Faj van de Loo

inflammation-inducible intra-articular production of human IL-1 receptor antagonist results in a more efficient inhibition of collagen-induced arthritis than does constitutive expression of the same transgene

Background Achieving biologically effective yet safe levels of recombinant anti-inflammatory proteins by gene therapy may require three regulatory features: 1) a basal level that is sufficiently low to avoid or minimise chronic immunosuppression 2) transcriptional regulation over a wide dynamic range, and 3) induced expression levels that are sufficiently high to achieve the desired biological effects. The C3-Tat/HIV promoter construct seems to have these properties.1 In this two-component expression system the complement factor 3 (C3) promoter regulates production of the HIV transactivator of transcription, and the Tat protein then stimulates expression of the desired transgene, which is regulated by the HIV-LTR promoter. Both C3 and HIV LTR promoters are inducible by proinflammatory cytokines. Objectives To achieve disease-inducible expression of recombinant anti-inflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. Methods We compared a disease-inducible, two component expression system (C3-Tat/HIV) with the constitutive immediate early cytomegalovirus (ieCMV) promoter in the polyarticular Collagen-Induced Arthritis (CIA) model in mice. DBA/I mice were immunised with bovine type II collagen and boostered on day 22. On day 22, mice without any clinical signs of arthritis were selected and adenoviral vectors (Ad. CMV-Luc, Ad. CMV-IL-1Ra, or Ad. C3-Tat/HIV-IL-1Ra) that contained luciferase or the human IL-1Ra gene under control of one of the two promoters were used to transfect the synovial lining of both knees. The injected knee joints and ipsilateral paws were then scored for signs of arthritis and at the end histology was taken. Results Inducible promoter-driven IL-1Ra expression resulted in significantly improved inhibition of CIA than did CMV-driven IL-1Ra production. Moreover, overexpression of IL-1Ra in the knee joints also prevented CIA in the ipsilateral paws. Conclusion Our data (1) demonstrate the feasibility of an inducible expression system for producing a recombinant transgene for treatment of arthritis and (2) show that this system is more effective than strong, constitutive transgene expression for preventing collagen-induced arthritis in mice. Reference Varley AW, Geiszler SM, Gaynor RB, Munford RS. A two-component expression system that responds to inflammatory stimuli in vivoin vivo. Nat Biotechnol. 1997;15(10):1002–6

IL-18 blockade is a potential disease-modifying therapy for rheumatoid arthritis

Interleukin-18 (IL-18) has been demonstrated as promoting the development of a TH1 response in vivo in synergy with IL-12. Significant levels of IL-18 and IL-12 have been detected in the joints of patients with rheumatoid arthritis (RA).

A4.20 Viral expression of TSG-6 can stimulate osteophyte formation in experimental osteoarthritis

