Jerzy Swierkot

Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis

INTRODUCTION Despite the fact that therapy with TNF-α inhibitors constitutes a breakthrough in rheumatoid arthritis management, no improvement is still achieved in approximately 30% of cases. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. METHODS Five SNPs within the TNF-α and TNF receptor encoding genes (TNFA: G-308A, G-238A, C-857T; TNFR1A G36A; TNFR1B T676G) were determined in 280 RA patients who had been treated with TNF-α inhibitors for at least 6 months or they stop therapy because of adverse events. The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. RESULTS At week 24, low disease activity or remission was achieved by 45% of the patients. After 6 months remission of the disease or low disease activity were more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P=0.04). At week 24 DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA-857T variant compared to the C allele carriers (P=0.045). The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. CONCLUSIONS Homozygosity for the TNFR1A 36A allele and the TNFA-875T variant could act as a genetic factor associated with better response to anti-TNF treatment.

Application of (1)H NMR-based serum metabolomic studies for monitoring female patients with rheumatoid arthritis.

Rheumatoid arthritis is a chronic autoimmune-based inflammatory disease that leads to progressive joint degeneration, disability, and an increased risk of cardiovascular complications, which is the main cause of mortality in this population of patients. Although several biomarkers are routinely used in the management of rheumatoid arthritis, there is a high demand for novel biomarkers to further improve the early diagnosis of rheumatoid arthritis, stratification of patients, and the prediction of a better response to a specific therapy. In this study, the metabolomics approach was used to provide relevant biomarkers to improve diagnostic accuracy, define prognosis and predict and monitor treatment efficacy. The results indicated that twelve metabolites were important for the discrimination of healthy control and rheumatoid arthritis. Notably, valine, isoleucine, lactate, alanine, creatinine, GPC  APC and histidine relative levels were lower in rheumatoid arthritis, whereas 3-hydroxyisobutyrate, acetate, NAC, acetoacetate and acetone relative levels were higher. Simultaneously, the analysis of the concentration of metabolites in rheumatoid arthritis and 3 months after induction treatment revealed that L1, 3-hydroxyisobutyrate, lysine, L5, acetoacetate, creatine, GPC+APC, histidine and phenylalanine were elevated in RA, whereas leucine, acetate, betaine and formate were lower. Additionally, metabolomics tools were employed to discriminate between patients with different IL-17A genotypes. Metabolomics may provide relevant biomarkers to improve diagnostic accuracy, define prognosis and predict and monitor treatment efficacy in rheumatoid arthritis.

Exogenous IL-2 controls the balance in Th1, Th17, and Treg cell distribution in patients with progressive rheumatoid arthritis treated with TNF-alpha inhibitors.

Interleukin-2 (IL-2) has been suggested to control Treg/Th17 balance. Recently, we reported a relationship of rheumatoid arthritis (RA) activity/progression with irreversible systemic Treg and Th1 defects including serum IL-2 shortage. Herein, we explore the role of in vitro stimulation with rIL-2 in the observed immune alterations reversal. Patients with stable or progressive RA were assigned to methotrexate (MTX) group or to TNF-alpha inhibitors (iTNF) group, respectively. Flow cytometric analyses were performed before and after 6 months of treatment. Circulating Th1, Th17, and Treg cells were determined before and after 72-h culture with anti-CD3 + rIL-2. Before therapy, 72-h stimulation restored recently observed phenotypic Th cell alterations, except for the enriched Th17 subset normalized as late as after therapy in all patients. Under 6-month therapy, anti-CD3 stimulation changed the Th cell distribution only in progressive RA; despite Th1 enrichment, it revealed Treg population defects, which were completely reversed by exogenous IL-2 added to the stimulating culture. Our paper shows that in aggressive RA patients exhibiting serum IL-2 shortage despite iTNF therapy, exogenous rIL-2 is capable of promoting Treg differentiation affected by chronic activation, thus supporting its use in the combined strategy of biologic treatment of the progressive form of RA.

The role of microRNA-5196 in the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterised by tissue fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from patients with SSc play an important role in early stages of SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP‐1), IL‐8 and reactive oxygen species (ROS) induction. However, the exact factors that contribute to chronic inflammation and subsequently fibrosis progression are still unknown.

