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Background:Axial spondyloarthritis (ax-SpA) corresponds to a group of chronic inflammatory disease mainly affecting the axial skeleton. TNFα and IL-17A have been identified as key inflammatory mediators driving the inflammatory process of ax-SpA. Epigenetics refers to different mechanisms that alter gene expression without involving changes in DNA sequence. The mechanisms of epigenetics include microRNA, histone modifications or DNA methylation. DNA methylation is associated with a repressed chromatin state and inhibition of gene expression. It is recognized that aberrant DNA methylation can result in immune cell autoreactivity.Objectives:Epigenetics have been rarely evaluated in ax-SpA. We previoulsy reported that patients with ankysloing spondylitis (AS) had an imbalance between HAT and HDAC activities. In this study, we aimed to evaluate the global DNA methylation of patients with ax-SpA.Methods:Case-control study (NCT03092583). Patients with radiographic (AS) or non radiographic (nr) ax-SpA (ASAS criteria) and healthy controls (HC) were evaluated. All the patients were biologic naïve and under NSAIDs. Disease activity was evaluated by BASDAI and ASDAS. CD4+T cells and CD14+ monocytes were isolated form peripheral blood and then DNA was extracted (E.Z.N.A. Blood DNA kit, Omega Bio-Tek). Global DNA methylation (5-mC) was determined using MethylAmp global DNA methylation quantification kit (Epigentek) using 150ng of total DNA.Results:25 patients with AS (18 M; mean age ± SEM: 48.9 ± 3.5 y; mean disease duration: 14.9 ± 2.2 y; B27+: 84%), 21 with nr-axSpA (11 M; age: 42 ± 3.3 y; disease duration: 7.9 ± 2.3 y; B27+: 68%) and 11 HC (7 M; age: 48.4 ± 3.9 y) were evaluated. Patients had active disease (BASDAI and ASDAS in AS and nr-axSpA: 5.1 ± 0.4 and 5.4 ± 0.5; 4.7 ± 0.4 and 5 ± 0.4, respectively). In CD4+ T lymphocytes, global DNA methylation was lower in the whole group of patients (AS and nr-ax-SpA) compared to HC (0.91 ± 0.26vs1.08 ± 0.19 % of 5-mC) (NS). Conversely, DNA methylation was higher in monocytes from patients compared to HC (1.43 ± 0.16vs1.15 ± 0.5 % of 5-mC) (NS). When analysing the results between ax-SpA subgroups, an hypomethylation was more evident in the CD4+T lymphocytes from patients with nr-axSpA compared to AS and HC, a result that was not observed in the monocyte subpopulation (Figures).Conclusion:A global DNA hypomethylation is observed in patients with ax-SpA, especially in the nr-axSpA subgroup. These results are more evident in T CD4+ lymphocytes. Additional analysis on a larger series of patients is required to confirm these preliminary results. In addition, we aim to examine the specific DNA methylation status of the TNF promoter gene.References:[1]Toussirot Eet al, PlosOne 2013Figure.global DNA methylation of CD4+ T lymphocytes and monocyctes from patients with ankylosing spondylitis (AS), non radiographic axial spondyloarthritis (nr-ax-SpA) and healthy controls (HC)Disclosure of Interests:None declared

Background Sirtuin 1 (Sirt1) is a nuclear enzyme from the class III histone deacetylases (HDACs) modulating gene expression, involved in the regulation of various biological processes (cell survival, apoptosis, gluconeogenesis, adipogenesis, lipolysis), local and systemic inflammation, as well as in bone and cartilage remodeling. Sirt-1 reduces pro inflammatory cytokines release. Objectives The main objective of the study was to evaluate Sirt1 activity in PBMC by venous blood aspiration in rheumatoid arthritis (RA) patients, compared to those of control patients, and to analyze the relationship between Sirt1 activity, disease activity and production of mediators of inflammation and cytokines (TNFalpha, IL-6, IL-8) by the cells, before and after ex vivo treatment with a Sirtuin activator, resveratrol. Methods A prospective and comparative monocentric study was performed in order to compare the activity of Sirt1 in patients with RA (according to ACR criteria) and controls. Disease activity was assessed by DAS28 (ESR) and CRP. PBMC were isolated from venous blood, and