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Objective: Tumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis., Methods: Bone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees., Results: TSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group., Conclusion: Gene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation., Competing Interests: The authors declare that they have no competing interests.

Objective: Rheumatoid arthritis (RA) is a chronic and progressive joint disease. It appears that anti-inflammatory feedback mechanisms that could restrain joint inflammation and restore homeostasis are insufficient to perform this control. In this study, we investigated the contribution of the MER tyrosine kinase-mediated anti-inflammatory response on arthritis and whether targeting MER could be a valid approach to treat RA., Methods: KRN serum transfer arthritis (KRN STA) was induced in either Mertk -deficient mice or in mice that adenovirally overexpressed Pros1 . Human synovial micromasses were treated with MER-specific antibodies or PROS1. Collagen-induced arthritis (CIA) mice were treated with MER-specific agonistic antibodies or by viral overexpression of Pros1 ., Results: Mertk -/- mice showed exacerbated arthritis pathology, whereas Pros1 overexpression diminished joint pathology in KRN STA. Human synovial micromasses challenged with MER-specific antibodies enhanced the secretion of inflammatory cytokines, whereas stimulating MER with PROS1 reduced the secretion of these cytokines, confirming the protective role of MER. Next, we treated CIA mice with MER-specific agonistic antibodies, and this unexpectedly resulted in exacerbated arthritis pathology. This was associated with increased numbers of apoptotic cells in their knee joints and higher serum levels of interleukin (IL)-16C, a cytokine released by secondary necrotic neutrophils. Apoptotic cell numbers and IL-16C levels were enhanced during arthritis in Mertk -/- mice and reduced in Pros1 -overexpressing mice., Conclusion: MER plays a protective role during joint inflammation and activating MER by its ligand PROS1 ameliorates disease. Treatment of mice with MER receptor agonistic antibodies is deleterious due to its counterproductive effect of blocking efferocytosis in the arthritic joint.

Osteoarthritis (OA) is a degenerative joint disease affecting cartilage and is the most common form of arthritis worldwide. One third of OA patients have severe synovitis and less than 10% have no evidence of synovitis. Moreover, synovitis is predictive for more severe disease progression. This offers a target for therapy but more research on the pathophysiological processes in the synovial tissue of these patients is needed. Functional studies performed with synovial tissue will be more approachable when this material, that becomes available by joint replacement surgery, can be stored for later use. We set out to determine the consequences of slow-freezing of human OA synovial tissue. Therefore, we validated a method that can be applied in every routine laboratory and performed a comparative study of five cryoprotective agent (CPA) solutions. To determine possible deleterious cryopreservation-thaw effects on viability, the synovial tissue architecture, metabolic activity, RNA quality, expression of cryopreservation associated stress genes, and expression of OA characteristic disease genes was studied. Furthermore, the biological activity of the cryopreserved tissue was determined by measuring cytokine secretion induced by the TLR ligands lipopolysaccharides and Pam3Cys. Compared to non frozen synovium, no difference in cell and tissue morphology could be identified in the conditions using the CS10, standard and CryoSFM CPA solution for cryopreservation. However, we observed significantly lower preservation of tissue morphology with the Biofreeze and CS2 media. The other viability assays showed trends in the same direction but were not sensitive enough to detect significant differences between conditions. In all assays tested a clearly lower viability was detected in the condition in which synovium was frozen without CPA solution. This detailed analysis showed that OA synovial tissue explants can be cryopreserved while maintaining the morphology, viability and phenotypical response after thawing, offering enhanced opportunities for human in vitro studies., Competing Interests: The authors have declared that no competing interests exist.

Background: Gene therapy has the potential to provide long-term production of therapeutic proteins in the joints of osteoarthritis (OA) patients. The objective of this study was to analyse the therapeutic potential of disease-inducible expression of anti-inflammatory interleukin-10 (IL-10) in the three-dimensional micromass model of the human synovial membrane., Methods: Synovial tissue samples from OA patients were digested and the cells were mixed with Matrigel to obtain 3D micromasses. The CXCL10 promoter combined with the firefly luciferase reporter in a lentiviral vector was used to determine the response of the CXCL10 promoter to tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and lipopolysaccharide (LPS). The effects of recombinant IL-10 on gene expression were determined by quantitative PCR. The production of IL-10 from the CXCL10p-IL10 vector and the effects on pro-inflammatory cytokine production were assessed by multiplex ELISA., Results: Micromasses made from whole synovial membrane cell suspensions form a distinct surface composition containing macrophage and fibroblast-like synoviocytes thus mimicking the synovial lining. This lining can be transduced by lentiviruses and allow CXCL-10 promoter-regulated transgene expression. Adequate amounts of IL-10 transgene were produced after stimulation with pro-inflammatory factors able to reduce the production of synovial IL-1β and IL-6., Conclusions: Synovial micromasses are a suitable model to test disease-regulated gene therapy approaches and the CXCL10p-IL10 vector might be a good candidate to decrease the inflammatory response implicated in the pathogenesis of OA.