Alterations in Both the Activatory and Inhibitory Potential of Peripheral Blood CD4+ T Cells in Rheumatoid Arthritis Patients Correlate with Disease Progression

The chronic nature of rheumatoid arthritis (RA) suggests immune dysfunction, including persistent systemic activation. Therefore, we evaluated the activatory and inhibitory potential as well as proliferative activity of peripheral blood (PB) CD4+ T cells from RA patients in different stages of the disease and after different therapeutic interventions. We found that CD4+ T cells from RA patients were activated in vivo concerning decreased CD28 expression and increase of CD40L, CD69, and CTLA-4 expression; however, the extent of stimulation was suboptimal when compared to healthy controls. Consequently, impaired proliferative activities of these cells were found in all patients irrespective of the active disease duration. Treatment with methotrexate (MTX) and/or inhibitors of TNF-alpha (iTNF) did not significantly influence systemic activation in RA patients, which corresponded with the maintenance of inflammation markers; however, partial restoration of CD28 and CTLA-4 expression as well as clinical improvement were observed. In patients with early disease (the MTX group), we noted higher capacity of CD4+ T cells for restoration of T cell function, whereas cells from the iTNF group with progressive disease remained with a proliferative defect after the treatment. In conclusion, our study demonstrates that the dysregulated expression of molecules interfering with CD4+ T cell signaling may result in functional impairment of the effector T cells and correlates with disease progression.

OP0071 Tnf-Alpha, TNF Receptor, HLA-E and NKG2A Gene Polymorphisms and Response to Anti-TNF-Alpha Treatement in Rheumatoid Arthritis

Background Despite the fact that therapy with TNF-alpha inhibitors constitutes a breakthrough in rheumatoid arthritis (RA) management, no improvement is still achieved in approximately 30% of cases. Objectives The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor, and HLA-E and NKG2A receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. Methods For these purpose 280 RA patients who had been treated with TNF-alpha inhibitors for at least 6 months or they stopped therapy because of adverse events were investigated and genotyped for 9 SNPs within the TNFA promoter (rs1800629 G>A; rs361525 G>A; rs1799724 C>T); TNF receptors (TNFR1A: rs767455 G>A; TNFR1B: rs1061622; T>G) while 89 patients were studied for the HLA-E (rs1264457 C>T; HLA-E*01:01, HLA-E*01:03, A>G) and NKG2A (rs7301582 C>T; rs2734440 A>G) genes using LightSNiP typing or Custom TaqMan® SNP Genotyping Assays. Results Among polymorphisms located within TNF-alpha and receptors genes only the TNFR1A (rs767455, G>A) and one of the TNFA (rs1799724, C>T) promoter polymorphisms were found to be associated with response to anti-TNF therapy. Significantly more patients with the homozygous TNFR1A AA genotype achieved a good EULAR response at 3 months compared to patients carrying the G allele (p=0.011). At week 24 DAS28 was significantly lower in patients homozygous for the TNFA T variant (DAS28 – 2.05) compared to the C allele carriers (p=0.045). As for HLA-E and NKG2A genes, after 3 months of anti-TNF treatment the significantly worse (EULAR DAS28) response was observed in patients carrying the HLA-E C allele (20/45 vs. 9/28, p=0.030), HLA-E*01:03/01:03 genotype (8/10 vs. 30/73, p=0.038), NKG2A-(rs7301582)-CC genotype (28/51 vs. 10/33, p=0.043) or NKG2A-(rs2734440)-AA genotype (15/26 vs. 15/50, p=0.026). At week 12 low disease activity or remission was not observed in any of the patients with the HLA-E CC genotype (p=0.09). Also treatment failure (inefficiency or loss of effectiveness of therapy) was more frequently observed in the HLA-E CC homozygous patients (5/8 vs. 12/61, p=0.018) as well as in those with the NKG2A-(rs2734440)-AA genotype (15/37 vs. 3/33, p=0.005). Conclusions These results imply that the polymorphisms within genes coding for TNF-alpha and its TNFR1 receptor as well as HLA-E and NKG2A affect the response to anti-TNF therapy in patients with RA. Acknowledgements Supported by the UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367 grants from the National Science Center. Disclosure of Interest J. Swierkot Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, M. Iwaszko Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, K. Gebura Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, B. Nowak Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, L. Korman Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, K. Kolossa: None declared, S. Jeka: None declared, P. Wiland: None declared, K. Bogunia-Kubik Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367 DOI 10.1136/annrheumdis-2014-eular.5416

