Maria Filkova

Association of circulating miR-223 and miR-16 with disease activity in patients with early rheumatoid arthritis

Background Identification of parameters for early diagnosis and treatment response would be beneficial for patients with early rheumatoid arthritis (ERA) to prevent ongoing joint damage. miRNAs have features of potential biomarkers, and an altered expression of miRNAs was shown in established rheumatoid arthritis (RA). Objective To analyse RA associated miRNAs in the sera of patients with ERA to find markers of early disease, clinical activity or predictors of disease outcome. Methods Total RNA was isolated from whole sera in ERA patients (prior to and after 3 and 12 months of therapy with disease modifying antirheumatic drugs), in patients with established RA and in healthy controls (HC) using phenol–chloroform extraction. Expression of miR-146a, miR-155, miR-223, miR-16, miR-203, miR-132 and miR-124a was analysed by TaqMan Real Time PCR. Results From all analysed miRNAs, levels of miR-146a, miR-155 and miR-16 were decreased in the sera of ERA patients in comparison with established RA. A change in circulating miR-16 in the first 3 months of therapy was associated with a decrease in DAS28 in long term follow-up in ERA (p=0.002). Levels of circulating miR-223 in treatment naïve ERA correlated with C reactive protein (p=0.008), DAS28 (p=0.031) and change in DAS28 after 3 months (p=0.003) and 12 months (p=0.011) of follow-up. However, neither miR-16 nor miR-223 could distinguish ERA from HC. Conclusions Differential expression of circulating miR-146a, miR-155 and miR-16 in the sera of ERA patients may characterise an early stage of the disease. We suggest miR-223 as a marker of disease activity and miR-16 and miR-223 as possible predictors for disease outcome in ERA.

MicroRNAs in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic, inflammatory, autoimmune disorder with progressive articular damage that may result in lifelong disability. Although major strides in understanding the disease have been made, the pathogenesis of RA has not yet been fully elucidated. Early treatment can prevent severe disability and lead to remarkable patient benefits, although a lack of therapeutic efficiency in a considerable number of patients remains problematic.MicroRNAs (miRNAs) are small, non-coding RNAs that, depending upon base pairing to messenger RNA (mRNA), mediate mRNA cleavage, translational repression or mRNA destabilization. As fine tuning regulators of gene expression, miRNAs are involved in crucial cellular processes and their dysregulation has been described in many cell types in different diseases. In body fluids, miRNAs are present in microvesicles or incorporated into complexes with Argonaute 2 (Ago 2) or high-density lipoproteins and show high stability. Therefore, they are of interest as potential biomarkers of disease in daily diagnostic applications. Targeting miRNAs by gain or loss of function approaches have brought therapeutic effects in various animal models.Over the past several years it has become clear that alterations exist in the expression of miRNAs in patients with RA. Increasing numbers of studies have shown that dysregulation of miRNAs in peripheral blood mononuclear cells or isolated T lymphocytes, in synovial tissue and synovial fibroblasts that are considered key effector cells in joint destruction, contributes to inflammation, degradation of extracellular matrix and invasive behaviour of resident cells. Thereby, miRNAs maintain the pathophysiological process typical of RA.The aim of the current review is to discuss the available evidence linking the expression of miRNAs to inflammatory and immune response in RA and their potential as biomarkers and the novel targets for treatment in patients with RA.

Citrullination enhances the pro-inflammatory response to fibrin in rheumatoid arthritis synovial fibroblasts

Objective Fibrin deposits are characteristic of the synovial tissues in rheumatoid arthritis (RA). Once citrullinated, fibrin becomes an autoantigen and is thought to contribute in this way to perpetuate the disease. Our study aimed to analyse the responses of RA synovial fibroblasts (RASF) to native and citrullinated fibrin. Methods The transcriptome induced by fibrin in RASF was approached with whole-genome-based gene expression arrays. The upregulation of selected pro-inflammatory genes by fibrin was confirmed in additional primary cell cultures using quantitative PCR and ELISA. Citrullination reactions were carried out with recombinant human peptidylarginine deiminases (PAD) 2 and 4. Results In the whole-genome array native fibrin was found to modulate the gene expression profile of RASF, particularly upregulating mRNA levels of several pro-inflammatory cytokines. The induction of interleukin (IL)-6 and IL-8 by fibrin was confirmed in additional samples at both the mRNA and the protein level. Blocking and knockdown experiments showed the participation of toll-like receptor (TLR)4 in the induction of both cytokines. As compared with the native macromolecule, PAD2-citrullinated fibrin induced significantly higher expression of the pro-inflammatory cytokines in these cells. Conclusions Our results suggest that fibrin mediates inflammatory responses in RASF via a TLR4 pathway. In this way, fibrin and particularly its citrullinated form may contribute to sustain the cytokine burst in RA.