Sirt1 activity was evaluated from cytoplasmic and nuclear compartments using a fluorometric assay (SIRT1 fluorimetric kit, BML-AK-555, Enzo Life Sciences, Villeurbanne, France) at the 15 minutes point. Culture supernatant levels of TNF alpha, IL-6, IL-8 were quantified before and after resveratrol (1 μmol and 5 μmol) ex vivo treatment, with commercial kits (Quantikine Kits, RD significance: p less than 0.05. Results Twenty two patients with RA (age 55±12 years, meanDAS28: 4.27;disease duration 15±10 years; 75% ACPA +; mean CRP: 12.7 mg/l) and 18 controls (age 54±13 years) were included. No differences were found in cytoplasmic or nuclear Sirt1 activity between patients and controls. Cytoplasmic and nuclear Sirt1 activity were correlated in patients and controls. Sirt1 activity (nuclear and cytoplasmic) was not correlated with DAS28, CRP or ESR, but cytoplasmic Sirt-1 activity was correlated to baseline IL-6 (p=0.02) and baseline TNF (p=0.04) in RA and controls, but not with IL-8. Sirt1 activity (cytoplasmic and nuclear) was lower in RA ACPA+ compared to ACPA neg, and nuclear activity was higher in patients treated with corticosteroids. After resveratrol treatment, no changes in TNF, IL-6 or IL-8 levels were found, Conclusions Sirt1 activity (cytoplasmic and nuclear) from PBMC was not different between RA patients and controls, nevertheless, the correlation in RA with Sirt1 activity and TNF and IL-6 levels, as well as reduced Sirt1 activity in ACPA + patients suggest an implication of this epigenetic regulation in RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1584

Background Sirtuin 1 (Sirt1) is a nuclear enzyme from the class III histone deacetylases (HDACs) modulating gene expression, involved in the regulation of various biological processes (cell survival, apoptosis, gluconeogenesis, adipogenesis, lipolysis), local and systemic inflammation, as well as in bone and cartilage remodeling. Sirt-1 reduces pro inflammatory cytokines release. Spondyloarthritis (SpA) is a multifactorial disease, but no epigenetic data are currently available in this disease. Objectives The main objective of the study was to evaluate Sirt1 activity in PBMC by venous blood aspiration in spondyloarthritis (SpA) patients, compared to those of control patients, and to analyze the relationship between Sirt1 activity, disease activity and production of mediators of inflammation and cytokines (TNFalpha, IL-6, IL-8) by the cells, before and after ex vivo treatment with a Sirtuin activator, resveratrol. Methods A prospective and comparative monocentric study was performed in order to compare the activity of Sirt1 in patients with SpA (according to ASAS criteria) and controls. Disease activity was assessed by BASDAI, ASDAS, ESR and CRP. PBMC were isolated from venous blood, and Sirt1 activity was evaluated from cytoplasmic and nuclear compartments using a fluorometric assay (SIRT1 fluorimetric kit, BML-AK-555, Enzo Life Sciences, Villeurbanne, France) at the 15 minutes point. Culture supernatant levels of TNF alpha, IL-6, IL-8 were quantified before and after resveratrol (1 μmol and 5 μmol) ex vivo treatment, with commercial kits (Quantikine Kits, RD significance: p less than 0.05. Results Forteen patients with SpA (age 48±14 years, mean BASDAI 43±18; ASDAS-CRP 3,1±0,9; disease duration 19±10 years; all were HLA-B27+; mean CRP: 19.1 mg/l) and 18 controls (age 54±13 years) were included. No differences were found in cytoplasmic or nuclear Sirt1 activity between patients and controls. Cytoplasmic and nuclear Sirt1 activity were correlated each other in patients and controls. Sirt1 activity (nuclear and cytoplasmic) was not correlated with ASDAS, BASFI, CRP or ESR, but a trend of correlation was found between nuclear Sirt-1 activity and BASDAI (r =0.5; p=0.07). No differences in Sirt1 activity was found between axial and peripheral forms of SpA. Sirt1 activity (cytoplasmic and nuclear) was higher in SpA with NSAIDs compared to patients without NSAIDs (p=0.005). No correlations were found between Sirt1 activity and cytokine levels at baseline, and no change after ex vivo treatment by resveratrol. Conclusions Sirt1 activity (cytoplasmic and nuclear) from PBMC was not different between SpA patients and controls in this study, these result do not argue for a major implication of Sirt1 in SpA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1744