Changes in MiRNA-5196 Expression as a Potential Biomarker of Anti-TNF-α Therapy in Rheumatoid Arthritis and Ankylosing Spondylitis Patients

In this study, we analysed the expression level of sera circulating miRNA-5196 in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients before and after tumor necrosis factor (TNF)-α therapy as biomarkers predicting positive treatment outcome. We enrolled 10 RA patients, 13 AS patients, and 12 healthy individuals in the study. The expression of miRNA-5196 was measured by real-time polymerase chain reaction before and after anti-TNF-α therapy. Disease activity of RA patients was assessed using disease activity score 28 (DAS28), whereas ankylosing spondylitis DAS (ASDAS) was used in AS patients. MiRNA-5196 expression was significantly higher in patients with RA and AS before TNF-α therapy than in those following anti-TNF-α therapy and healthy controls. Changes in miRNA-5196 expression positively correlated with delta DAS28 or delta ASDAS, respectively, following TNF-α therapy. In contrast, changes in C-reactive protein (CRP) levels in RA and AS patients did not positively correlate with DAS28 or ASDAS changes. Receiver-operating characteristic analysis showed better diagnostic accuracy of miRNA-5196 expression both in RA (area under curve (AUC) = 0.87, p = 0.055) and AS patients (AUC = 0.90, p = 0.050) compared to CRP levels in RA (AUC = 0.75, p = 0.201) and AS patients (AUC = 0.85, p = 0.086) upon biologic therapy treatment. Finding novel biomarkers, including miRNA-5196 which allow to predict and monitor anti-TNF-α response, would be of clinical value especially during the early phase of RA or AS development.

AB0006 Mir-26a polymorphism is associated with susceptibility of rheumatoid and psoriatic arthritis

Background Serum levels of miR-26a has been reported to act as potential biomarker of rheumatic diseases. Objectives The aim of the study was to analyse the genetic variation and expression of miR-26a as potential diagnostic and/or prognostic markers of rheumatoid diseases. Methods The miR-26a polymorphism was examined in 111 patients with rheumatoid arthritis (RA), 86 patients with psoriatic arthritis (PsA) and 162 healthy blood donors that served as a control group. Genotyping for miR-26a rs7372209 was performed using a LightSNiP assay. For analysis of the miR-26a expression, RNA was isolated from sera of 15 RA patients (before and 3 months after anti-TNF treatment) and 10 controls (NucleospinmiRNA Plasma; MACHEREY-NAGEL GmbH and Co. KG) followed by cDNA synthesis (TaqMan MicroRNA Reverse Transcription Kit; Applied BiosystemsTM by Life Technologies) and Real-time PCR amplifications with hsa-miR-26a TaqMan specific and U6 snRNA control primers for each probe. The results were analysed using the (ΔΔCt) calculations. Results It was found that the presence of miR-26a TT genotype (rs7372209) more than 5 times increases the risk of RA (OR=5.28, p=0.003) while the presence of CC homozygotes is associated with the risk of PsA (OR=1.77, p=0.037). There was no significant difference in the miR-26a serum levels between patients and controls. Also miR-26a serum levels did not significantly differed between RA patients before, 3 and 6 months after the implementation of biological therapy with TNF-alpha inhibitors. Conclusions These results imply that miR-26a rs7372209 allelic variants differentially affect the risk of rheumatoid and psoriatic arthritis while anti-TNF biological treatment seems not to affect the miR-26a expression in RA patients. Disclosure of Interest None declared

A1.20 Epigenetic modificationsand TLR8 signalling contribute to systemic sclerosis pathogenesis by ros and profibrotic genes induction