Background and objectives Tumour necrosis factor-inducible gene 6 (TSG-6, also named TNFAPI6) is upregulated during experimental arthritis in mice (Geurts et al. 2009). The therapeutic efficacy of TSG-6 has been shown in multiple animal models for rheumatoid arthritis, reducing both inflammation and cartilage damage. Recent studies suggest that inflammation can also play a role in early osteoarthritis (OA) pathogenesis (Ayral et al. 2005). Therefore, TSG-6 therapy might also be an effective treatment in inflammatory OA. In this study, we analysed the expression of TSG-6 during experimental arthritis and explored the effects of TSG-6 gene therapy in a murine model of inflammatory osteoarthritis. Materials and methods TSG-6 gene expression was determined in microarrays of collagenase-induced osteoarthritis at day 7 and day 14 after induction. The murine TSG-6 gene was cloned in a lentiviral and adenoviral vector. Freshly isolated bone marrow-derived dendritic cells (BMDCs) were transduced with the lentiviral vector and subsequently differentiated into osteoclasts on bone slices with M-CSF and RANKL. After 10 days, bone slices were washed and stained using Coomassie Blue and bone resorption was determined using the Leica Application Suite software. To study the effects of TSG-6 in experimental knee osteoarthritis, mice received two intra-articular injections of collagenase. Four days prior to and 20 days after arthritis induction, mice were injected intra-articularly with TSG-6 adenovirus. At day 7, inflammation was assessed using fluorescent Prosense probes. At day 42, knee joints were analysed by X-ray and histological assessment. Results TSG-6 was upregulated in collagenase-induced OA (2,6x at day 7 (P < 0.05) and 2.5x at day 14 (P = 0.55)). BMDCs transduced with TSG-6 lentivirus showed strong expression of TSG-6. Bone resorption by BMDC-derived osteoclasts was significantly reduced (20.1% surface erosion with control virus to 10.4%, p = 0.01), providing a possible mechanism for the therapeutic effects in rheumatoid arthritis models. At day 7 of collagenase-induced osteoarthritis, no difference in inflammation was detected using the Prosense probes. At day 42, no improvement on cartilage damage was seen, but X-ray analysis showed strong osteophyte formation at the femur/tibia region in the knee joint. Histological analysis showed that the osteophytes contained both bone and cartilage. Conclusions Viral expression of TSG-6 can reduce the bone resorption activity by BMDC-derived osteoclasts. The expression of TSG-6 by synovial cells during experimental osteoarthritis results in the formation of osteophytes. These results imply a causative role for TSG-6 in osteophyte formation, supporting the recent finding that TSG-6 activity is associated with radiographic progression of OA (Wisniewski et al. 2014).

A4.6 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?

Background and objectives Pain is the main problem for patients with OA. Pain is linked to inflammation, but in OA a subset of patients suffer from pain without inflammation, indicating an alternative source of pain. NGF inhibition very efficiently blocks pain during OA, but the exact source of NGF is unclear. NGF is elevated in animal models of OA with cartilage damage without inflammation. TGF-beta is released from cartilage upon damage. We investigated whether TGF-beta could contribute to NGF expression in the joint and which signalling route is involved. Materials and methods 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 IL-1β as a positive control for NGF induction. We analysed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1 (oxozeaenol), Smad2/3 (SB-505124) or Smad1/5/8 (LDN-193189) signalling to identify the TGF-β pathway inducing NGF. Results TGF-beta consistently induced NGF expression at higher levels than IL-1 in all of our experimental models: murine, bovine and human origin, in cell lines, primary chondrocytes and explant cultures. We initially expected TAK1 to be dominant and pivotal in NGF induction due to involvement in both IL-1 and TGF-beta signalling. TAK1 inhibition consistently reduced (but not blocked) 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. Blocking the Smad1/5/8 pathway did not affect NGF expression. Human OA primary chondrocytes had a large variation in basal NGF levels (mRNA and histology). TGF-β consistently induced a higher level of NGF induction than IL-1 in these chondrocytes despite variable basal levels of NGF when stimulating human OA cartilage explants with TGF-beta, NGF expression increased, which could be fully blocked by inhibiting ALK5-Smad2/3 signalling. Conclusions This is the first time that it is shown that TGF-β induces NGF expression in (primary) chondrocytes. Moreover, we found that TGF-beta consistently induced NGF at higher levels than IL-1. In addition, IL-1-induced NGF could be fully abrogated by TAK1 inhibition, but this only reduced TGF-beta-induced NGF expression. In contrast blocking signalling via ALK5-Smad2/3 fully abrogated the TGF-beta-induced NGF expression. Clearly, our data show that there are sources of NGF other than inflammation in the joint and that this should be taken into account when studying pain in OA.

A1.14 Synovial macrophages promote TGF-β signalling but protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidised low-density lipoproteins

Background and objectives LDL in inflamed synovium is oxidised and taken-up by macrophages, leading to an activated macrophage phenotype. In this study, we investigate whether injection of oxLDL directly into a murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Materials and Methods Synovium was obtained from end-stage OA patients. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (PBS). This procedure was repeated in mice depleted of synovial lining macrophages by intraarticular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. Results Synovial macrophages and fibroblast of OA patients showed extensive accumulation apolipoprotein B, the main protein present in LDL and oxLDL. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of specifically oxLDL in macrophage-depleted knee joints led to increased synovial thickening. Furthermore, protein and RNA levels of CCL2 and CCL3 were significantly upregulated in macrophage-depleted joints after oxLDL injections and 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 active TGF-β was measured in macrophage-depleted joints. Conclusions Synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity in combination with increased influx of monocytes and neutrophils.

