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Background:Geranylgeranyltransferase type I (GGTaseI) is the enzyme responsible for the prenylation/ lipidation of the RhoA family proteins, which keeps them attached to the cell membrane. We reported that GGTaseI-deficient (GLC) mice develop a spontaneous and age-dependent arthritis, reproducing the pathology of RA1. Targeting GGTaseI activates RhoA proteins.Objectives:To study which of the activated Rho proteins is responsible for development of arthritis, we deleted individual RhoA, Rac1 or Cdc42 genes in GLC mice. We study consequences of GGTaseI deficiency for lymphocyte function.Methods:Double deficient mice that lack Rac1 (GLC Rac1fl/fl), RhoA (GLC RhoAfl/fl) and Cdc42 (GLC Cdc42fl/fl) were developed by Cre-technology using the LysM-promotor, and were on a mixed genetic background (129Ola/Hsd-C57BL/6)2. Joints of the hind paws were assessed for signs of arthritis histologically and by micro CT at age of 16 weeks. Phenotype of spleen CD4 and CD8 T cells was analysis by flow cytometry. Proliferation and cytokine production was assessed in spleen cultures by ELISA. Gene expression profile was analyzed by RT-PCR.Results:Deletion of Rho proteins had divergent effect on development of arthritis in GLC mice. We observed a reduction of the arthritis index in GLC Rac1fl/fl (n=19, p=0.027) and GLC RhoAfl/fl (n=4, p=0.007) mice compared to GLC (n=16), while GLC Cdc42fl/fl (n=4) had no change in arthritis development. GLC RhoAfl/fl mice increased the bone mass compared to GLC (p=0.029).Flow cytometry analysis showed that RA-prone GLC and GLC Cdc42fl/fl mice had lower number of CD4 cells in spleen. CD4 cells of RA-prone GLC and GLC Cdc42 mice had significantly higher subsets of the regulatory FoxP3+ and FOXp3+CD25+ cells (p=0.016-0.029 and p=0.016-0.029 respectively) compared to control and GLC RhoAfl/fl mice. Additionally, RA-prone mice had higher expression of receptors to extracellular matrix proteins collagen (α2β2) and fibronectin (α5β1) compared to control mice (p=0.016 and p=0.011 resp) and to RA-protected mice (GLC Rac1fl/fl and GLC RhoAfl/fl, p=0.0004 and p=0.011, resp). In total, both the number of FoxP3+ CD4 cells and the expression of α5β1 receptors on CD4 cells correlated strongly with the synovitis score (r=0.72, p=0.0017 and r=0.59, p=0.012, respectively).GGTaseI gene lays under the control of HOX proteins essential for cell homing. Importantly, HOX regulate the expression of integrins. Studying the expression of HoxA genes in spleen, we found that RA prone GLC and GLC Cdc42 mice tended to have lower expression of HoxA2 and higher expression of HoxA9 compared to RA-protected GLC Rac1 and GLC RhoA and to control mice. The Hoxa9/Hoxa2 ratio was significantly higher in RA prone mice compared to RA-protected mice (p=0.0085) and control mice (p=0.019). This ratio correlated with α5β1 receptors (r=0.55, p=0.0084), FOXP3+ CD4 cells (r=0.50, p=0.017), and the arthritis index (r=0.50, p=0.033).Conclusion:Taken together this study shows that Rho proteins play divergent role in development of arthritis. Activation of Rac1 and RhoA by GGTaseI deletion changes the pattern of HOXA proteins and increases expression of integrin receptors, which facilitates leukocyte influx in the paw joints. Deletion of Rac1 and RhoA has RA-protective effect in GLC mice.References:[1]Khan, O.M., et al.J Clin Invest121, 628 (2011).[2]Akula, M.K., et al.Nat Commun10, 3975 (2019).Disclosure of Interests:None declared

Background:The insulin-like growth factor 1 receptor (IGF1R) signalling mediates numerous developmental processes acting through downstream adaptor molecules IRS1/2, which activate Akt and inhibit the family of forkhead box class O (FoxO). Inhibition of IGF1R signalling alleviates rheumatoid arthritis (RA) (Erlandsson et al., 2017), however, the role of IGF1R signalling in the regulation of immune function is poorly understood.Objectives:To investigate the link between IGF1R signalling and antigen presentation in experimental arthritis.Methods:Arthritis was induced by immunising Balb/c mice with methylated bovine serum albumin (mBSA, n=18) and DBA/1 mice with type II collagen (CII, n=18). The mice were treated with a synthetic IGF1R inhibitor NT157 or with short hairpin RNA targeting IGF1R (shIGF1R) from the day prior to immunisation. Controls were treated with cyclodextrine vehicle/ non-targeting (nt)RNA, respectively. Flow cytometry was used for spleen cell phenotype. Antibody levels were measured by ELISA. Immunohistochemistry (IHC) of spleen was performed for assessment of marginal zone (MZ) and location of pS612IRS1+ and pS256FoxO1+ cells. IHC images were acquired by fluorescent confocal microscopy, and analysed using ZEN2009 and Cell Profiler soft ware.Results:The inhibition of IGF1R resulted in an 80% increase in MZ area in