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.

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.

TGF-ß dampens IL-6 signaling in articular chondrocytes by decreasing IL-6 receptor expression.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) is an important homeostatic regulator of cartilage. In contrast, interleukin-6 (IL-6) is a pro-inflammatory cytokine implicated in cartilage degeneration. Cross-talk between TGF-ß and IL-6 is reported in tissues other than articular cartilage. Here, we investigated regulation of IL-6 signaling by TGF-ß in articular chondrocytes. DESIGN: Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with TGF-ß1 or interleukin-1ß (IL-1ß). Expression of IL-6 and IL-6 receptor (IL-6R) was determined on mRNA and protein level. TGF-ß regulation of IL-6 signaling via phosho-STAT3 (p-STAT3) was determined using Western blot, in presence of inhibitors for IL-6R, and Janus kinase(JAK)- and activin receptor-like kinase ALK)5 kinase activity. Furthermore, induction of STAT3-responsive genes was used as a read-out for IL-6 induced gene expression. RESULTS: TGF-ß1 increased IL-6 mRNA and protein expression in both G6 and primary chondrocytes. Moreover, TGF-ß1 stimulation clearly induced p-STAT3), which was abolished by inhibition of either IL-6R, JAK- or ALK5 kinase activity. However, TGF-ß1 did not increase expression of the STAT3-responsive gene SOCS3 and pre-treatment with TGF-ß1 even inhibited induction of p-STAT3 and SOCS3 by rhIL-6. Interestingly, TGF-ß1 potently decreased IL-6R expression. In contrast, IL-1ß did increase IL-6 levels, but did not affect IL-6R expression. Finally, addition of recombinant IL-6R abolished the inhibitory effect of TGF-ß1 on IL-6-induced p-STAT3 and downstream SOCS3, BCL3, SAA1 and MMP1 expression. CONCLUSIONS: In this study we show that TGF-ß decreases IL-6R expression, thereby dampening IL-6 signaling in chondrocytes. This reveals a novel effect of TGF-ß, possibly important to restrict pro-inflammatory IL-6 effects to preserve cartilage homeostasis.

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.

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.

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.

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.

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.

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.

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.

Gene expression analysis in RA: towards personalized medicine.

Gene expression has recently been at the forefront of advance in personalized medicine, notably in the field of cancer and transplantation, providing a rational for a similar approach in rheumatoid arthritis (RA). RA is a prototypic inflammatory autoimmune disease with a poorly understood etiopathogenesis. Inflammation is the main feature of RA; however, many biological processes are involved at different stages of the disease. Gene expression signatures offer management tools to meet the current needs for personalization of RA patients' care. This review analyses currently available information with respect to RA diagnostic, prognostic and prediction of response to therapy with a view to highlight the abundance of data, whose comparison is often inconclusive due to the mixed use of material source, experimental methodologies and analysis tools, reinforcing the need for harmonization if gene expression signatures are to become a useful clinical tool in personalized medicine for RA patients.

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.

Therapeutic efficacy of Tyro3, Axl, and Mer tyrosine kinase agonists in collagen-induced arthritis.

OBJECTIVE: Hyperactivation of innate immunity by Toll-like receptors (TLRs) can contribute to the development of autoinflammatory or autoimmune diseases. This study evaluated the activation of Tyro3, Axl, Mer (TAM) receptors, physiologic negative regulators of TLRs, by their agonists, growth arrest-specific protein 6 (GAS-6) and protein S, in the prevention of collagen-induced arthritis (CIA). METHODS: Adenoviruses overexpressing GAS-6 and protein S were injected intravenously or intraarticularly into mice during CIA. Splenic T helper cell subsets from intravenously injected mice were studied by flow cytometry, and the knee joints of mice injected intravenously and intraarticularly were assessed histologically. Synovium from mice injected intraarticularly was evaluated for cytokine and suppressor of cytokine signaling (SOCS) expression. RESULTS: Protein S significantly reduced ankle joint swelling when overexpressed systemically. Further analysis of knee joints revealed a moderate reduction in pathologic changes in the joint and a significant reduction in the number of splenic Th1 cells when protein S was overexpressed systemically. Local overexpression of GAS-6 decreased joint inflammation and joint pathology. Protein S treatment showed a similar trend of protection. Consistently, GAS-6 and protein S reduced cytokine production in the synovium. Moreover, levels of messenger RNA for interleukin-12 (IL-12) and IL-23 were reduced by GAS-6 and protein S treatment, with a corresponding decrease in the production of interferon-γ and IL-17. TAM ligand overexpression was associated with an increase in SOCS-3 levels, which likely contributed to the amelioration of arthritis. CONCLUSION: This study provides the first evidence that TAM receptor stimulation by GAS-6 and protein S can be used to ameliorate arthritis when applied systemically or locally. TAM receptor stimulation limits proinflammatory signaling and adaptive immunity. This pathway provides a novel strategy by which to combat rheumatoid arthritis.

