M.B. Bennink

Adenoviral delivery of IL-18 binding protein C ameliorates collagen-induced arthritis in mice.

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.

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1β 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 (106u2009ffu) 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.

Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-β.

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.

Oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models.

SCOPEnThis study shows the effect of bovine milk derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra-deficient mice and collagen-induced arthritis.nnnMETHODS AND RESULTSnBMEVs 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 microRNAs (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.nnnCONCLUSIONnThis 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.

A tropism-modified adenoviral vector increased the effectiveness of gene therapy for arthritis

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 107 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.

In vivo molecular imaging of cathepsin and matrix metalloproteinase activity discriminates between arthritic and osteoarthritic processes in mice

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.

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

OBJECTIVEnPain 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.nnnDESIGNnMurine 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.nnnRESULTSnNGF 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.nnnCONCLUSIONnWe 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.

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-18Rβ) with unknown function has recently been identified. This study examined the ability of sIL-18Rβ to inhibit IL-18 biological activities and to modulate immune responses during collagen-induced arthritis (CIA). Methods: Adenoviruses encoding sIL-18Rβ 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-18Rβ in collagen-immunised mice led to enhanced transgene expression in splenic antigen-presenting cells (APC). A co-culture of these sIL-18Rβ-transduced APC with purified splenic CD3+ T cells led to a marked inhibition of IL-18-induced IFNγ, IL-4 and IL-17 production by CD3+ T cells. Remarkably, systemic treatment with Ad5.sIL-18Rβ 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 IFNγ (−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-18Rβ as a novel IL-18 inhibitor, which promotes CIA after intravenous overexpression by affecting Treg levels and supporting a T helper type 17 response.

Complementary action of granulocyte macrophage colony-stimulating factor and interleukin-17A induces interleukin-23, receptor activator of nuclear factor-κB ligand, and matrix metalloproteinases and drives bone and cartilage pathology in experimental arthritis: rationale for combination therapy in rheumatoid arthritis

IntroductionType 17xa0T helper cells and interleukin (IL)-17 play important roles in the pathogenesis of human and murine arthritis. Although there is a clear link between IL-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) in the inflammatory cascade, details about their interaction in arthritic synovial joints are unclear. In view of the introduction of GM-CSF and IL-17 inhibitors to the clinic, we studied how IL-17 and GM-CSF orchestrate the local production of inflammatory mediators during experimental arthritis.MethodsTo allow detection of additive, complementary or synergistic effects of IL-17 and GM-CSF, we used two opposing experimental approaches: treatment of arthritic mice with neutralising antibodies to IL-17 and GM-CSF and local overexpression of these cytokines in naive synovial joints. Mice were treated for 2xa0weeks with antibodies against IL-17 and/or GM-CSF after onset of collagen-induced arthritis. Naive mice were injected intraarticularly with adenoviral vectors for IL-17 and/or GM-CSF, resulting in local overexpression. Joint inflammation was monitored by macroscopic scoring, X-rays and histology. Joint washouts, synovial cell and lymph node cultures were analysed for cytokines, chemokines and inflammatory mediators by Luminex analysis, flow cytometry and quantitative polymerase chain reaction.ResultsCombined therapeutic anti-IL-17 and anti-GM-CSF ameliorated arthritis progression, and joint damage was dramatically reduced compared with treatment with anti-IL-17 or anti-GM-CSF alone. Anti-IL-17 specifically reduced synovial IL-23 transcription, whereas anti-GM-CSF reduced transcription of matrix metalloproteinases (MMPs) and receptor activator of nuclear factor κB ligand (RANKL). Overexpression of IL-17 or GM-CSF in naive knee joints elicited extensive inflammatory infiltrate, cartilage damage and bone destruction. Combined overexpression revealed additive and synergistic effects on the production of MMPs, RANKL and IL-23 in the synovium and led to complete destruction of the joint structure within 7xa0days.ConclusionsIL-17 and GM-CSF differentially mediate the inflammatory process in arthritic joints and show complementary and local additive effects. Combined blockade in arthritic mice reduced joint damage not only by direct inhibition of IL-17 and GM-CSF but also by indirect inhibition of IL-23 and RANKL. Our results provide a rationale for combination therapy in autoinflammatory conditions, especially for patients who do not fully respond to inhibition of the separate cytokines.

Disease-regulated local IL-10 gene therapy diminishes synovitis and cartilage proteoglycan depletion in experimental arthritis

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 4u2005days, 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.