Anita Fischer

Animal Models of Rheumatoid Arthritis (I): Pristane-Induced Arthritis in the Rat

Background To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. Methods We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. Results Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. Conclusions PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.

Anticarbamylated protein antibodies can be detected in animal models of arthritis that require active involvement of the adaptive immune system

A key characteristic of rheumatoid arthritis (RA), is the occurrence of antibodies against post-translationally modified proteins. Citrullination and anti-citrullinated protein antibodies (ACPAs) have been studied extensively.1 Carbamylation is another type of post-translational modification. During carbamylation, isocyanic acid reacts with the amine group of an amino acid. This mostly results in the conversion of lysine into homocitrulline.2 A subset of patients with RA harbour anticarbamylated protein (anti-CarP) antibodies and the presence of these antibodies is predictive of worse disease progression in ACPA-negative patients.3 Anti-CarP antibodies can be present in patients with arthralgia and their presence predicts the development of RA.4 A cornerstone of biomedical research is the use of animal models to explore basic pathophysiological mechanisms. Therefore, it is important to know whether antibodies against post-translationally modified proteins are present in these models. Although ACPAs were initially reported to be present in collagen-induced arthritis (CIA),5 ,6 this topic is now debated. Little is …

Inhibition of Inflammation and Bone Erosion by RNA Interference-Mediated Silencing of Heterogeneous Nuclear RNP A2/B1 in Two Experimental Models of Rheumatoid Arthritis.

The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up‐regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA.

4.8 Role of Toll-like receptor 9 in the pathogenesis of inflammatory autoimmune arthritis and osteoclast activation

Background and Objectives There is some evidence that release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions that might be important in the initiation of RA. Nucleic acid-sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenic autoimmune processes, particularly in SLE, but their role in RA is less clear. Data recently obtained in rats with pristane-induced arthritis (PIA) suggested involvement of TLR9, because antagonizing this receptor in the initiation phase of the disease led to an improved clinical outcome of arthritis. To gain more insight into the role of TLR9 in autoimmune arthritis we have extended our studies to the K/BxN serum-transfer arthritis model which is reflecting the effector phase of erosive autoimmune arthritis. Materials and Methods Arthritis was induced in C57BL/6 mice by two subsequent injections of arthritogenic serum. Antagonists for TLR7 and TLR9 or an agonist for TLR9, respectively, a non-inhibitory control sequence or PBS as placebo was applied every third day. The first treatment was given one day before disease induction. To further investigate TLR9 involvement, arthritis was also induced in TLR9 knock-out mice. Furthermore, the effect of TLR9 on osteoclast differentiation and activation was investigated in an in vitro osteoclast formation assay. Results Neither inhibitor affected arthritis onset and severity in the serum transfer model, which is independent of the adaptive immune system, in contrast to PIA. Furthermore, arthritis severity was not changed in mice lacking a functional tlr9 gene in comparison to wild type animals. In contrast, treatment with a TLR9 agonist led to an improved clinical outcome. Remarkably, in vitro formation of osteoclasts appeared to be slightly enhanced in cells isolated from TLR9-/- mice compared to those from wild type animals. Moreover, osteoclastogenesis was reduced by ~60% in cells from wild type animals grown in the presence of the TLR9 agonist whereas, as expected, no effect was seen in cells from TLR9-/- animals. Conclusions These results suggest that TLR9 might play a beneficial regulatory role in the effector phase of erosive autoimmune arthritis by negatively affecting differentiation and activation of bone resorbing osteoclasts. Nevertheless, the precise role of TLR9 in the different stages of arthritis needs to be further elucidated.

The involvement of Toll-like receptor 9 in the pathogenesis of erosive autoimmune arthritis

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9−/− mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.

Non-classical monocytes as mediators of tissue destruction in arthritis

Objectives Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive. Methods We investigated CCR2−/− mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis. Results We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL. Conclusion Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage.

OP0194 Involvement of toll-like receptor 9 in the pathogenesis of erosive autoimmune arthritis and during osteoclastogenesis