NT157-treated mice compared to controls (p=0.0001). This was supported by a significant increase of CD21+ (p=0.034) and CD23+ cell populations (p=0.00059), both among the CD19+ B cells and antigen-presenting MHCII+CD19- cells, implying that IGF1R expression regulates the populations of MZ and follicular cells. Additionally, there was a strong positive correlation between the decrease of IGF1R+ and ICOSL+ population on CD21+ cells (r=0.70, p=0.0071), which retained them in the MZ and prolonged communication with macrophages. Insufficient feedback from ICOSL- B cells limited expression of CXCR5 on CD4 cells. The IHC analysis displayed that, IGF1R inhibition led to abundance of inactivate pS612IRS1+ and pS256FoxO1+ cells within the MZ, compared with controls (p=0.0002). Alongside the increase of IgM+ B cell population (p=0.0022), we observed significant increase in number of antigen-presenting F4/80+ cells (p=0.043) and MARCO expression (p=0.043) after IGF1R intervention. Finally, the NT157- treated mice displayed a significant pleiotropic increase in IgM autoantibody production, with anti-CCP IgM (p=0.027), RF-IgM (p=0.0085), anti-DNA IgM (p=0.066) and in total IgM (p=0.027) levels, which correlated positively with pS256FoxO1+ cells (r=0.51, p=0.03). Levels of IgG were not changed.Conclusion:We show that IGF1R signalling is important for immune cell communication after antigen challenge. IGF1R controls ICOSL dependent trafficking of B cells through the MZ and facilitates interaction with T cells. Retention of B cells in the MZ tips the balance from T cell to macrophage-dependent processes, which permits the formation of autoantibody producing B cells.References:[1]Erlandsson, M., et.al., 2017. IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1863(9), pp.2158-2170.Disclosure of Interests:None declared

Background:Interferon-gamma (IFNg) producing effector T cells play the leading role in triggering and perpetuation of inflammation in rheumatoid arthritis. Inflammation leads to metabolic reprogramming of T cells and high energy consumption supporting proliferation and IFNg production. Being a part of chromosomal passenger complex, oncoprotein survivin is essential for cell proliferation. It has also been identified as a marker of severe therapy-resistant rheumatoid arthritis. Thus, we aimed to explore the association between survivin and IFNg producing phenotype of CD4 T cells.Objectives:We study if survivin mediates the glucose dependent mechanism of IFNg production in CD4 T cells.Methods:CD4 cells were sorted from the peripheral blood of RA patients and healthy controls, activated with aCD3, cultured in presence of survivin inhibitor YM155 and subjected to RNA sequencing (Illumina, Life Science). IFNg levels in supernatants were measured by ELISA. To study glucose uptake in presence of YM155, CD4 cells were treated with IFNg+aCD3 overnight followed by 2NBD-glucose challenge for 30 min. Uptake of fluorescent 2NBD-glucose probe was measured by flow cytometry. Statistical analysis of RNAseq was performed in R-studio using the Bioconductor package DESeq2.Results:Comparison of the whole-genome transcription profile of CD4 cells different by levels of BIRC5, coding for survivin, demonstrated that the BIRC5hi group expressed significantly higher levels of IFNg (mRNA, p=10-26 and protein, p=10-4). Also, BIRC5hi CD4 cells had higher expression of glucose transporter GLUT1 (SLC2A1, p=0.0064) and of glycolytic enzymes glucose-6-phosphate dehydrogenase (G6PD, p=10-6), pyruvate kinase (PFKP, p=10-6), and lactate dehydrogenase (LDHA, p=10-14). On the contrary, expression of the key regulator of glycolysis 6-phosphofructo-2-kinase (PFKFB3) was significantly lower in the BIRC5hi group (p=4.4x10-5). Notably, expression of glycolytic enzymes G6PD and PFKFB3 correlated strongly to IFNg (r=0.880 and -0.698, respectively), TBX21 (r=0.811 and -0.698) and perforin (r=0.781 and -0.698). To demonstrate functional relevance of the connection between BIRC5 and glucose metabolism, survivin was inhibited in CD4 cell cultures. Survivin inhibition resulted in significant increase of PFKFB3 (p=7x10-6) and LDHA (p=0.0089), leading to inhibition of phosphoglycerate mutase PGAM1 and ATF citrate lyase ACLY (p=0.021 and p=0.0074, respectively), which dignify the restoration of aerobic glycolysis. Importantly, inhibition of survivin decreased 2NBD-glucose uptake by CD4 cells (p=0.031) and reduced expression of GLUT1 (p=0.034). These changes in glucose metabolism were followed by decreased IFNg production in supernatants (p=0.037).Conclusion:The study demonstrates a strong connection between IFNg production and glucose metabolism in CD4 cells. Survivin emerges as an important regulator of glycolysis acting through expression of glycolytic enzymes and glucose transport.Disclosure of Interests:None declared.