AB0114 Comparative Analysis of the Expression of Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2) in Autoimmune Disease

Background Protein Tyrosine Phosphatase Non-receptor Type 2 (PTPN2) is a phosphatase that plays an important role in inhibiting T-cell activation. The PTPN2 gene contains SNPs that are risk factors for the development of rheumatoid arthritis (RA), Crohns disease and type 1 diabetes. We have previously shown that PTPN2 expression is elevated in RA synovial fibroblasts (RASF) compared to osteoarthritis synovial fibroblasts (OASF). Objectives PTPN2 expression in peripheral blood mononuclear cells (PBMC) from patients with RA, OA, ankylosing spondylitis (AS) and systemic lupus erythematosus (SLE) was analyzed and compared to those from healthy subjects. Furthermore, we aim to analyze the inflammation-induced expression of PTPN2 in synovial fibroblasts from patients with RA and compare it with the expression in the TNF transgenic mouse model. Methods PBMC were isolated using Ficoll-Paque (healthy controls n=5, OA n=1, RA n=5, AS n=6, SLE n=7)and either lysed directly or cultured and stimulated with tumor necrosis factor α (TNFα, 10 ng/ml, 24 h). Synovial fibroblasts were obtained from RA patients undergoing surgery and from the TNF transgenic mouse model. SF were stimulated with TNFα (10 ng/ml, 24 h or 1 week). Levels of PTPN2 mRNA were measured using TaqMan Real-time PCR. Western blot was used to measure PTPN2 protein levels. Results PTPN2 is expressed in PBMC, however, no significant changes were found in the expression of PTPN2 in PBMC from autoimmune diseases (dCt ± SD, healthy controls 3.98±0.97, OA 4.00±0.0, RA 4.28±0.39, AS 4.06±0.54, SLE 4.05±0.48) In PBMC from healthy controls, stimulation with TNFα (10 ng/ml, 24h) did not increase the levels of PTPN2 (n=4, dCt ± SD unstimulated 4.18±0.3, TNF 4.23±0.8). Basal levels of PTPN2 were higher in RASF compared to OASF (mRNA: 1.6 fold, p<0.01; protein: 2.0 fold, p<0.05). After 24h of stimulation with TNFα, PTPN2 expression increased on the mRNA level (3.1 fold, p<0.05), as well as on the protein level (1.7 fold, p<0.05, n=7). Moreover, PTPN2 protein levels were also upregulated after long term stimulation with TNFα (10 ng/ml, 1 week, 2.4 fold, n=3). Increased protein levels were observed in both the 45 kDa and 48 kDa isoforms of PTPN2 after the short and long-term stimulation with TNFα. Most interestingly, SF from the TNFα transgenic mouse model show a higher expression of PTPN2 compared to wild type mice (TNFα transgenic mice n=4, wildtype mice n=5, ddCt of medians=-0.96). Conclusions In the current study we show that PTPN2 expression is elevated in RASF but it is not changed in PBMC. Since we have previously shown that PTPN2 negatively regulates inflammation in RASF, we conclude that PTPN2 is a local regulator in the affected inflamed joints but not a modulator of systemic inflammation. Acknowledgements IMI BTCure, IAR, EURO-TEAM Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4212

A9.13 TNF-Induced- Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) as a Negative Regulator of Inflammation in Rheumatoid Arthritis