Disease-inducible promoters for the treatment of rheumatoid arthritis (RA) have the potential to provide regulated expression of therapeutic proteins in arthritic joints. In this study, we set out to identify promoters of human genes that are upregulated during RA and are suitable to drive the expression of relevant amounts of anti-inflammatory interleukin (IL)-10. Microarray analysis of RA synovial biopsies compared with healthy controls yielded a list of 22 genes upregulated during RA. Of these genes, CXCL10 showed the highest induction in lipopolysaccharide-stimulated synovial cells. The CXCL10 promoter was obtained from human cDNA and cloned into a lentiviral vector carrying firefly luciferase to determine the promoter inducibility in primary synovial cells and in THP-1 cells. The promoter activation was strongest 8-12 hr after stimulation with the proinflammatory cytokine tumor necrosis factor (TNF)-α and was reinducible after 96 hr. In addition, the CXCL10 promoter showed a significant response to RA patient serum, compared with sera from healthy individuals. The luciferase gene was replaced with IL-10 to determine the therapeutic properties of the CXCL10p-IL10 lentiviral vector. Primary synovial cells transduced with CXCL10p-IL10 showed a great increase in IL-10 production after stimulation, which reduced the release of proinflammatory cytokines TNF-α and IL-1β. We conclude that the selected proximal promoter of the CXCL10 gene responds to inflammatory mediators present in the serum of patients with RA and that transduction with the lentiviral CXCL10p-IL10 vector reduces inflammatory cytokine production by primary synovial cells from patients with RA. CXCL10 promoter-regulated IL-10 overexpression can thus provide disease-inducible local gene therapy suitable for RA.

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

Objectives: Rheumatoid arthritis is a chronic destructive autoimmune disease, but the course is unpredictable in individual patients. An attractive treatment would provide a disease-regulated therapy that offers personalised drug delivery. Therefore, we expressed the anti-inflammatory interleukin-10 (IL-10) gene under the control of inflammation-dependent promoters in a mouse model of arthritis., Methods: Proximal promoters of S100a8, Cxcl1, Mmp13, Saa3, IL-1b and Tsg6 were selected by whole-genome expression analysis of inflamed synovial tissues from arthritic mice. Mice were injected intraarticularly in knee joints with lentiviral vectors expressing a luciferase reporter or the therapeutic protein IL-10 under control of the Saa3 or Mmp13 promoter. After 4 days, arthritis was induced by intraarticular injection of streptococcal cell walls (SCW). At different time points after arthritis induction, in vivo bioluminescent imaging was performed and knee joints were dissected for histological and RNA analysis., Results: The disease-regulated promoter-luciferase reporter constructs showed different activation profiles during the course of the disease. The Saa3 and Mmp13 promoters were significantly induced at day 1 or day 4 after arthritis induction respectively and selected for further research. Overexpression of IL-10 using these two disease-inducible promoters resulted in less synovitis and markedly diminished cartilage proteoglycan depletion and in upregulation of IL-1Ra and SOCS3 gene expression., Conclusions: Our study shows that promoters of genes that are expressed locally during arthritis can be candidates for disease-regulated overexpression of biologics into arthritic joints, as shown for IL-10 in SCW arthritis. The disease-inducible approach might be promising for future tailor-made local gene therapy in arthritis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Scope: This study shows the effect of bovine milk derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra-deficient mice and collagen-induced arthritis., Methods and Results: BMEVs were isolated from semi-skimmed milk by ultracentrifugation and the particle size was around 100 nm by dynamic light scattering and electron microscopy. BMEVs expressed exosome marker CD63, immunoregulatory microRNA's (miR-30a, -223, -92a), and milk-specific beta-casein and beta-lactoglobulin mRNA. In vitro, PKH-67-labeled BMEVs were taken up by RAW264.7, splenocytes, and intestinal cells as determined by flow cytometry and confocal microscopy. IL-1Ra(-/-) mice received BMEVs by daily oral gavage starting at wk 5 till 15 after birth and collagen-induced arthritis mice via their drinking water starting 1 wk before immunization till day 40. Macroscopically, BMEV treatment delayed the onset of arthritis and histology showed diminished cartilage pathology and bone marrow inflammation in both models. BMEV treatment also reduced the serum levels of MCP-1 and IL-6 and their production by splenic cells. BMEV treatment diminished the anticollagen IgG2a levels, which was accompanied by reduced splenic Th1 (Tbet) and Th17 (RORγT) mRNA., Conclusion: This is the first report that oral delivery of BMEVs ameliorates experimental arthritis and this warrants further research to determine whether this beneficial effect can be seen in rheumatoid arthritis patients., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Superparamagnetic Iron Oxide Nanoparticles (SPION) are used in diagnostic imaging of a variety of different diseases. For such in-vivo application, an additional coating with a polymer, for example polyvinyl alcohol (PVA), is needed to stabilize the SPION and prevent aggregation. As the particles are foreign to the body, reaction against the SPION could occur. In this study we investigated the effects that SPION may have on experimental arthritis after intra-articular (i.a.) or intravenous (i.v.) injection., Methods: PVA-coated SPION were injected either i.a. (6 or 24 μg iron) or i.v. (100 μg or 1 mg iron) into naïve Toll-like receptor-4 deficient (TLR4-/-) or wild-type C57Bl/6 mice, or C57Bl/6 mice with antigen-induced arthritis. As control, some mice were injected with PVA or PBS. MR imaging was performed at 1 and 7 days after injection. Mice were sacrificed 2 hours and 1, 2, 7, 10 and 14 days after injection of the SPION, and RNA from synovium and liver was isolated for pro-inflammatory gene expression analysis. Serum cytokine measurements and whole knee joint histology were also performed., Results: Injection of a high dose of SPION or PVA into naïve knee joints resulted in an immediate upregulation of pro-inflammatory gene expression in the synovium. A similar gene expression profile was observed after SPION or PVA injection into knee joints of TLR4-/- mice, indicating that this effect is not due to LPS contamination. Histological analysis of the knee joints also revealed synovial inflammation after SPION injection. Two hours after i.v. injection of SPION or PVA into naïve mice, an upregulation of pro-inflammatory gene expression was detected in the liver. Administration of SPION or PVA into arthritic mice via i.a. injection did not result in an upregulation in gene expression and also no additional effects were observed on histology. MR imaging and histology showed long-term retention of SPION in the inflamed joint. However, 14 days after the injections no long-term effects were evident for gene expression, histology or serum cytokine concentrations., Conclusions: Injection of SPION, either locally or systemically, gives an acute inflammatory response. In the long term, up to 14 days after the injection, while the SPION reside in the joint, no further activating effects of SPION were observed. Hence, we conclude that SPION do not aggravate arthritis and can therefore be used safely to detect joint inflammation by MR imaging.