Background Release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions important in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). Nucleic acid sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenic autoimmune processes, particularly in systemic lupus erythematosus, but their role in RA is less clear. Results previously obtained in rats with pristane-induced arthrits (PIA) suggested involvement of TLR9 in the pathogenesis of this arthritis model (1). Interestingly, rats with PIA develop autoantibodies associated with RA including rheumatoid factor, anti-RA33 and antibodies to carbamylated proteins (2). Objectives To gain more insight into the role of TLR9 in the pathogenesis of autoimmune arthritis by investigating the effects of TLR9 inhibition in rats with PIA. Methods Arthritis was induced in DA rats with the mineral oil pristane. Rats were treated with a TLR9 antagonist every other day, starting one before disease induction. Arthritis was scored using established scoring systems, inflammation and bone erosion were quantified by histological analysis. Expression of TLR9 and other nucleic acid sensing TLRs was quantified by RT-PCR and Western blotting; activation (phosphorylation) of various signal transduction molecules was determined by Western blotting. Furthermore, the role of TLR9 in osteoclast differentiation and activation was investigated in vitro. Results The TLR9 antagonist significantly reduced clinical signs of arthritis by approximately 50%. Histological analyses revealed diminshed inflammation, cartilage degradation, bone erosion and significantly reduced numbers of osteoclasts in animals treated with the TLR9 antagonist. However, when treatment was started after onset of arthritis TLR9 inhibition had no effect on arthritis development and severity. IL-6 serum levels were greatly diminished in animals treated with the TLR9 antagonist and expression and activation of NF-kB in lymph nodes was reduced. Remarkably, mRNA levels of TLR7 and TLR9 strongly differed in the course of in vitro osteoclastogenesis. Whereas TLR7 expression did not change throughout osteoclastogenesis, expression of TLR9 was higher in precursor cells than in mature osteoclasts and stimulation with a TLR9 agonist (CpG) completely inhibited osteoclastogenesis. Conclusions Taken together, the results suggest an important role for TLR9 in the T cell-dependent initiation phase of PIA and thus important involvement of endogenous DNA released during apoptosis, necrosis or netosis in the initiation of autoimmune arthritis and during osteoclastogenesis. The possible relevance of these findings for human RA needs to be further elucidated in future experiments. References Hoffmann MH, Skriner K, Herman S, Baumann C, Steiner CW, Ospelt C, Meyer B, Gleiss A, Pfatschbacher J, Niederreiter B, Tuncel J, Zanoni G, Steiner G. Nucleic acid-stimulated antigen presenting cells trigger T cells to induce disease in a rat transfer model of inflammatory arthritis. J Autoimmun. 2011; 36:288–300. Stoop JN, Fischer A, Hayer S, Hegen M, Huizinga TW, Steiner G, Trouw LA, Toes RE. Anticarbamylated protein antibodies can be detected in animal models of arthritis that require active involvement of the adaptive immune system.Ann Rheum Dis. 2015; 74(5):949–50. Disclosure of Interest None declared

08.27 Impact of toll-like receptor 9 in inflammatory arthritis and osteoclastogenesis

Background Release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions important in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). Nucleic acid sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenic autoimmune processes, but their role in RA is less clear. To gain more insight into the role of TLR9 in autoimmune arthritis, rats with pristane-induced arthritis (PIA) were treated with a TLR9 antagonist. To further investigate TLR9 involvement, serum transfer arthritis was induced in TLR9 knock-out mice. Materials and methods Arthritis was induced in mice with arthritogenic K/BxN serum and in rats with the mineral oil pristane. Rats were treated with a TLR9 antagonist every other day, starting one day before disease induction. Arthritis was scored using established scoring systems, inflammation and bone erosion were quantified by histological analysis. Furthermore, the role of TLR9 in osteoclast differentiation and activation was investigated in vitro. Results In the serum transfer model, which is independent of the adaptive immune system, arthritis severity was not changed in mice lacking a functional tlr9 gene. In PIA, which is T cell-dependent, the TLR9 antagonist reduced arthritis severity by ~50%. TLR9 inhibition led to reduced inflammation, bone erosion and cartilage degradation. However, when treatment was started after the onset of arthritis TLR9 inhibition did not influence disease severity. Treatment with the TLR9 antagonist influenced downstream signalling, mainly via NFκBp65, in lymph nodes. Remarkably, mRNA levels of TLR7 and TLR9 strongly differed in the course of in vitro osteoclastogenesis. Whereas TLR7 expression did not change throughout osteoclastogenesis, expression of TLR9 was higher in precursor cells than in mature osteoclasts and stimulation with a TLR9 agonist (CpG) completely inhibited osteoclastogenesis. Conclusions Taken together, the results suggest a role for TLR9 in the T cell-dependent initiation phase of PIA and thus an important involvement of the DNA (CpG) recognising TLR9 in the initiation of autoimmune arthritis and during osteoclastogenesis. The precise role(s) of TLR9 in the different stages of arthritis needs to be further elucidated in future experiments.