Background Acetylation or deacetylation of the histone proteins are controlled by histone acetyltransferase (HAT) and histone deacetylase (HDAC), respectively. The equilibrium between HAT and HDAC gives insights into the level of gene transcription, including genes coding for inflammatory cytokines such as TNFa. We previously reported an imbalance between HAT and HDAC in patients with rheumatoid arthritis (RA) or ankylosing spondylitis (AS) (ref). The influence of biological treatments, given for these inflammatory rheumatic diseases, on HAT/HDAC ratio is currently unknown. Objectives to determine whether biological agents used in routine clinical practice in RA or AS may influence the HAT/HDAC ratio. Methods seven patients with RA and five with AS were evaluated. All were biologic-naive. Two patients with RA and five with AS received a TNFa inhibitor (etanercept, n=4; infliximab, n=2; adalimumab, n=1) and five patients with RA received rituximab (RTX). Patients were evaluated at baseline and after three months of anti-TNFa or four months of RTX. HAT/HDAC activity was evaluated in PBMC nuclear extracts using colorimetric assay (HAT: Enzo Life Sciences, Koropi, Greece; HDAC: EpiQuik™, Epigentek, Farmingdale, NY, USA). Activity was measured prior to, and after ex vivo exposure of PBMCs to the HDAC inhibitors (HDACi) trichostatin A (TSA) and sirtinol (Sirt) Results all patients responded to treatment. With TNFa inhibitors, HAT activity increased considerably in patients with RA (+236%) or AS (+198%). Similar changes were observed for HAT with TSA (+114.5%) or Sirt (+38%) added to PBMC nuclear extracts in RA. In AS, there were no changes for HAT in PBMCs measured with TSA or Sirt. For HDAC, there was a decrease in RA (-41%) while in AS, no variation was observed. With HDACi, we found a decrease in the levels of HDAC in RA (TSA: -22.7%; Sirt: -41.5%) while in AS, only TSA induced a decline in HDAC (-43.6%). Under RTX, both HAT and HDAC increased (+445.2% and +73.9%). The addition of TSA or Sirt to PBMC nuclear extracts increased the levels of HAT (+15.6% and +109.8%) while HDAC tended to decrease (- 32.6% and -17.9%). Conclusions Our results indicate a clear increase of the HAT/HDAC ratio in RA and AS during anti-TNFa therapy, while RTX increased both HAT and HDAC. HDACi have been reported to limit production of proinflammatory cytokines including TNFa, and expression of the sirtuin 1 (SIRT1) gene is under the control of NF-kB, which is activated by TNFa. TNFa inhibitors via deactivation of NF-kB could decrease SIRT1 gene expression and activity, and ultimately enhance HAT/HDAC ratio in PBMCs of patients. Our results suggest that an anti-inflammatory biological profile was expressed under the influence of anti-TNFa therapy that increases HAT/HDAC ratio and therefore may contribute to the disease improvement. References 1. Toussirot E et al. PlosOne 2013, 8:e70939 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2164

We report a case of hepatitis B virus (HBV) reactivation following the use of anti-tumour necrosis factor α (TNFα) antibodies that illustrates the need for careful viral monitoring and pre-emptive antiviral treatment in such patients. A 35 year old white woman presented with a history of chronic hepatitis B without an increase in serum alanine aminotransferase (ALT) or detectable HBV DNA by a hybridisation technique since its diagnosis (in 1993); she was thus considered to be an asymptomatic HBV carrier. Her serological status was as follows: hepatitis B surface antigen positive, hepatitis B e antigen negative, hepatitis B e antibody positive, suggesting HBV precore mutant. Her rheumatological history began in September 2001 with oligoarthritis, inflammatory low back pain, limitation of motion, and anterior chest wall involvement. Symptoms improved incompletely with non-steroidal anti-inflammatory drugs. Biological inflammation (erythrocyte sedimentation rate 62 mm/1st h, C reactive protein 53 mg/l), positive HLA-B27 …