A10.08 Detailed analysis of the effect of cryopreservation on the viability and cytokine release of human synovial tissue

Background and objectives Human synovial tissue derived from joint surgery of osteoarthritis (OA) and rheumatoid arthritis (RA) patients is valuable material for studying fundamental research questions. It would be ideal to store and collect all this valuable tissue for later use. In this study cryopreservation solutions are validated for their capability to preserve the tissue as freshly obtained material. Materials and methods Human synovial OA tissue was derived (n = 4), with informed consent, from joint surgery and frozen as 3 mm biopsies. Cryosections were made to prove synovial origin. Biopsies were cryopreserved by slow freezing using several cryoprotectant (CPA) solutions (Cryostor CS2, Cryostor CS10, CryoSFM, Biofreeze and standard freezing medium containing 10% DMSO and 10% FCS). For slow freezing an isopropanol container was used assuring a temperature decrease of 1°C/min. Frozen tissues were stored for 7 days in liquid nitrogen. Thawing was performed at 37°C, followed by quick removal of the CPA solution. Results To asses general viability after thawing the tissue was cultured overnight. Both the ATP and XTT assays showed viability rates up to 90% of non frozen tissue. However, decreased viability (60%) was observed with higher concentrations of DMSO (CS10 and standard media, XTT) and the complete absence of DMSO (Biofreeze, ATP). Next, the RNA integrity number (RIN) was determined 24 h after thawing to ensure the RNA is suitable for gene array experiments. A high RIN (˜8) was found in all conditions. Stress genes (CASP3, HSPA1A, HSP27, MCL1, BAX, CD95, p53) described to be up/downregulated after cryopreservation did not show deviations from the non frozen tissue in the four donors tested. However, studying OA characteristic gene expression (TIMP3, CCL18, MMP9) showed that the Biofreeze medium had most changes (up/down) compared to non frozen tissue. Furthermore, we showed that the tissue was able to respond to inflammatory stimuli (Pam3Cys/LPS) as cytokine secretion (IL1b, TNFa, IL6, IL8) was highly upregulated. Conclusion Evaluation by two viability assays, stress – and disease characteristic gene expression, and cytokine secretion showed that during a short-time culture of human synovial tissue the disease characteristic phenotype was unchanged after cryopreservation.

A1.05 The mer tyrosine kinase receptor plays a protective role in joint inflammation by mediating efferocytosis

Background and objective Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by a hyper-inflammatory response in synovial joints. During arthritis, increased levels of pro-inflammatory mediators are present in the joint cavity attracting leukocytes of which many undergo apoptosis. One receptor involved in uptake of apoptotic cells is the Mer receptor tyrosine kinase. In addition, the Mer receptor is involved in an anti-inflammatory natural feedback mechanism of Toll-like receptor signalling. The main ligand for Mer is Protein S (Pros1), acting as a bridge between apoptotic cells and the Merreceptor on phagocytic cells. Our objective was to elucidate the role of Mer in experimental arthritis. Materials and methods KRN serum transfer arthritis was induced in either Mer-deficient mice or in wild type mice that overexpress Pros1 in their knee joints. Mice were macroscopically scored and at day 7 or 14, respectively, knee joints were taken for histology. In collagen-induced arthritis (CIA), mice were intravenously injected with a commercially available Mer-specific antibodies or an adenovirus overexpressing Pros1. At day 30 or 31, respectively, knee joints were taken for histology and immunohistochemical staining of active Caspase-3. Results In Mer-deficient mice, induction of passive KRN arthritis enhanced the macroscopic score in both knee and ankle joints compared to wild type. In addition, increased cartilage and bone erosion was found in knee joints of Mer-/- mice. Intra-articular overexpression of Pros1 before KRN serum injections markedly reduced arthritis severity in wild type mice. CIA mice treated with Mer-specific antibodies showed increased incidence and higher macroscopic knee score compared to those receiving an isotype control (IgG). Histological analysis showed significantly enhanced synovial inflammation and bone erosion. In addition, significantly more active Caspase-3 positive cells were present, a marker for apoptotic cells. This indicates reduced efferocytosis in the anti-Mer treated animals. In concordance with these data, CIA mice which systemically overexpressed Pros1 showed a significant reduction in knee inflammation. Conclusion These data indicate the importance of Mer in controlling the severity and resolution of inflammation in experimental arthritis. Our data suggest that promoting Mer-mediated uptake of apoptotic cells in the arthritic joint might be therapeutically beneficial.