Background and objectives Systemic sclerosis (SSc) is a chronic multisystem autoimmune disease characterised by skin and internal organs fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from SSc patients play a role in SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction, which contribute to fibrosis progression and chronic inflammation. The exact factors driving TIMP-1, IL-8 and ROS secretion are still unknown. The aim of this study was to investigate the expression pattern of profibrotic IL-8, TIMP-1, AP1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist (ssRNA) stimulation. Materials and methods The expression of Fra2, IL-8 and TIMP-1 and anti-oxidant superoxide dismutase 1 (SOD1) was measured by qRT-PCR in stimulated and unstimulated HC (n = 14) and SSc (n = 17) monocytes. Generation of ROS was determined using luciferase based assay. Fra2 DNA-binding activity was measured in AP-1 transcription assay in monocytic U937 cell line following epigenetic and TLR8 modifications. The level of anti-fibrotic miRNA-5196, which is predicted to bind and inhibit 3’UTR of Fra-2 gene, was also determined in HC and SSc monocytes. Results Combination of DZNep+TLR8 enhanced Fra2 (2-fold, p = 0.02), TIMP-1 (2-fold) and IL-8 (7.87-fold, p < 0.001) expression in SSc monocytes. Fra2 DNA-binding activity was 1.5-fold increased upon stimulation. Secreted level of TIMP-1 was 1.46-fold higher in SSc monocytes compared to unstimulated cells. Generated ROS was 2.21-fold (p = 0.0395) higher following DZNep+TLR8 stimulation in monocytic U937 cells. In contrast, miRNA-5196 expression was 2.13-fold decreased in SSc monocytes upon DZNep+TLR8 stimulation. Also the level of SOD1 was decreased in HC and SSc monocytes following stimulation, 2.16-fold (p = 0.025) and 1.56-fold, respectively. Conclusions These data suggest that DZNep and TLR8 agonist are able to enhance pro-fibrotic TIMP-1, IL-8 and oxidative stress generation. As opposed by the decrease of anti-fibrotic miRNA-5196 and anti-oxidant SOD1 expression in SSc monocytes, which might be used as a potential modulators of fibrogenesis in SSc. Supported by Homing Plus grant/2013–8/4 from Foundation for Polish Science and UMO-2015/16/S/NZ6/00041 from National Science Centre, Poland.

A3.1 Epigenetic and TLR8-dependent regulation of profibrotic genes expression in monocytes in systemic sclerosis and its implication on fibroblasts trans-differentiation

Background and objectives To investigate whether epigenetic changes induced by 3’deazaneplanocin - DZNep and TLR signalling pathway can modulate monocytes to produce tissue-inhibitor of metalloproteinase-1 (TIMP-1) via Fra2 (Fos-related antigen 2) activity, a novel downstream mediator promoting fibrogenesis. Methods AP-1 and TIMP-1 expression following TLR8 treatment was measured by qRT-PCR in monocytes from Systemic sclerosis (SSc) patients (n = 13) and healthy controls (HC) (n = 13). TIMP-1 promoter activity was measured in U397 monocytic cell line using luciferase reporter assay. The effect of DZNep treatment on inhibition of tri-methylation of lysine 27 on histone 3 was analysed by Western Blot. Expression of TIMP-1 and Fra2 was determined in response to DZNep. The functional effect of TLR8 and DZNep-treated HC monocytes was studied on dermal fibroblasts’ trans-differentiation. Results Increased Fra2 and TIMP-1 expression was correlated in SSc monocytes (p = 0.021), but not in HC monocytes upon TLR8 stimulation. In contrast, the expression of anti-fibrotic Fra1 was significantly (p = 0.037) reduced in SSc monocytes compared to HC. Inhibiting AP-1 activity reduced TIMP-1 production in TLR8 stimulated HC and SSc monocytes. Also, TLR8 stimulation induced significant (p = 0.015) TIMP-1 promoter activity in monocytic U937 cells. Combination of DZNep plus TLR8 enhanced Fra2 (4.1 times) and TIMP-1 (4.7 times) expression in HC monocytes. However, the reverse effect on Fra2 and TIMP-1 expression was observed in SSc monocytes following stimulation. Finally, DZNep plus TLR8-treated HC monocytes induced strong production of collagen and a-SMA in dermal fibroblasts reflecting their trans-differentiation, which is a key event in the pathogenesis of SSc. Conclusions These data demonstrate that histone modification induces by DZNep has an opposite effect of Fra2-mediated TIMP-1 production on HC versus SSc monocytes. Therefore, DZNep cloud be used as a selective regulator of downstream mediators, which orchestrates SSc development.