Background:Deficiency in geranylgeranyltransferase type I (GGTase-I) results in accumulation of active Rho family proteins RhoA, Rac1 and Cdc42, responsible for cell communication and migration. We reported that mice with GGTase-I deficient macrophages (GLC mice) develop a spontaneous and age-dependent arthritis, reproducing pathology of RA [1].Objectives:We study how GGTase-I deficiency in Mø changes T cell phenotype to facilitate their translocation to joints and the development of arthritis.Methods:GLC mice were developed on a mixed genetic background (129Ola/Hsd-C57BL/6) by Cre-technology using LysM-promotor to knockout the Pggt1b gene in Mø[2]. CD4+ cells were isolated from spleen and lymph node (LN) of 16 weeks-old mice (GLC n=7, wt n=5) expected to have high prevalence of arthritis. RNA was extracted to measure expression of the Rho proteins and signature genes to characterize differences in Th-subtypes and migration abilities of CD4+ cells between GLC and wt mice. Furthermore, Illumina RNAseq analyzed the transcriptome of LN CD4+ cells. In a separate experiment we treated GLC mice with CTLA4-FP (n=12) or PBS (n=11) for 20 weeks from the age of 5 weeks. Rationale was to disrupt Mø/T cell contact to prevent arthritis. To study Rho-protein dependent phenotype in human RA, we performed RNAseq of sorted CD4+ cells of RA patients.Results:RNAseq showed that CD4+ cells in LN of GLC mice had IFN-γ dependent cytotoxic profile and upregulated numerous pro-inflammatory genes including Eomes, Cxcr3, Tigit, Tnfsf10, Il-1rl1, Stat1, Jak3, Irf7, Irf5, Ptpn13. Furthermore, the over-represented genes often depended on the IRF family in their transcription.GLC mice overexpressed Cdc42 and Rac1 in spleen CD4+ compared to wt (p=0.005 and p=0.048 resp.). Spleen GLC CD4+ cells had higher levels of α5β1 and α2β2 integrins, strongly correlating to Cdc42 (r= 0.61 p=0.0027 and r=0.50, p=0.018) and arthritis (r=0.64, p=0.0015 and r=0.69, p=0.0004). Importantly, Cdc42, Rac1, and RhoA were higher expressed in LN CD4+ compared to spleen (p=0.016, p=0.031 and p=0.016). In addition, Itgb1 coding for β1 integrin, was upregulated in GLC CD4+ cells of both spleen and LN (p=0.003 p=0.03, resp.), suggesting Rho proteins are important for migration of CD4+ cells to the joint draining LN and for arthritis development. CD4+ cells that migrated to the LN had high proportion of Foxp3+ cells. This also correlated to the expression of Itgb1 (r=0.84, p=0.0012) presenting a plausible mechanism for increased influx of Tregs into joints. Several observations are in favor of this notion. First, GLC mice expressed more Foxp3 in LN compared to spleen CD4+ cells (p=0.016). Second, transcription of Foxp3 in LN CD4+ cells was higher in GLC mice compared to wt (p=0.015). Third, this high Foxp3 coexisted with low transcription of Lef1 (p=0.03), required for Treg immunosuppression. Last, Foxp3 correlated negatively to both Lef1 (r=-0.72, p=0.017), and its cofactor Tcf1 (r=-0.75, p=0.01).CTLA4-FP reduced inflammation in GLC mice evident as lower IFN-γ, IL-6 and TNF-α production (p=0.0002, p+CD4+cells in spleen (p=0.027). In contrast, we observed increased IL-17A production (p=0.056). However, CTLA4-FP treatment did not affect migration of CD4+ cells enriched with Rho-protein into draining LN nor alleviate arthritis.Similar to the GLC mice, CD4+ cells of RA patients with high expression of RhoA, Rac1 and Cdc42 demonstrated enrichment for Th1 signature genes including IFNG, TBX21, Eomes, IL2RA, IL2RB, IL12RB2, TNF, IL18RAP (all, adj. pConclusion:This study shows that accumulation of Rho-proteins in CD4+ cells results in pro-inflammatory IFN-γ dependent phenotype in mice and human RA. Accumulation of RhoA, Rac1 and Cdc42 proteins trigger the migration of CD4+ cells into joint draining LN and facilitates arthritis. Inhibiting Mø/T cell contact in GLC mice did not suffice to prevent migration of Rho-protein expressing cells and arthritisReferences:[1]Khan, O.M., et al. J Clin Invest, 2011. 121(2): p. 628-39.[2]Akula, M.K., et al. Nat Commun, 2019. 10(1): p. 3975.Disclosure of Interests:None declared