Scope: Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells., Methods and Results: Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. Milk-derived extracellular vesicles were easily taken up by murine macrophages in vitro. Furthermore, we found that they can facilitate T cell differentiation towards the pathogenic Th17 lineage. Using a (CAGA)12-luc reporter assay we showed that these extracellular vesicles carried bioactive TGF-β, and that anti-TGF-β antibodies blocked Th17 differentiation., Conclusion: Our findings show that commercial milk contains stable extracellular vesicles, including exosomes, and carry immunoregulatory cargo. These data suggest that the extracellular vesicles present in commercial cow milk remains intact in the gastrointestinal tract and exert an immunoregulatory effect.

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-β (TGF-β), 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-β1 and/or Interleukin-1 (IL-1)β. 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-β pathway inducing NGF., Results: NGF expression was consistently induced in higher levels by TGF-β 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-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-β exposure led to a consistent high level of NGF induction., Conclusion: We show for the first time that TGF-β induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA., (Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Rheumatoid arthritis (RA) and osteoarthritis (OA) are serologically and clinically distinctive, but at the local level, both diseases have many molecular pathways in common. In vivo molecular imaging can unravel the local pathologic processes involved in both diseases. In this study, we investigated matrix metalloproteinase (MMP) and cathepsin activity during cartilage destruction, in an RA and an OA mouse model, using biophotonic imaging of substrate-based probes. Mice with collagen-induced arthritis (CIA) or destabilization of the medial meniscus (DMM) were imaged using near-infrared fluorescent probes, activated by several cathepsins or MMPs. Fluorescence signal intensity was compared to synovial gene expression, histology, and cartilage staining of a neoepitope of aggrecan cleaved by MMPs with the amino acids DIPEN. Increased cathepsin and MMP activity was seen during CIA, whereas the DMM model only showed increased MMP activity. DIPEN expression was seen only during CIA. A possible explanation can be differences in gene expressions; MMP3 and -13, known to produce DIPEN neoepitopes, were upregulated in the CIA model, whereas MMP12, known to be involved in elastin degradation and chemokine inhibition, was upregulated in the DMM model. Thus, molecular imaging showed no cathepsin activity at the time of cartilage damage in the DMM model, whereas both cathepsins and MMPs are active in the CIA model during disease progression.

Objective: A prominent role of Toll-like receptor 4 (TLR4) in arthritis is emerging. TLR4 is functional in immune cells and stromal cells. The aim was to investigate the involvement of TLR4 in bone marrow (BM)-derived and resident cells in arthritis., Methods: Reciprocal sex-mismatched BM transplantation was performed between IL-1Ra(-/-)TLR4(+/+) and IL-1Ra(-/-)TLR4(-/-) double knockout animals in Balb/c background. Arthritis was assessed macroscopically and by histopathology. Immunity was evaluated by splenic cytokine production and flow cytometry in draining lymph node (DLN) cells., Results: Arthritis progression was reduced to a similar extent in animals lacking TLR4 on BM-derived, resident cells or both. Histology revealed that joint inflammation was partially TLR4-dependent in either BM-derived or resident cells. TLR4 plays an additive role in BM-derived and resident cells in promoting cartilage erosion. By contrast, TLR4 was equally important in BM-derived and resident cells in mediating bone erosion. Systemically, TLR4 in both BM-derived and resident cells contributed to IL-17 production by splenic T-cells, whereas in the DLNs of arthritic joints this was not the case. Interestingly, in DLN, the dominant cells producing IL-17 were CD4 negative, and cell numbers were determined by TLR4 in the BM-derived cells., Conclusions: TLR4 is necessary in both BM-derived and resident cells for full-blown joint swelling, inflammation and bone erosion. Furthermore, TLR4 on BM-derived and tissue-resident cells show an additive effect in cartilage destruction. Interestingly, TLR4 on BM-derived and tissue-resident cells are both required for IL-17 production in spleen, but only in BM-derived cells in DLN.

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., (Copyright © 2013 by the American College of Rheumatology.)