Patient involvement in basic rheumatology research at Nijmegen: a three year's responsive evaluation of added value, pitfalls and conditions for success.

BACKGROUND: Empirical evidence for effective patient-researcher collaboration in basic research is lacking. This study aims to explore good working models and impact of patient involvement in basic rheumatology research and to identify barriers and facilitators. METHOD: A responsive evaluation of a three years' participatory research project in a basic and translational laboratory research setting. Several working models for patient involvement were piloted and adapted if considered necessary. The study comprised surveys, interviews, training days, meeting reports, Q-sort exercises and field notes, and regular reflective team sessions with participant involvement. A qualitative analysis using thematic coding focused on impact, barriers and facilitators. RESULTS: Thirteen patient research partners (PRPs) and fifteen basic researchers participated. PRPs experienced basic research as fascinating though complex to understand. Their initial role was mostly listening and asking questions. After several meetings equal and more meaningful relationships emerged. Researchers' motivation increased by listening to patient stories. They learned about disease impact on daily life and to speak in understandable language. This enabled PRPs to learn about research and the pathogenesis of their disease. It inspired them to stay involved over a longer period. After three years, both parties preferred 1:1 contacts over collaboration in team meetings. A common language and respectful communication were important facilitators. Limitations were the complexity of disease processes for patients and the time commitment for researchers. Impact was reported as a sincere dialogue with multiple advantages for patients and researchers, and to a lesser extent than expected on the research process and outcomes. CONCLUSION: Patient involvement contributes to motivating young scientists in performing basic research projects. Patients and researchers valued the benefits of long-term one-on-one collaboration. These benefits outweigh the lack of direct impact on basic research goals and performance. A plain language summary of the abstract is available (as) online Additional file 1.

The natural soluble form of IL-18 receptor beta exacerbates collagen-induced arthritis via modulation of T-cell immune responses.

OBJECTIVE: IL-18 is a pluripotent cytokine that has been implicated in the development of rheumatoid arthritis. A soluble form of the IL-18 receptor accessory protein (sIL-18Rbeta) with unknown function has recently been identified. This study examined the ability of sIL-18Rbeta to inhibit IL-18 biological activities and to modulate immune responses during collagen-induced arthritis (CIA). METHODS: Adenoviruses encoding sIL-18Rbeta were administered intravenously in type II collagen-immunised DBA/1 mice. Humoral responses were analysed by determining anti-bovine collagen type II (BCII) antibody levels by ELISA. Cytokine production by splenic T cells and cytokine levels in serum were measured by Luminex multi-analyte technology. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) were measured by flow cytometry. RESULTS: Intravenous delivery of Ad5.sIL-18Rbeta in collagen-immunised mice led to enhanced transgene expression in splenic antigen-presenting cells (APC). A co-culture of these sIL-18Rbeta-transduced APC with purified splenic CD3(+) T cells led to a marked inhibition of IL-18-induced IFNgamma, IL-4 and IL-17 production by CD3(+) T cells. Remarkably, systemic treatment with Ad5.sIL-18Rbeta caused an exacerbation of arthritis, and histological evaluation of knee joints showed increased cartilage and bone erosion. No significant differences were observed in anti-BCII antibodies, but the aggravation was accompanied by decreased IFNgamma (-30%) and IL-4 (-44%) and increased IL-17 (+84%) production by splenic CD3(+) T cells. In addition, reduced circulating levels of CD4(+)CD25(+)Foxp3(+) Treg and anti-inflammatory IL-10 were shown. CONCLUSION: This study identifies sIL-18Rbeta as a novel IL-18 inhibitor, which promotes CIA after intravenous overexpression by affecting Treg levels and supporting a T helper type 17 response.

TGFß-mediated expression of TGFß-activating integrins in SSc monocytes: disturbed activation of latent TGFß?

INTRODUCTION: The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFß signaling. TGFß is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFß in serum of SSc patients and investigated if this correlates with integrin expression on monocytes. METHODS: A TGFß/SMAD3- or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFß and BMP levels respectively. A pan-specific TGFß1/2/3 neutralizing antibody was used to confirm TGFß signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFß1 or a small molecule inhibitor of TGFBR1: SB-505124. RESULTS: SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFß levels were not different. Addition of a pan-specific TGFß1/2/3 neutralizing antibody fully inhibited SMAD3-reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFß1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFß receptor inhibitor SB-505124 had the opposite effect. CONCLUSION: Total TGFß serum levels are not different between SSc patients and controls, but TGFß activity is. This coincides with a reduced expression of TGFß-activating integrins in monocytes of SSc patients. Because TGFß regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.

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.

Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti-IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-alpha, IL-6, and IFN-gamma secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.

Corrigendum: Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

This corrects the article DOI: 10.1038/srep43923.