Background and Objectives Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) is a protein tyrosine phosphatase that plays a role in the development of autoimmune diseases. PTPN2 function has not been studied in rheumatoid arthritis (RA), although single nucleotide polymorphisms within the gene have been described to be associated with RA in genome wide association studies. Considering the involvement of PTPN2 in the regulation of key inflammatory pathways, our aim was to analyse the expression and function of PTPN2 in RA synovial fibroblasts (RASF). Materials and Methods The expression of PTPN2 was assessed in synovial tissue and fibroblasts (passage 4–10) from patients with RA and osteoarthritis (OA) using immunohistochemistry, real-time PCR (w/o tumour necrosis factor α (TNFα), IL1β, LPS and hypoxia) and Western blotting. PTPN2 was silenced with silencing RNA. Levels of IL-6 and IL-8 expression were measured by commercially available ELISA in cell culture supernatants after silencing PTPN2 in RASF w/o stimulation with tumour necrosis factor α (TNFα). Apoptosis of RASF was evaluated by AnnexinV staining using flow cytometry after stimulation with TNF-related apoptosis-inducing ligand (TRAIL, 20 ng/ml) for 24 hours. Results In RA synovial tissue, compared with OA, we observed a stronger staining of ptpn2 in both the lining and the sublining layer by immunohistochemistry. On mRNA level we confirmed this overexpression in RA synovial tissue (2.0 fold, n = 4–5). In isolated RASF the constitutive mRNA level of PTPN2 was higher than in OASF (1.6 fold, p < 0.01, n = 10–16). Levels of PTPN2 were further upregulated in RASF after stimulation with inflammatory cytokines such as TNFα (10 ng/ml, 24 hours, 3.1 fold, p < 0.05, n = 4), TNFα and IL-1β (1 ng/ml, 2.3 fold, n = 5), LPS (100 µg/ml, 24 hours, 1.9 fold, n = 5) and by 1% hypoxia (1.3 fold, n = 3). Accordingly, basal PTPN2 protein expression was 2.0 fold higher in RASF than in OASF (n = 4) and TNFα upregulated levels of PTPN2 (1.7 fold). ptpn2-deficient RASF produced 2.4 times more IL-6 than scrambled siRNA transfected cells (mean ± SD pg/ml 11412 ± 6313 versus 28133 ± 12734, n = 3). On the other hand, levels of IL-8 were not affected (35800 pg/ml versus 24330 pg/ml, n = 3). Furthermore, after silencing, 34% increase in TRAIL-induced apoptosis was detected in RASF (n = 5) compared to scrambled controls. Conclusions Our findings indicate that PTPN2, known to be involved in the pathogenesis of several autoimmune diseases, could be an important negative regulator of inflammation in RASF. Acknowledgement This work was supported by IMI BTCure, IAR and Masterswitch-FP7.

THU0071 Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) is an Important Regulator of Inflammation and Autophagy in Rheumatoid Arthritis

Background Recently we found that Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) is involved in the apoptosis resistance of rheumatoid arthritis synovial fibroblasts (RASF). Objectives Autophagy is strongly connected to apoptosis, and is activated via a series of phosphorylation events. We wanted to investigate whether PTPN2 plays a role in autophagy and inflammation in RASF. Methods PTPN2 expression was analysed in synovial tissues by immunohistochemistry and real-time PCR. Transcription levels of PTPN2 were detected with or without tumor necrosis factor α (TNFα 10 ng/ml, 24 h), IL1β (1 ng/ml), LPS (100 µg/ml) and hypoxia (1%). PTPN2 protein levels were assessed by Western blotting. After silencing PTPN2 in RASF, commercially available ELISA was used to measure IL-6 and IL-8 in culture supernatants. TNF-related apoptosis-inducing ligand (TRAIL, 20 ng/ml, 24 h) induced apoptosis was measured after staining with AnnexinV using flow cytometry. Autophagy was induced by Thapsigargin (5 µM, 24h). Western blot was used to measure LC3B-I and LC3B-II. Results In the lining and the sublining layer of RA synovial tissue, a stronger PTPN2 staining was detected by immunohistochemistry compared to OA (n=7). That overexpression could also be confirmed on mRNA level (2.0 fold, RA tissue n=4, OA tissue n=5). Moreover, in RASF, the constitutive mRNA level of PTPN2 was higher than in OA synovial fibroblasts (OASF) (1.6 fold, p<0.01, n=10-16). Stimulation with inflammatory cytokines such as TNFα (3.1 fold, p<0.05, n=4), TNFα and IL-1β (2.3 fold, n=5), LPS (1.9 fold, n=5) and hypoxia (1.3 fold, n=3) resulted in a further increase in PTPN2 mRNA levels. We found a 2.0 fold higher PTPN2 protein expression in RASF compared to OASF (n=4), and a 1.7±0.4 fold induction of PTPN2 in RASF after TNFα stimulation (n=4) by densitometric analysis of Western blot signals. IL-6 production increased 2.1 times in PTPN2 deficient cells (mean±SD pg/ml 11159±5179 vs. 24400±12801, n=4) compared to scrambled control. We could not see this effect concerning IL-8 production (35450±20619 pg/ml vs. 24148±24450 pg/ml, n=4). TRAIL-induced apoptosis increased by 34% after PTPN2 silencing (n=5). The level of autophagy (LC3B-II) did not differ basally and after TNFα stimulation in scrambled control and PTPN2 silenced cells. However, in scrambled control transfected cells, the addition of Thapsigargin induced the expression of LC3B-II protein (2.07±0.97 fold, n=5), indicative for autophagy. PTPN2 silencing reduced the induction of autophagy to 0.8±0.2 fold (n=5) compared to scrambled control. Conclusions Since levels of autophagy are lower in PTPN2 silenced RASF compared to scrambled control, it is indicated that increased PTPN2 protein expression in RASF induces inflammation and elevated autophagy in RASF. Acknowledgements This work was supported by IMI BTCure, IAR, Masterswitch-FP7 and ZIHP. Disclosure of Interest None Declared