Antigen presenting cells (APCs) play an important role in arthritis and APC specific gene therapeutic targeting will enable intracellular modulation of cell activity. Viral mediated overexpression is a potent approach to achieve adequate transgene expression levels and lentivirus (LV) is useful for sustained expression in target cells. Therefore, we studied the feasibility of lentiviral mediated targeting of APCs in experimental arthritis. Third generation VSV-G pseudotyped self-inactivating (SIN)-LV were injected intravenously and spleen cells were analyzed with flow cytometry for green fluorescent protein (GFP) transgene expression and cell surface markers. Collagen-induced arthritis (CIA) was induced by immunization with bovine collagen type II in complete Freund's adjuvant. Effect on inflammation was monitored macroscopically and T-cell subsets in spleen were analyzed by flow cytometry. Synovium from arthritic knee joints were analyzed for proinflammatory cytokine expression. Lentiviruses injected via the tail vein preferentially infected the spleen and transduction peaks at day 10. A dose escalating study showed that 8% of all spleen cells were targeted and further analysis showed that predominantly Ly6C+ and F4/80+ cells in spleen were targeted by the LV. To study the feasibility of blocking TAK1-dependent pathways by this approach, a catalytically inactive mutant of TAK1 (TAK1-K63W) was overexpressed during CIA. LV-TAK1-K63W significantly reduced incidence and arthritis severity macroscopically. Further histological analysis showed a significant decrease in bone erosion in LV-TAK1-K63W treated animals. Moreover, systemic Th17 levels were decreased by LV-TAK1-K63W treatment in addition to diminished IL-6 and KC production in inflamed synovium. In conclusion, systemically delivered LV efficiently targets monocytes and macrophages in spleen that are involved in autoimmune arthritis. Moreover, this study confirms efficacy of TAK1 targeting in arthritis. This approach may provide a valuable tool in targeting splenic APCs, to unravel their role in autoimmune arthritis and to identify and validate APC specific therapeutic targets.

Objective: To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences., Methods: Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. To ascertain the role of SOCS-3 in the chondrocyte response to interleukin-1β (IL-1β) or lipopolysaccharide (LPS), the expression of SOCS3 was either reduced by small interfering RNA or enhanced by viral transduction., Results: The expression of SOCS-3 mRNA (but not that of SOCS-1 mRNA) was significantly enhanced in chondrocytes obtained from OA cartilage (mean ± SD ΔC(t) 3.4 ± 1.0) and RA cartilage (ΔC(t) 3.4 ± 1.4) compared with cartilage obtained from patients with femoral neck fracture (ΔC(t) 5.3 ± 1.2). The expression of SOCS3 correlated significantly with that of other genes known to be expressed in arthritic chondrocytes, such as MMP13 (r = 0.743), ADAMTS4 (r = 0.779), and ADAMTS5 (r = 0.647), and an inverse relationship was observed with COL2A1 (r = -0.561). Up-regulation of SOCS-3 by IL-1 in G6 chondrocytes and its spontaneous expression in OA chondrocytes were reduced by mithramycin, a specific inhibitor of transcription factor Sp-1. Overexpression of SOCS-3 in bovine chondrocytes reduced IL-1- and LPS-induced nitric oxide production and insulin-like growth factor 1-induced proteoglycan synthesis. Interestingly, a similar impairment of function was observed in OA chondrocytes, which was partially restored by SOCS-3 gene knockdown., Conclusion: This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology., (Copyright © 2012 by the American College of Rheumatology.)

Objective: Suppressor of cytokine signalling (SOCS) proteins constitute a class of intracellular proteins that are key physiological regulators of immune cell function. It has previously been shown that antigen-presenting cells (APCs) overexpressing SOCS3 steer T helper immune responses and protect against experimental arthritis. A study was undertaken to investigate the contribution of SOCS3 in regulating invariant natural killer T (iNKT) cell responses during collagen-induced arthritis (CIA)., Methods: DBA/1 mice were immunised with type II collagen and adenoviruses encoding SOCS3 were administered intravenously before the clinical onset of arthritis. Murine APCs overexpressing SOCS3 were co-cultured with an iNKT cell hybridoma and interleukin 2 (IL-2) release was measured by Luminex multi-analyte technology. The frequency and activation of primary iNKT cells was assessed by flow cytometry. Murine APCs were analysed for cytokine and CD1d expression following viral SOCS3 gene transfer., Results: Viral overexpression of SOCS3 in APCs resulted in reduced activation of the iNKT cell hybridoma. Importantly, during initiation of CIA, adenovirus-mediated overexpression of SOCS3 in hepatic and splenic APCs inhibited iNKT cell expansion in both organs. The iNKT cell population from SOCS3-treated mice showed low expression of the early activation marker CD69 and primary liver iNKT cells produced less interferon γ and IL-4 upon α-galactosylceramide stimulation. No differences in CD1d surface expression were observed, but SOCS3-transduced APCs produced decreased levels of proinflammatory cytokines and increased levels of IL-10., Conclusion: These results demonstrate a critical role for SOCS3 in controlling the immunostimulatory capacities of APCs, which has direct implications for the effector function of iNKT cells during arthritis.

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium., (2011 Elsevier B.V. All rights reserved.)