Background Sirtuin 1 (Sirt1) is a nuclear enzyme from the class 3 histone deacetylases (HDACs) modulating gene expression, involved in the regulation of diverse biological processes ( cell survival, apoptosis, gluconeogenesis, adipogenesis, lipolysis), local and systemic inflammation, as well as in bone and cartilage remodeling. Objectives The main objective of the study was to evaluate Sirt1 activity in PBMC by venous blood aspiration in patients with osteoporosis (OP), compared to those of control patients, and to analyze the relationship between Sirt1 activity and production of mediators of inflammation and cytokines (TNFalpha, IL-6, IL-8) by the cells after ex vivo treatment with a Sirtuin activator, resveratrol, the most potent natural compound able to activate Sirt1. Methods A prospective and comparative monocentric study was performed in order to compare the activity of Sirt1 in patients with OP and controls. OP was defined by low BMD and at least one vertebral fracture. Secondary causes of osteoporosis were ruled out. PBMC were isolated from venous blood, and Sirt1 activity was evaluated from cytoplasmic and nuclear compartments using a fluorometric assay (SIRT1 fluorometric kit, BML-AK-555, Enzo Life Sciences, Villeurbanne, France) at the 15 minutes point. Culture supernatant levels of TNF alpha, IL-6, IL-8 were quantified before and after resveratrol ( 1 µmol and 5 µmol) ex vivo treatment, with commercial kits (Quantikine Kits, RD significance : p less than 0.05. Results Eighteen patients with symptomatic OP (age 68 ± 9 years) and 18 controls (age 54 ± 13 years) were included. No differences were found in cytoplasmic or nuclear Sirt1 activity between patients and controls. Cytoplasmic and nuclear Sirt1 activity were significantly correlated in patients and controls. Sirt1 activity did not correlate with bone biologic remodeling biomarkers or biologic markers of inflammation (CRP). After resveratrol treatment, no changes in TNF alpha, IL-6 or IL-8 levels were found, compared to pre treatment values, or between patients and controls. Conclusions Sirt1 activity (cytoplasmic and nuclear) from PBMC was not different between OP patients and controls. These results suggest that there is no implication of Sirt regulation of pro inflammatory cytokines or chemokines in PBMC from patients with osteoporosis. Disclosure of Interest None Declared

Background Sirtuin 1 (Sirt1) is a nuclear enzyme from the class 3 histone deacetylases (HDACs) modulating gene expression, involved in the regulation of diverse biological processes (cell survival, apoptosis, gluconeogenesis, adipogenesis, lipolysis), local and systemic inflammation, as well as in bone and cartilage remodeling. Objectives The main objective of the study was to evaluate Sirt1 activity in PBMC by venous blood aspiration in osteoarthritis (OA) patients, compared to those of control patients, and to analyze the relationship between Sirt1 activity and production of mediators of inflammation and cytokines (TNFalpha, IL-6, IL-8) by the cells after ex vivo treatment with a Sirtuin activator, resveratrol. Methods A prospective and comparative monocentric study was performed in order to compare the activity of Sirt1 in patients with osteoarthritis and controls. OA was defined according to the ACR criteria, with radiological grading (K-L); symptoms were quantified with Lequesne’s index. PBMC were isolated from venous blood, and Sirt1 activity was evaluated from cytoplasmic and nuclear compartments using a fluorometric assay (SIRT1 fluorimetric kit, BML-AK-555, Enzo Life Sciences, Villeurbanne, France) at the 15 minutes point. Culture supernatant levels of TNF alpha, IL-6, IL-8 were quantified before and after resveratrol (1 µmol and 5 µmol) ex vivo treatment, with commercial kits (Quantikine Kits, RD significance : p less than 0.05. Results Nineteen patients with symptomatic knee OA (age 64 ± 9 years, mean Lequesne’s index : 8.4; grade II or III of the