Background:Survivin is an integral part of the Chromosomal Passenger Complex (CPC) which plays a vital role in mitosis. Experiments have demonstrated that survivin can physically bind to DNA. Crystallographic studies show that survivin binds to Threonine-3 of histone H3. In patients with autoimmune diseases, increased survivin expression contributes to an aggravated disease phenotype. Thus, functional, and mechanistic data point to a potential chromatin regulatory role for survivin, possibly in combination with the established gene regulatory function carried out by histone epigenetic modifications (EM).Objectives:The objective of the study was to analyse the co-localization of chromatin bound survivin with three histone H3 epigenetic modifications – acetylated lysine 27 (K27ac) and trimethylated lysine 4 (K4me3) and lysine-27 (K27me3). The second objective was to analyse if survivin-bound DNA sequences overlapped with sequences in the vicinity of 106 GWAS SNPs that are associated with a risk of developing rheumatoid arthritis (RA).Methods:Chromatin from CD4 T cells of 14 female subjects was immunoprecipitated with survivin antibodies and each of the histone H3 antibodies, and coupled with sequencing (ChIPseq, Hiseq2000, Illumina). After mapping the annotations of sequenced regions to the human reference genome hg38, enriched peaks were identified through Homer software. The identified survivin ChIP peaks were analysed for colocalization with peaks of the three histone H3 EMs and with RA risk loci, using the Bioconductor package ‘ChIPPeakAnno’ through RStudio.Results:Among the total of ~13,000 individual survivin ChIP-peaks, 33% colocalized with histone H3 EM peaks. The overlapping peaks show a linear increase in average peak size compared with the peaks showing no colocalization with any H3 EM peak. A maximum of 5.5-fold increase in average peak size was observed when survivin bound peaks overlap with peaks of all three H3 EMs. A major proportion (86%) of top RA risk SNPs was associated with either binding of survivin or H3 EMs. In this subset, 63% of RA risk SNPs were found within an area of 100 kilobases from survivin ChIP-peaks, with preferential enrichment of high-scoring peaks when survivin colocalizes with all 3 H3 EMs. Survivin was bound to risk SNPs annotated to, among others, the major immunological genes CD83, IRF4, CD28, ICOS and IL2RAConclusion:This study presents experimental evidence that survivin binding to DNA preferentially occurred in regions with high density of histone EMs. The increased aggregation of survivin around histone H3 EMs point to its potential regulatory function in gene transcription. Since regions around RA risk SNPs overlap with survivin peaks, survivin’s nuclear function could have immunologically important effects in mechanisms of autoimmune diseases.Disclosure of Interests:None declared

Background:Survivin, coded by BIRC5 gene, is a multitasking protein essential for cell renewal and homeostasis. In autoimmune conditions as rheumatoid and psoriasis arthritis, survivin was associated with inflammation severity and joint damage. Importantly, inhibition of survivin alleviated experimental arthritis in mice. We have recently shown survivin to be essential for T cell differentiation and micro-RNA processing. The known anti-apoptotic and proliferation facilitating functions of survivin does not explain the nuclear localization of survivin in interphase.Objectives:We aimed to uncover nuclear functions of BIRC5/survivin in CD4 cell of RA patients and healthy.Methods:CD4 T cells were isolated from the peripheral blood using positive selection on magnetic beads (EasySep) and activated for 48h with ConA+LPS. Chromatin immunoprecipitation (ChIP) with polyclonal anti-survivin antibodies was done in four independent samples of healthy donors (n=5), healthy smokers (n=3), rheumatoid arthritis (n=3) and breast cancer (n=2). Pooled libraries were constructed for each group and ChIPseq was carried out (Illumina). For comparative RNAseq analysis, activated CD4 T cells were incubated with or without survivin inhibitor (YM155) for 24h. State-of-the-art bioinformatics pipelines were applied for NGS data and the survivin-binding