Introduction: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that mainly affects synovial joints. Biologics directed against tumor-necrosis-factor (TNF)-alpha are efficacious in the treatment of RA. However, the role of TNF receptor-1 (TNFR1) in mediating the TNFalpha effects in RA has not been elucidated and conflicting data exist in experimental arthritis models. The objective is to investigate the role of TNFR1 in the synovial lining cells (SLC) and the reticuloendothelial system (RES) during experimental arthritis., Methods: Third generation of adenovirus serotype 5 were either injected locally in the knee joint cavity or systemically by intravenous injection into the retro-orbital venous sinus to specifically target SLC and RES, respectively. Transduction of organs was detected by immunohistochemistry of the eGFP transgene. An adenoviral vector containing a short hairpin (sh) RNA directed against TNFR1 (HpTNFR1) was constructed and functionally evaluated in vitro using a nuclear factor-kappaB (NF-kappaB) reporter assay and in vivo in streptococcal cell wall-induced arthritis (SCW) and collagen-induced arthritis (CIA). Adenoviruses were administered before onset of CIA, and the effect of TNFR1 targeting on the clinical development of arthritis, histology, quantitative polymerase chain reaction (qPCR), cytokine analyses and T-cell assays was evaluated., Results: Systemic delivery of Ad5.CMV-eGFP predominantly transduced the RES in liver and spleen. Local delivery transduced the synovium and not the RES in liver, spleen and draining lymph nodes. In vitro, HpTNFR1 reduced the TNFR1 mRNA expression by three-fold resulting in a 70% reduction of TNFalpha-induced NF-kappaB activation. Local treatment with HpTNFR1 markedly reduced mRNA and protein levels of interleukin (IL)-1beta and IL-6 in SLC during SCW arthritis and ameliorated CIA. Systemic targeting of TNFR1 in RES of liver and spleen by systemic delivery of Ad5 virus encoding for a small hairpin RNA against TNFR1 markedly ameliorated CIA and simultaneously reduced the mRNA expression of IL-1beta, IL-6 and Saa1 (75%), in the liver and that of Th1/2/17-specific transcription factors T-bet, GATA-3 and RORgammaT in the spleen. Flow cytometry confirmed that HpTNFR1 reduced the numbers of interferon (IFN)gamma (Th1)-, IL-4 (Th2)- and IL-17 (Th17)-producing cells in spleen., Conclusions: TNFR1-mediated signaling in both synovial lining cells and the reticuloendothelial system independently played a major pro-inflammatory and immunoregulatory role in the development of experimental arthritis.

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.

Background: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products., Methodology/principal Findings: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior., Conclusion/significance: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients.

The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define proximal-promoters from a gene expression profiling study of murine experimental arthritis. Synovium expression profiles from progressing stages of collagen-induced arthritis (CIA) were classified into six distinct groups using k-means clustering. Using an algorithm based on local over-representation and comparative genomics, we identified putatively functional transcription factor-binding sites (TFBS) in TATA-dependent proximal-promoters. Applying a filter based on spacing between TATA box and transcription start site (TSS) combined with the presence of over-represented nuclear factor kappaB (NFkappaB), AP-1, or CCAAT/enhancer-binding protein beta (C/EBPbeta) sites, 382 candidate murine and human promoters were reduced to 66, corresponding to 45 genes. In vitro, 9 out of 10 computationally defined promoter regions conferred cytokine-inducible expression in murine cells and human synovial fibroblasts. Under these conditions, the serum amyloid A3 (Saa3) promoter showed the strongest transcriptional induction and strength. We applied this promoter for driving therapeutically efficacious levels of the interleukin-1 receptor antagonist (Il1rn) in a disease-regulated fashion. These results demonstrate the value of bioinformatics for guiding the selection of endogenous promoters for transcriptionally targeted gene therapy.

Objective: Members of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS-3 in splenic antigen-presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS-3., Methods: DBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen-induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi-analyte technology. Th cell populations were analyzed by flow cytometry., Results: Systemic delivery of adenovirus encoding SOCS-3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin-23 (IL-23), IL-6, and tumor necrosis factor alpha, but significantly higher production of antiinflammatory IL-10, by these cells. Fluorescence-activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS-3 treatment. In the presence of SOCS-3-transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen-specific proliferation and a significant reduction in the production of interferon-gamma (-43%), IL-4 (-41%), and IL-17 (-70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction., Conclusion: This study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS-3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.

The application of disease-regulated promoters in local gene therapy for rheumatoid arthritis potentiates the development of a sophisticated treatment that relies on a restricted and fine-tuned supply of biologicals. Although several studies have investigated regulated promoters for achieving effective transgene expression during arthritis, none have explored their potential for minimizing deleterious effects arising from constitutive overexpression of transgenes under naive conditions. Using naive and collagen-induced arthritic mice, we examined the applicability of a hybrid interleukin-1 enhancer/interleukin-6 proximal promoter for achieving efficacious murine interleukin-4 gene therapy under arthritic conditions, while minimizing interleukin-4-induced inflammation under naive conditions. We found strong upregulation of transgene expression in virally transduced knee joints under arthritic conditions compared to levels in naive animals. Besides its responsiveness, the promoter strength proved sufficient for generating therapeutically efficacious levels interleukin-4, as demonstrated by the successful protection against cartilage erosion in collagen-induced arthritis. Most importantly, promoter-mediated restriction of the potent chemotactic interleukin-4 in naive animals strongly reduced the amounts of inflammatory cell influx. This study suggests the suitability of the interleukin-1 enhancer/interleukin-6 proximal promoter for the development of a local gene therapy strategy for rheumatoid arthritis that requires fine-tuned and restricted expression of transgenes with a pleiotrophic nature.