A7.15 in vitro silencing of HNRNP-A2/B1 in synovial fibroblasts reveals involvement in regulation of several signal transduction pathways

Background and objectives The heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in post-transcriptional regulation of gene expression. It has been shown to be highly upregulated in synovial tissue of patients with rheumatoid arthritis (RA). In addition, autoantibodies and T cells directed against hnRNP-A2/B1 can be found in RA patients. Recently, it was shown that silencing of hnRNP-A2/B1 in two animal models of RA, namely Collagen-induced arthritis and K/BxN serum transfer arthritis, led to reduction of arthritis severity.1 To further elucidate the role of hnRNP-A2/B1 in RA, we sought of analysing the signalling pathways affected by silencing of hnRNP-A2/B1 in human fibroblast-like synoviocytes (FLS). Materials and methods siRNA-mediated silencing of hnRNP-A2/B1 in FLS was achieved by lipofectamine-based transfection. After three days, successful reduction of hnRNP-A2/B1 expression was analysed by real-time quantitative polymerase chain reaction (RT-qPCR). The role of hnRNP-A2/B1 in FLS was investigated by activating cells with TNF-α. Proteome Profiler Arrays were used to analyse cytokine production and phosphorylation of various signal transduction molecules. Interleukin (IL) -6 and IL-8 secretion was assayed using enzyme-linked immunosorption assay (ELISA). Results Silencing of hnRNP-A2/B1 led to a reduction of phosphorylation of AKT and mammalian target of rapamycin (mTOR) in TNF-α stimulated cells and a slight reduction in phosphorylation of p70 S6 kinase, which is a downstream signalling component of mTOR. Moreover, down-regulation of hnRNP-A2/B1 led to reduced levels of phosphorylated MAPK14 (p38α), and a reduction of MSK2 phosphoryl ation. Analysis of supernatants revealed reduced levels of CCL5 (RANTES), CXCL10 (IP-10), CCL20 (MIP-3α) and Serpine E1, but increased levels of ICAM-1. In addition, reduced secretion of Dickkopf-1 (DKK-1) or IGFBP-3 could be detected in silenced cells. Surprisingly, secretion of IL-6 and IL-8 was slightly increased in hnRNP-A2/B1 silenced FLS. Conclusions hnRNP-A2/B1 seems to play an important role in regulation of several signalling pathways, mainly the mTOR pathway, which is involved in translation and cell growth. Further analyses will be needed to fully understand the role of hnRNP-A2/B1 in signalling pathways operative in FLS and other inflammatory cell types involved in the pathogenesis of RA. Reference Herman S, et al. Inhibition of Inflammation and Bone Erosion by RNA Interference-Mediated Silencing of Heterogeneous Nuclear RNP A2/B1 in Two Experimental Models of Rheumatoid Arthritis. Arthritis Rheumatol 2005;67:2536–46

A6.38 Toll-like receptor 7 and 9 in the pathogenesis of inflammatory autoimmune arthritis

Background and objectives There is evidence that release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions important in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). Nucleic acid sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenetic autoimmune processes, particularly in systemic lupus erythematosus, but their role in RA is less clear. We aimed to study the role of TLR7 and TLR9 in the pathogenesis of inflammatory arthritis by antagonising or stimulating them in rats with pristane-induced arthritis (PIA). Materials and methods Arthritis was induced in Dark Agouti rats with the mineral oil pristane. Antagonists or agonists, respectively, for TLR7 and TLR9, a non-inhibitory control sequence or PBS as placebo were applied every other day. Treatment was started before disease induction. Arthritis was scored using established scoring systems, inflammation and bone erosion were quantified by histological analysis. Serum cytokine levels were measured by ELISA. Results While the control sequence showed no effect on arthritis development and severity, the TLR9 antagonist reduced arthritis severity significantly in PIA. In contrast, a slight aggravation of disease severity was observed in animals treated with the TLR7 antagonist. Inhibition of TLR9 led to strongly reduced bone erosion, whereas it appeared moderately aggravated in animals treated with the TLR7 inhibitor. Furthermore, IL-6 serum levels were reduced in animals treated with the TLR9 antagonist. However, these effects were only seen when the inhibitor was applied before disease onset. When treatment with the antagonists was started at disease-onset neither disease severity nor bone erosion were affected. Treatment with agonists for TLR9 or TLR7 showed no significant effect on disease severity in animals treated with the TLR9 agonist. In contrast, disease was significantly aggravated in animals treated with the TLR7 agonist and this effect was more pronounced than that observed in previous experiments with the TLR7 antagonist. Conclusions Inhibition of TLR9 in rats with PIA significantly reduced inflammation and bone erosion whereas stimulation of TLR7 aggravated disease severity. Therefore, these results suggest different roles for TLR7 and TLR9 in the T cell-dependent initiation phase of PIA and thus an important involvement of the DNA (CpG) recognising TLR9 and the RNA recognising TLR7 in the initiation of autoimmune arthritis which needs to be further elucidated in ongoing and future experiments.