Background Sirtuin 1 (SIRT1) is a class III histone deacetylase which could play a critical role in several biological functions including lifespan, stress, and inflammation. Objectives Our main objective was to evaluate SIRT1 expression in peripheral blood mononuclear cells (PBMCs) isolated from rats with adjuvant induced arthritis (AIA) treated or not with low and high doses of glucocorticoids (GCs). Our second aim was to determine the production of proinflammatory cytokines such as tumour necrosis factor alpha (TNF), interleukin-1 beta (IL-1) and interleukin-6 (IL-6) in tissues (liver and spleen) of these rats, and to assess a potential correlation between SIRT1 expression and the production of cytokines in tissues of rats with AIA treated or not with low and high doses of glucocorticoids. Methods We performed a study on rats with AIA treated with low and high doses of prednisolone or left untreated. The SIRT1 expression was quantified in PBMCs using western blotting. Levels of proinflammatory cytokines TNF, IL-1 and IL-6 were assessed in liver and spleen from rats with AIA treated with GCs or left untreated using an ELISA assay. Results SIRT1 expression was increased in PBMCs isolated from rats with AIA treated with LD-GCs and HD-GCs compared to untreated animals. In culture, resveratrol enhanced the SIRT1 expression in PBMCs isolated from rats with AIA in a dose-dependent manner. We observed increased levels of TNF alpha in liver (correlated with SIRT1 expression) and decreased IL-6 levels in spleens (negatively correlated with SIRT1 expression) from rats with AIA treated with LD-GCs and HD-GCs compared to controls. Conclusions SIRT1 expression is increased in PBMCs isolated from rats with AIA and treated with GCs parallel to decreased levels of IL-6 in spleen and increased levels of TNF in liver. This study shows that the SIRT 1 expression is measurable in circulating PBMCs of rats with AIA and is increased under GC treatment. The potential epigenetic effect of GCs on production of proinflammatory cytokines in tissues, namely liver and spleen, is also studied. Disclosure of Interest None declared

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of Nef and of the translation elongation factor eEF1A in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.

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, R&D Systems, Minneapolis, MN). Statistical analysis used Wilcoxon and t tests; 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, R&D Systems, Minneapolis, MN). Statistical analysis used Wilcoxon and t tests; 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 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, R&D Systems, Minneapolis, MN). Statistical analysis used Wilcoxon and t tests; 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 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, R&D Systems, Minneapolis, MN). Bone remodeling markers (Ca, P, Vitamin D, sCTX I), and inflammation (CRP, ESR) were evaluated at the same time. Statistical analysis used Wilcoxon and t tests; 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 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

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of the Nef/eEF1A (eukaryotic translation elongation factor 1-alpha) complex in nucleocytoplasmic shuttling in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t, occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin-A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.

The introduction of highly active antiretroviral therapy (HAART) has been an important breakthrough in the treatment of HIV-1 infection and has also a powerful tool to upset the equilibrium of viral production and HIV-1 pathogenesis. Despite the advent of potent combinations of this therapy, the long-lived HIV-1 reservoirs like cells from monocyte-macrophage lineage and resting memory CD4+ T cells which are established early during primary infection constitute a major obstacle to virus eradication. Further HAART interruption leads to immediate rebound viremia from latent reservoirs. This paper focuses on the essentials of the molecular mechanisms for the establishment of HIV-1 latency with special concern to present and future possible treatment strategies to completely purge and target viral persistence in the reservoirs.