Objective: An important mechanism contributing to cartilage destruction in arthritis is chondrocyte desensitization toward its main anabolic factor, insulin-like growth factor 1 (IGF-1). In this study, we sought to determine the role of suppressor of cytokine signaling 3 (SOCS-3) in the induction of IGF-1 desensitization of murine chondrocytes., Methods: Chondrocyte responsiveness to IGF-1 was assessed by 35S-sulfate incorporation into proteoglycans (PGs), via aggrecan messenger RNA expression, using quantitative real-time polymerase chain reaction or insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (Western blot analysis). IGF-1 desensitization of patellar chondrocytes was studied in zymosan-induced arthritis. IGF-1 desensitization was induced in patellar cartilage explants or the H4 chondrocyte cell line, exposed to interleukin-1alpha (IL-1alpha). SOCS-3 protein expression was assessed by immunohistochemistry or by Western blot analysis of protein extracts. The role of SOCS-3 in IGF-1 signaling was elucidated by adenoviral overexpression., Results: Exposure of murine articular cartilage to IL-1 caused a significant decrease in IGF-1-induced PG synthesis. This effect also occurred in inducible nitric oxide synthase-knockout mice, revealing the involvement of a secondary IL-1-induced factor other than nitric oxide. We showed that IL-1 significantly up-regulated SOCS-3 transcription and protein synthesis in H4 chondrocytes. In contrast, IL-18 was unable to induce SOCS-3 expression and failed to induce chondrocyte IGF-1 desensitization. Histologic analysis of samples from arthritic knee joints revealed high expression of SOCS-3 in chondrocytes. Through adenoviral overexpression of SOCS-3, we obtained direct evidence that SOCS-3 inhibits IGF-1-mediated cell signaling, since IRS-1 phosphorylation was reduced., Conclusion: This study demonstrates that IL-1-induced SOCS-3 expression is a novel mechanism of IGF-1 desensitization in chondrocytes; in conjunction with nitric oxide it can contribute to cartilage damage during arthritis.

Objective: To discern the mode of interleukin-1 (IL-1) inhibition of soluble IL-1 receptor accessory protein (sIL-1RAcP) by comparison with IL-1 receptor antagonist (IL-1Ra) in arthritis., Methods: Adenoviral vectors encoding either sIL-1RAcP or IL-1Ra were administered systemically before onset of collagen-induced arthritis in DBA/1 mice. Anti-bovine type II collagen IgG and IL-6 were quantified in serum. Proliferative response of splenic T cells was determined in the presence of sIL-1RAcP or IL-1Ra. The effect on IL-1 inhibition of recombinant sIL-1RAcP and IL-1Ra was further examined in vitro, using NF-kappaB luciferase reporter cell lines. Quantitative polymerase chain reaction was used to determine the relative messenger RNA expression of the IL-1 receptors., Results: Adenoviral overexpression of both sIL-1RAcP and IL-1Ra resulted in amelioration of the collagen-induced arthritis. Both IL-1 antagonists reduced the circulating levels of antigen-specific IgG2a antibodies, but only IL-1Ra was able to inhibit lymphocyte proliferation. By using purified lymphocyte populations derived from NF-kappaB reporter mice, we showed that sIL-1RAcP inhibits IL-1-induced NF-kappaB activity in B cells but not T cells, whereas IL-1Ra inhibited IL-1 on both cell types. A study in a panel of NF-kappaB luciferase reporter cells showed that the sIL-1RAcP inhibits IL-1 signaling on cells expressing either low levels of membrane IL-1RAcP or high levels of IL-1RII., Conclusion: We show that the sIL-1RAcP ameliorated experimental arthritis without affecting T cell immunity, in contrast to IL-1Ra. Our results provide data in support of receptor competition by sIL-1RAcP as an explanation for the different mode of IL-1 antagonism in comparison with IL-1Ra.

Myotonic dystrophy protein kinase (DMPK) is a Ser/Thr-type protein kinase with unknown function, originally identified as the product of the gene that is mutated by triplet repeat expansion in patients with myotonic dystrophy type 1 (DM1). Alternative splicing of DMPK transcripts results in multiple protein isoforms carrying distinct C termini. Here, we demonstrate by expressing individual DMPKs in various cell types, including C(2)C(12) and DMPK(-/-) myoblast cells, that unique sequence arrangements in these tails control the specificity of anchoring into intracellular membranes. Mouse DMPK A and C were found to associate specifically with either the endoplasmic reticulum (ER) or the mitochondrial outer membrane, whereas the corresponding human DMPK A and C proteins both localized to mitochondria. Expression of mouse and human DMPK A-but not C-isoforms in mammalian cells caused clustering of ER or mitochondria. Membrane association of DMPK isoforms was resistant to alkaline conditions, and mutagenesis analysis showed that proper anchoring was differentially dependent on basic residues flanking putative transmembrane domains, demonstrating that DMPK tails form unique tail anchors. This work identifies DMPK as the first kinase in the class of tail-anchored proteins, with a possible role in organelle distribution and dynamics.

Objective: Interleukin-6 (IL-6) is expressed in osteoarthritic joints but its function in osteoarthritis (OA) is unknown. To study this, spontaneous and experimental OA were evaluated in IL-6 deficient (IL-6(-/-)) mice., Design: Histology of knees of 18-23-month-old wild type (wt) and IL-6(-/-) mice was compared for signs of OA. Cartilage proteoglycan (PG) density was measured by image analysis on safranin-O stained whole knee sections. Chondrocyte PG synthesis was measured ex vivo by (35)S-sulfate incorporation. Knee bone mineral density (BMD) was measured by dual energy x-ray absorptiometry. In young mice (3 months), OA was induced by intra-articular injection of collagenase., Results: The incidence of extensive cartilage loss at both lateral and medial sides was markedly higher in old IL-6(-/-) males, but not in females, as compared to their wt controls. Compared to age-matched wt mice, reduced ex vivo PG synthesis was found during aging in IL-6(-/-) males, without affecting their cartilage PG density. IL-6(-/-) males showed more extensive extracellular matrix deposition in the collateral ligaments and subchondral bone sclerosis, predominantly at the medial side. Total knee BMD decreased more in IL-6(-/-) (-23%) than in wt (-10%) males during aging. Collagenase-induced OA showed a similar degree of joint pathology in both strains, implying that OA susceptibility was not different at younger age., Conclusions: Upon aging, IL-6(-/-) male mice developed more severe spontaneous OA. Reduced PG synthesis and BMD values might be indicative for an impaired repair response in IL-6(-/-) mice. This suggests a protective role for IL-6 in age-related OA in male mice.