K-L classification) and 18 controls (age 54 ± 13 years) were included. No differences were found in cytoplasmic or nuclear Sirt1 activity between patients and controls. Cytoplasmic and nuclear Sirt1 activity were correlated in patients and controls. Sirt1 activity (nuclear and cytoplasmic) was correlated to baseline IL-6 (p = 0.002) and baseline TNF (p = 0.004), but not with IL-8. Sirt1 activity did not correlate with clinical activity (Lequesne’s index) or biologic inflammation. After resveratrol treatment, no changes in TNF or IL-8 levels were found, but a significative, resveratrol-dose-dependent increase in IL-6 levels was demonstrated in OA patients (5.17 ± 0.89 pg/ml at baseline, 6,72 ± 1.31 after 1 µmol ex vivo treatment, and 7.64 ± 1.35 after 5 µmol ex vivo treatment; p = 0.02), not found in controls. Conclusions Sirt1 activity (cytoplasmic and nuclear) from PBMC was not different between OA patients and controls, nevertheless, ex vivo treatment of these PBMC with resveratrol, a Sirt1 activator, was unexpectedly associated with increased IL-6 levels in a dose-dependent relation only in OA patients, suggesting that IL-6 expression could be specifically regulated via Sirt1 in OA. Disclosure of Interest None Declared

Background TNFa is a major cytokine involved in conditions such as ankylosing spondylitis (AS) and rheumatoid arthritis (RA). Epigenetic regulation corresponds to different processes including modifications in histone proteins. These mechanisms regulate the transcription of genes coding for inflammatory cytokines such as TNFa. The acetylation of histone proteins (dependent on histone acetyl transferase-HAT-) promotes gene transcription while deacetylation (controlled by histone deacetylase) prevents this reaction. Limited data are available on HAT and HDAC activities in AS or RA. HDAC inhibitors (HDACi) are currently in development and may be of interest in modulating TNFa production in RA or AS. Objectives To determine the levels of HAT and HDAC activities in patients with AS or RA compared to healthy controls (HC) and to evaluate the ex vivo effects of HDACi (trichostatin A-TSA- and sirtinol -Sirt-) on HAT and HDAC activities and TNFa production by PBMC. Methods 21 patients with AS (New York criteria, 18 M, mean age ± SEM 44.3 ± 3.2 years, disease duration 14.1 ± 2.2 years), 52 patients with RA (ACR 1987 criteria, 16 M, mean age 56.9 ± 1.6, disease duration 11.3 ± 1.2) and 38 healthy controls (HC) (12 M, mean age: 34.6 ± 1.8) were evaluated. No patient received biologics. HAT and HDAC activities were assessed on nuclear extracts of PBMC isolated by Ficoll hypaque using a colorimetric assay (EpiQuick HAT Activity/inhibition Assay kit, and EpiQuick HDAC Activity/inhibition Assay kit, Epigentek). These activities were measured prior and after ex vivo treatment of PBMC by HDAC inhibitors. TNFa was evaluated in PBMC culture supernatants after 1 and 3 days (TNFalpha Quantikine ELISA kit, R&D Systems). Results HAT activity was decreased in patients with AS compared to HC (68.2 ± 8.1 vs 111.3 ± 15.5 ng/h/mg) (p= 0.05) while RA patients had increased HAT activity (126.8 ± 16.4 vs 111.3 ± 15.5 ng/h/mg; NS). Compared to HC, HDAC activity was decreased in both AS (p= 0.01) and RA (NS) (HC vs AS vs RA: 4778.9 ± 752 vs 1984. 6 ± 249 vs 3915.9 ± 790 pmol/min/mg). No correlation was observed between clinical disease assessment in RA or AS and HAT or HDAC activity. Ex vivo addition of TSA or Sirt to PBMC reduced HDAC activity by 51.1% in HC and by 37.7% in RA but had no effect in AS.HAT activity was not modulated by HDACi. TNFa production by PBMC was down regulated by the addition of TSA or Sirt to cell culture in HC and RA but this regulation was only obtained with Sirt in PBMC culture from patients with AS. Conclusions HAT and HDAC activities are dysregulated in AS and RA with a balance between HDAC and HAT favoring HAT activity and promoting gene transcription. Ex vivo treatment of PBMC by HDAC inhibitors may regulate HDAC activity and TNFa production especially in HC and RA but seems less effective in AS. Disclosure of Interest None Declared