A8.15 ProS and Gas6 - two structurally related proteins with therapeutic effects in experimental arthritis, but via two different mechanisms

Protein S (ProS) and growth-arrest-specific protein 6 (Gas6) are both ligands for TAM receptors, a family of receptor tyrosin kinases consisting of Axl, Mertk and Tyro3. Activation of these receptors induces a negative feedback loop by inhibiting TLR- and cytokine receptor signalling. ProS, next to its function as a TAM receptor ligand, plays a role in coagulation where it acts as a cofactor of the anticoagulant Protein C. Due to the anti-inflammatory effects of Gas6 and ProS, they could be a promising treatment for different autoimmune disease such as rheumatoid arthritis (RA). In RA joints, activation of non-immunological pathways also takes place, like the coagulation cascade and factors of the coagulation cascade are described to cleave and activate complement factors. In this way the anti-coagulative function of ProS could positively affect RA pathology. We therefore investigated the therapeutic effect of Gas6 and ProS in experimental arthritis. Adenoviruses for overexpressing Gas6 or Pros1 genes were injected locally into the knee joints of mice in two different RA models: one day after the booster injection in the T-cell dependent collagen-induced arthritis (CIA) model or one day before the second injection of serum in the anti-GPI autoantibody driven KRN serum transfer model. Mice were sacrificed at day 31 or day 14, respectively and knee joints were taken for histology and synovial biopsies for cytokine profiling. In the CIA model, mice receiving adenoviruses encoding for either Pros1 or Gas6 showed reduced joint inflammation, bone and cartilage erosion in which the effect of Gas6 was more prominent than that of Pros1. Furthermore, levels of pro-inflammatory cytokines in the synovium such as IL-6 and IL-1b and also MMP-9 and MMP-13 were reduced whereas expression of SOCS1 and protein levels of SOCS3, downstream molecules of TAM receptor signalling, were increased indicating a TAM receptor mediated mechanism. In KRN arthritis, inflammation, bone and cartilage erosion and osteophyte formation was strongly reduced in Pros1 treated mice, whereas Gas6 was completely ineffective in this model. Expression of cytokines like TNFa, IL-1b and MMP-13 in the synovium were decreased in mice receiving Pros1 whereas SOCS1 and SOCS3 expression remains unchanged, indicating a TAM receptor independent effect. In conclusion, Gas6 and ProS appear to interfere with the pathology of arthritis on two different levels. In CIA, both of them can induce anti-inflammatory TAM receptor signalling whereas in the KRN model, ProS presumably blocks the coagulation cascade and thereby prevents complement C5 activation.

A8.2 Oral administration of bovine milk-derived extracellular vesicles diminishes cartilage pathology in two arthritis models

