Borbala Aradi

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.

Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.

Protein Tyrosine Phosphatase Nonreceptor Type 2: An Important Regulator of lnterleukin‐6 Production in Rheumatoid Arthritis Synovial Fibroblasts

To investigate the role of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in the pathogenesis of rheumatoid arthritis (RA).

Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2), an Important Regulator of IL-6 Production in Rheumatoid Arthritis Synovial Fibroblasts

To investigate the role of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in the pathogenesis of rheumatoid arthritis (RA).

The role of citrullination of an immunodominant proteoglycan (PG) aggrecan T cell epitope in BALB/c mice with PG-induced arthritis

The P70-84 peptide (also called 5/4E8 epitope) of the human cartilage proteoglycan (PG) aggrecan is the dominant/arthritogenic epitope in both humans and arthritis-prone BALB/c mice (PG-induced arthritis, PGIA). An elevated T cell reactivity was demonstrated to a citrullinated version of the P70-84 epitope in most of the patients with rheumatoid arthritis (RA). The goal of this study was to understand better how a T cell epitope, if citrullinated, may affect antigenicity/arthritogenicity in PGIA, a murine model of RA. T cell reactivity to differentially citrullinated versions of either the human PG aggrecan P70-84 peptide or the corresponding mouse sequence was assessed in peptide or aggrecan-immunized and arthritic BALB/c mice as well as in T cell receptor transgenic mice specific for peptide P70-84 sequence. Peripheral T cell responses were induced by priming BALB/c mice with either the human wild-type or its citrullinated versions. Unexpectedly, priming with the citrullinated self-peptide induced a higher T cell response compared to the wild-type sequence (p<0.001), and the citrullination of the human peptide abolished T cell reactivity in PGIA. Our data suggest that T cells reactive to the citrullinated P70-84 peptide escaped thymic selection and are present in the peripheral T cell repertoire. Results of this study provide evidence that citrullination of an immunodominant T cell epitope may substantially alter, either increase or abolish, T cell recognition at the periphery in an experimental model of arthritis.

Infection and autoimmunity: Lessons of animal models

While the key initiating processes that trigger human autoimmune diseases remain enigmatic, increasing evidences support the concept that microbial stimuli are among major environmental factors eliciting autoimmune diseases in genetically susceptible individuals. Here, we present an overview of evidences obtained through various experimental models of autoimmunity for the role of microbial stimuli in disease development. Disease onset and severity have been compared in numerous models under conventional, specific-pathogen-free and germ-free conditions. The results of these experiments suggest that there is no uniform scheme that could describe the role played by infectious agents in the experimental models of autoimmunity. While some models are dependent, others prove to be completely independent of microbial stimuli. In line with the threshold hypothesis of autoimmune diseases, highly relevant genetic factors or microbial stimuli induce autoimmunity on their own, without requiring further factors. Importantly, recent evidences show that colonization of germ-free animals with certain members of the commensal flora [such as segmented filamentous bacteria (SFB)] may lead to autoimmunity. These data drive attention to the importance of the complex composition of gut flora in maintaining immune homeostasis. The intriguing observation obtained in autoimmune animal models that parasites often confer protection against autoimmune disease development may suggest new therapeutic perspectives of infectious agents in autoimmunity.

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

SAT0565 The Expression and Regulation of the Argonaute Protein Family Member PIWIL4 in RA

Background Aberrant expression of PIWIL (P-element induced wimpy testis like RNA-mediated gene silencing) 1-4 genes, members of the Argonaute gene family, was reported in cancer. PIWIL proteins build complexes with piwi-interacting RNAs (piRNAs) to silence gene expression. PIWIL4 is the only member of the PIWIL family, which is ubiquitously expressed in human tissues.[1] Objectives To analyze the expression of PIWIL4 in peripheral blood mononuclear cells (PBMC) in systemic autoimmune diseases and in synovial cells from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Methods Expression of PIWIL4 was analyzed by TaqMan RealTime-PCR in PBMC isolated from 12 RA, 7 systemic lupus erythematosus (SLE), 6 spondyloarthritis (SpA) patients, 12 healthy controls (HC), in synovial tissues and isolated synovial fibroblasts (SF) from RA and OA patients (n=5 each) and in cancer cell lines (HEK293, HeLa). RASF and OASF (n=5-12 each) were stimulated or not for 24 hours either with Poly(I:C) (10ug/ml) or LPS (5ug/ml) or TNFα (10ng/ml) and IL1β (1ng/ml). Protein expression was analyzed by Western blot. Proliferation after silencing of PIWIL4 with siRNA was assessed by cell counting. Results In PBMC the mRNA for PIWIL4 was detected. The expression of PIWIL4 in PBMC of RA patients was significantly higher (mean dCt=2.18) than in HC (mean dCt=2.64, p<0.05), but significantly lower than in patients with SLE (mean dCt=1.42, p<0.001). There was no difference between PBMC from RA and SpA patients (mean dCt=2.30). In synovial fibroblasts from RA and OA patients, PIWIL4 could be detected. The basal expression in synovial tissues and SFs was similar in RA and OA. PIWIL4 mRNA was inducible in both RASF and OASF by Poly(I:C) 2.9-fold (p=0.003)/3.4-fold (p=0.013); LPS 2.1-fold (p=0.026)/2.6-fold (p=0.025) and TNFα in combination with IL1β 1.9-fold (p=0.003)/1.7-fold (p=0.007). The PIWIL4 transcript levels in SF were similar to that in various cancer cell lines (RASF mean dCt=4.23, HEK293 dCt=10.00, HeLa dCt=5.49), however, in comparison with cancer cells, the protein expression in SF was very low. PIWIL4 silencing decreased cell proliferation in RASF (n=5) stimulated with TNFα and IL1β (to 0.7-fold, p=0.035), but not upon stimulation with Poly(I:C) (to 0.8-fold, p=0.143). Conclusions We have demonstrated, that PIWIL4 is present in PBMC and synovial cells from patients with RA. The induction of PIWIL4 by Toll-like receptor ligands and proinflammatory cytokines and the regulation of proliferation suggest a role of PIWIL4 in the activation of synovial fibroblasts in RA. References Sugimoto, K., et al., The induction of H3K9 methylation by PIWIL4 at the p16Ink4a locus. Biochem Biophys Res Commun, 2007. 359(3): p. 497-502. Acknowledgements OSTEOIMMUNE, BT Cure, TeamEU, IAR, MHCR project 023728. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3575

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