destruction during experimental arthritis by inhibiting protease expression and activity in the inflamed synovium.