Development of rheumatoid arthritis (RA) is associated with environmental factors and several studies show a connetion with diet. Recently, exosomes are identified in both bovine and human breast milk. Exosomes are small (30–200 nm) membrane-derived extracellular vesicles that carry immunoregulatory microRNAs, proteins and lipids, and mediate intercellular communication. In this study, we investigated the effect of oral intake of bovine milk-derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra deficient mice and on collagen-induced arthritis (CIA) in DBA1/J mice. BMEVs were isolated from skimmed milk by ultracentrifugation and characterised by nanoparticle tracking analysis, electron microscopy, anti-CD63 staining, and PCR. BMEVs were labelled with PKH-67 to determined the uptake by cells using FACS and confocal microscopy. TGFb levels were measured with a CAGA-fLuc reporter construct. Naïve T cells were cultured for 5 days with an inflammatory cocktail in the presence of milk exosomes, to induce Th17 differentiation as assessed by RORgT and IL-17 mRNA expression. In IL-1Ra-/- mice, BMEVs were administered daily by oral gavage starting at week 5 till 15 after birth and arthritis was scored macroscopically. In CIA, mice received BMEVs via their drinking water starting 1 week before immunisation till day 40. Arthritis was scored macroscopically and on histology. To determine the effect on immunity, serum IgG levels were measured by ELISA, T-cell specific gene expression (T-bet, RORgT, GATA-3) in LPS stimulated splenocytes by Luminex and RT-qPCR. The BMEV particle size was around 120 nm and expressed the exosome markers tetraspanin CD63, miRNA’s (miR-let-7a, -16, -30a, -92a, -223), and milk specific beta-casein and beta-lactoglobulin mRNA. Acidification, up to gastric acid level (pH 2) left the BMEVs intact, and did not alter their inhibitory effect on NFkB-activation. Active TGFb was detected on BMEVs and incubation of naïve T cells with BMEVs induced significant Th17 differentiation to a similar extent as TGFb. However, BMEVs treatment of mice showed a delayed onset of arthritis in both the IL-1Ra-/- and CIA model. Diminished cartilage pathology and bone marrow inflammation was observed in both models. BMEVs also reduced the circulation levels of MCP1 and IL-6 levels and their production by splenic cells. In BMEV treated CIA, circulating anti-collagen type II IgGa level was reduced and this was accompanied by reduced splenic Th1 and Th17 numbers. Bovine milk-derived extracellular vesicles are bioactive, probably can withstand the harsh conditions of the gut, and oral delivery ameliorates disease in two RA models.

A1.35 Cryopreservation of human synovial tissue for biobanking purposes

Background and objectives Human synovial tissue derived from joint surgery of osteoarthritis (OA) and rheumatoid arthritis (RA) patients is valuable material for studying fundamental research questions. However, due to improved treatment options for these patients less joint replacement surgery is needed and less articular material becomes available for research. Additionally, performing a standardised comparative experiment with fresh tissue is difficult as material is scarce and infrequently available. It would be ideal to store and collect all this valuable tissue for later use. Therefore, a cryopreservation method has to be developed and importantly, the cryopreserved tissue should perform as freshly obtained material. Materials and methods We used bovine metacarpophalangeal synovial tissue as a model. The inner layers of the joint capsule are isolated and 3 mm biopsies are taken. Human synovial tissue is derived, with informed consent, from joint surgery and also frozen as 3 mm biopsies. Cryosections are made to prove synovial origin. Bovine and human biopsies are cryopreserved by slow freezing using several cryoprotectant (CPA) solutions (Cryostor CS2 and standard freezing medium containing 10% DMSO and 10% FCS). For slow freezing a Mr. Frosty is used assuring a temperature decrease of 1°C/min. Thawing has been performed at 37°C, followed by quick removal of the CPA solution. Viability has been determined by MTT, XTT and ATP assays. Results Histologically, the bovine synovial lining layer is comparable to normal human synovium. No differences were observed between freshly isolated and frozen synovium by HE staining on paraffin sections. Culturing cryopreserved bovine synovial tissue showed viability of up to 70% as determined by MTT, XTT and ATP assays. Furthermore, the standard CPA solution gives slightly higher viability values compared to the Cryostor medium. A first test with human RA tissue showed even higher viability, almost reaching the fresh values, as determined by the XTT assay. When comparing the viability assays, negative control values (tissue frozen-thawed several times without CPA solution) are lowest for the ATP assay indicating this assay could be most optimal for determining viability. Conclusion The first results of the viability testing after cryopreservation are promising. However, further testing, also with additional CPA solutions is required to validate the cryopreservation method. Next to general viability, preservation of the pathologic response is important. Experiments are ongoing to determine cytokine secretion after cryopreservation and this will shed some more light on the ability to preserve and biobank human synovial tissue.