Diets enriched in the (n-3) PUFAs, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and their precursor alpha-linolenic acid (ALA), were evaluated for efficacy in ameliorating the development of IgA nephropathy (IgAN) induced in mice by the mycotoxin deoxynivalenol (DON). The effects of DON were compared in mice that were fed for 18 wk with AIN-93G diets containing 1) 10 g/kg corn oil plus 60 g/kg oleic acid (control); 2) 10 g/kg corn oil plus 35 g/kg oleic acid and 25 g/kg DHA-enriched fish oil (DHA); 3) 10 g/kg corn oil plus 33 g/kg oleic acid and 27 g/kg EPA-enriched fish oil (EPA); and 4) 10 g/kg corn oil plus 37 g/kg oleic acid and 23 g/kg DHA + EPA (1:1) enriched fish oil (DHA + EPA). The DHA, EPA and DHA + EPA diets attenuated induction by dietary DON (10 mg/kg) of serum IgA and IgA immune complexes, kidney mesangial IgA deposition, and ex vivo IgA secretion by spleen cells. Consumption of the DHA + EPA diet for 8 wk significantly abrogated the DON-induced gene expression of interleukin (IL)-6, a requisite cytokine for DON-induced IgA nephropathy, in spleen and Peyer's patches. Finally, incorporation of ALA-containing flaxseed oil up to 60 g/kg in the AIN-93G diet did not affect DON-induced IgA dysregulation in mice. Taken together, both DHA and EPA, but not ALA, ameliorated the early stages of IgAN, and these effects might be related to a reduced capacity for IL-6 production.

Objective: STAT proteins play an important role in cytokine signaling. Some investigators have reported preferential activation of STAT-1, and others have reported preferential activation of STAT-3, in response to endogenous interleukin-6 (IL-6), in patients with rheumatoid arthritis. The present study was undertaken to investigate synovial STAT-1 and STAT-3 activation in an experimental animal model of arthritis., Methods: Zymosan was injected intraarticularly into naive wild-type (WT), IL-6(-/-), and STAT-1(-/-) mice to induce arthritis. Western blots of synovial lysates were probed with phosphospecific antibodies to detect STAT-1/STAT-3 activation. Inflammation was assessed histologically. Synovial gene expression of the STAT-induced feedback inhibitors suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 in WT and STAT-1(-/-) mice was investigated by reverse transcriptase-polymerase chain reaction., Results: STAT-3 was activated in inflamed synovium of WT mice throughout the course of disease, whereas activated STAT-1 was observed only during the chronic phase. In IL-6(-/-) mice, STAT activation was limited to STAT-3 on day 1. Although macrophage influx was not inhibited, disease went into remission after day 7 in IL-6(-/-) mice. STAT-1 deficiency resulted in exacerbation of chronic joint inflammation and granuloma formation. In STAT-1(-/-) mice, STAT-3 activation in the inflamed joints was unaltered as compared with WT mice. However, synovial SOCS-1, but not SOCS-3, gene expression was markedly reduced in STAT-1(-/-) mice., Conclusion: The results in the IL-6(-/-) mice suggest that STAT-3 is involved in the chronicity of ZIA. Exacerbation of arthritis in STAT-1(-/-) mice suggests an opposing effect of STAT-1, i.e., suppression of joint inflammation. The expression of SOCS-1 could be the underlying mechanism by which STAT-1 controls joint inflammation.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1 beta enhancer region (-3690 to -2720) upstream of the human IL-6 promoter region (-163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (10(6) ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6(-/-) and IL-1(-/-) gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.

Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-L-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox(-/-) and gp91(-/-) mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-alpha, interleukin-1alpha, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-kappaB ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage.

Objective: To investigate whether the soluble form of interleukin-1 (IL-1) receptor accessory protein (sIL-1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL-1 signaling in vitro, and to evaluate its applicability in collagen-induced arthritis (CIA)., Methods: Soluble IL-1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL-1RAcP or a stable-transfected NIH3T3 fibroblast cell line. The ability of affinity-purified sIL-1RAcP to inhibit IL-1 signaling was tested on NF-kappaB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL-1RAcP was both locally (knee joint) and systemically overexpressed in collagen-immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL-6 and anti-bovine type II collagen (BCII) antibody levels., Results: Incubation of the NF-kappaB reporter fibroblast with purified sIL-1RAcP protein showed a marked reduction of IL-1-induced, but not tumor necrosis factor-induced, NF-kappaB activation. This showed a novel role for sIL-1RAcP as a specific inhibitor of IL-1 signaling. Local transplantation of sIL-1RAcP-producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL-1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL-1RAcP had no profound effect on serum levels of IL-6 and anti-BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL-1RAcP markedly ameliorated CIA in all joints., Conclusion: In this study we demonstrated that sIL-1RAcP is a biologically active and innovative inhibitor of IL-1, and treatment of mice with sIL-1RAcP had a profound prophylactic effect on collagen-induced arthritis.