SAT0105 Circulating MIR-223 is downregulated in SERA of patients with early RA after treatment with methotrexate

Background MiRs are involved as fine tuning regulators of gene expression in crucial cellular processes and their dysregulation has been described in many diseases. Incorporated in microvesicles, in complexes with Ago2 or high density lipoproteins, miRs show high stability in body fluids. Therefore, studying circulating miRs as potential biomarkers is of big interest. Objectives To analyze the profile of circulating miRs known to be associated with the pathogenesis of rheumatoid arthritis (RA) before and after treatment in patients with early RA (ERA) and established RA, their association with clinical activity and response to therapy. Methods Treatment naïve patients with ERA (n=42, median disease duration 3 months, 59.52% RF+, 47.61% ACPA+), fulfilling 2010 ACR/EULAR classification criteria for RA, who started therapy with methotrexate (MTX) and active patients with established RA (n=17, 76.47% RF+, 52.94% ACPA+), fulfilling 1987 ACR criteria, who were treated with TNFα blockers (Adalimumab, n=6; Infliximab, n=6; Etanercept, n=5) were included in this study. Sera/plasma were obtained before and 3 months after initiation of treatment. Total RNA was isolated by phenol-chloroform extraction and miRs were reverse transcribed using specific primers with equal input of 12.5ng RNA. The expression of miR-124a, 132, 146a, 155 and 223 were analyzed by TaqMan Real-Time PCR. MiR-let7a was used as an endogenous control. Data were calculated with dCt method. Results In ERA sera, miR-223 significantly decreased following the treatment with MTX (p=0.002), while levels of miR-132, 146a and 155 did not change. Expression of miR-223 positively correlated with DAS28 (p=0.022), CRP (p=0.010) and the number of leukocytes before (p=0.004) and after treatment (p=0.007). MiR-146a and 155 showed no association with disease activity at baseline or after MTX treatment. The baseline expression of miR-132 and 155 was higher (p=0.05) in patients with good (n=22) compared to moderate (n=11) clinical response to therapy but did not significantly differ from non-responders (n=4). In plasma of patients with established RA, the expression of miR-132, 146a, 155 and 223 did not correlate with DAS or CRP at baseline or after treatment. The expression of these miRs was not affected by TNFα blocking therapy and was comparable in responders or non-responders prior treatment. MiR-124a was not detectable in any of the samples. Conclusions MiR-223 correlated with the disease activity in treatment naïve patients with early RA and was significantly downregulated after MTX. MiR-223 is known to be overexpressed in T-lymphocytes of RA patients (Sebastini GD et al. 2011) and its presence in circulation may reflect pathogenic mechanisms in RA. We hypothesize that the expression of circulating miRs might serve as a potential tool to monitor disease activity and response to treatment, particularly in early stages of the disease. Acknowledgement This work was supported by IMI BTCure, IAR Epalinges, project No. 00023728 and MH CR-grant project No.10065-4. Disclosure of Interest None Declared

7th meeting of the global arthritis research network

Last October, the 7th meeting of the Global Arthritis Research Network was held in Zurich, Switzerland. European and American experts who have made major recent contributions to molecular biology got together to provide insights into novel technologies and approaches useful for biomedical research, especially for research on arthritis and related conditions.