Elevated concentrations of interleukin-18 (IL-18) are found in both serum and synovial fluid of patients suffering from rheumatoid arthritis (RA) and this cytokine has recently been implicated in the development of experimental arthritis. In this present study, we developed an IL-18 neutralizing intervention and examined its efficacy for local intra-articular treatment of experimental arthritis. To this end we constructed an adenoviral vector containing the murine IL-18 binding protein isoform c gene (AdCMVIL-18BPc). The constructed adenoviral vector was validated on replication deficiency, transfection efficacy and ability to express biological functional IL-18BPc. Intra-articular overexpression of IL-18BPc significantly reduced incidence of collagen-induced arthritis (CIA) in treated kneejoints. Affected kneejoints of IL-18BPc-treated mice showed less severe arthritis, characterized by reduction of inflammation and destruction of bone and cartilage. Local intra-articular IL-1BPc treatment in both knees provided additional protection against CIA incidence and severity in distal paws. Measurement of serum levels of specific collagen type (CII) Abs revealed a moderate reduction of circulating IgG2a anti-CII Abs, while IgG1 anti-CII Abs remained at similar level. The present study underlines the involvement of IL-18 as an important proinflammatory cytokine in onset of experimental arthritis. Furthermore, it shows that endogenous IL-18 can be blocked efficiently through local adenoviral overexpression of IL-18BPc, indicating that treatment with IL-18BPc might contribute to joint protection in RA.

Objective: To investigate the involvement of proinflammatory and destructive mediators in oncostatin M (OSM)-induced joint pathology, using gene-deficient mice., Methods: An adenoviral vector expressing murine OSM was injected into the joints of naive wild-type mice and mice deficient for interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNFalpha), or inducible nitric oxide synthase (iNOS). Reverse transcription-polymerase chain reaction was used to study gene expression. Inflammation and cartilage proteoglycan (PG) depletion were assessed by histology. OSM and IL-1 levels in synovial fluid from patients with juvenile idiopathic arthritis (JIA) were measured by enzyme-linked immunosorbent assay., Results: Adenoviral expression of murine OSM led to joint inflammation, bone apposition, chondrophyte formation, articular cartilage PG depletion, and VDIPEN neoepitope expression in wild-type mice. A unique and consistent observation was the focal PG depletion and disorganization of the growth plate cartilage during the first week of inflammation. Synovial IL-1beta, IL-6, TNFalpha, and iNOS gene expression was strongly induced. Of these factors, only deficiency in IL-1 markedly reduced inflammation and PG depletion and completely prevented growth plate damage. In addition, this is the first study in which OSM was detected in JIA synovial fluid. Most samples were also IL-1beta positive., Conclusion: IL-1, but not IL-6, TNFalpha, or iNOS, plays an important role in joint disease induced by intraarticular gene transfer of OSM in mice. The effect of OSM on murine connective tissue and the presence of OSM in human synovial fluid make involvement of OSM in human arthropathies very likely.

Oncostatin M (OSM) has been described as a bone-remodeling factor either stimulating osteoblast activity or osteoclast formation in vitro. To elucidate the in vivo effect of OSM on bone remodeling, we injected an adenoviral vector encoding murine OSM in knee joints of mice. OSM strongly induced interleukin (IL)-6 gene expression, a known mediator of osteoclast development. We investigated the OSM effect in wild-type and IL-6-deficient mice and found a similar degree of OSM-induced joint inflammation. Within the first week of inflammation, the periosteum along the femur and tibia increased in cell number and stained positive for the osteoblast marker alkaline phosphatase. At these sites bone apposition occurred in both strains as demonstrated by Goldner and Von Kossa staining. In vitro OSM enhanced the effect of bone morphogenetic protein-2 on osteoblast differentiation. Immunohistochemistry demonstrated expression of receptor activator of nuclear factor-kappa B ligand (RANKL) and its receptor, receptor activator of nuclear factor-kappa B (RANK), in the periosteum but osteoclasts were not detected at sites of bone apposition. Induced mRNA expression for the receptor activator of nuclear factor-kappa B ligand inhibitor osteoprotegerin probably controlled osteoclast development during OSM overexpression. Our results show that OSM favors bone apposition at periosteal sites instead of resorption in vivo. This effect was not dependent on or inhibited by IL-6.

Adenoviral vectors (AdV) are used for anti-inflammatory cytokine therapy in experimental arthritis. Cell entry of AdV is dependent on the initial recognition of the coxsackie-adenovirus receptor (CAR) on cells. Recently, an Arg-Gly-Asp (RGD) motif was introduced in the HI loop of the fiber knob, this enables the adenovirus to bypass CAR and mediate cell entry via RGD binding integrins. In this study, we explored the transduction efficiency of the RGD-modified adenovirus in synovium and compared the RGD-modified with the conventional adenoviral vector for their effectiveness to modulate the murine collagen-induced arthritis (CIA) model when used to overexpress mIL-1Ra in the knee joint. Twenty-four hours after intra-articular injection of 10(7) fluorescent forming units (ffu) virus, luciferase (luc) activity in Ad5LucRGD-injected joints was up to 38 times higher than in AdCMVLuc-injected joints, and in arthritic joints the transduction efficiency was up to 69 times higher for the Ad5LucRGD viruses. Transduction of the synovial lining by the RGD-modified adenovirus containing the mIL-1Ra transgene, markedly improved the inhibition of CIA compared with the conventional virus in both a prophylactic and therapeutic treatment protocol. These results show that targeting integrins with the RGD-modified AdV improved the outcome of gene therapy for arthritis.