peaks were used for comparison with genes, chromatin state annotation and functional gene- and regulatory regions-based functional analysis. Co-localization of peaks in the whole genome and in vicinity of the differentially expressed genes (DEG) was done using ReMap integrated ChIPseq datasets for all human cells and tissues.Results:We identified 13 thousands non-overlapping survivin ChIP-peaks (>3000 peaks were present in at least 3 samples). Survivin-bound regions were enriched near the genes and promoters (p=e-30 and p=e-8), which implied that survivin role in transcription could be mediated by known transcription factors. Thus, we analyzed survivin peaks vs binding regions of 1135 transcription regulators (TR) available in ReMap.Potential partner proteins of survivin were selected based on the enrichment of the overlapping peaks in the whole genome and in CD4-active regulatory areas. Both, strict overlaps and location within 10 and 100kb survivin peak vicinity were analyzed. This approach allowed us to select >150 TRs enriched in all tests. The enriched TRs were involved in immunity and RA-relevant pathways including cytokine response and production, JAK-STAT signaling, etc. Among the TRs co-localized with survivin were CHD8, MAX, EP300, BRD2, CTCF and RAD21, all responsible for chromatin architecture. Several TRs were massively enriched in the vicinity of DEGs after survivin depletion including MAX, AR, CTCF, MYC and IRF1. Search for TR binding motifs in survivin peaks supported over-representation of binding sites for IRFs (p=e-5) and several proteins of the bZIP-family (p=e-5).Conclusion:Analysis of the survivin bound DNA in CD4 cells demonstrated the nonrandom distribution with specific enrichment within the regulatory elements of the genes and co-localizeation with protein partners to regulate their transcription.Disclosure of Interests:None declared

Background:Interferon gamma (IFNg) signalling and downstream effects make important contribution in pathogenesis of rheumatoid arthritis (RA). Here, we propose a mechanism by which oncoprotein survivin participates in development of IFN-dependent repertoire of T cells in RA patients.Objectives:We study the role of survivin in the phenotype of CD4 T cells of RA patients.Methods:CD4 cells of RA patients and healthy controls were purified from blood, activated and subjected to RNAseq, ChIPseq with antibodies to survivin (BIRC5) was performed on CD4+ cells. Histone H3 ChIPseq was performed using antibodies to H3K27ac, H3K27me3 and H3K4me3. Statistical analysis was performed In R-studio using the Bioconductor package DESeq2, clustering using Spearman and Ward.D2.Results:Unsupervised clustering of CD4 samples by expression of 48 core Th cell markers identified subsets of CD28hiCD27hiIFNnegcentral memory cells (Tcm), CD28loCD27loIFNloeffector memory cells (Tem) and CD28nullCD27nullIFNhiterminal effector cells (Tte). Tte cells showed classical features of Th1 cells including high levels of TBX21, TNFa and IL32 and signs of activation in IFN signalling machinery. Interestingly, they combined the features of peripheral Tregs CD25hiFoxp3hiIKZF2negand IL10 producing cells together with type 1 regulatory cells, which rely on transcription factors BATF and IRF1 for the differentiation and produced high amounts of perforin and granzyme B. Importantly, Tte CD4 cells had also high transcription of BIRC5 (p=1.15e-18).To study if BIRC5 is a part of IFN signalling, CD4 cells were cultured with survivin inhibitor YM155 and activated with IFNg. RNAseq analysis revealed 2033 (FCEncouraged by the survivin ChIPseq results, we wanted to know its relation to histone marks. We observed that 50% of survivin peaks containing both IRF:bZIP and IRF:ETS motifs are co-localized with the H3K27ac marks. In total, 16 of 48 core Th cell markers used for patients clustering were identified by survivin ChIPseq, co-localized with IRF composite motifs and histone marks. They were also dependent of survivin for expression.Conclusion:his study showed that survivin binds to DNA and regulates the core gene expression contributing to maturation and function of the IFNg producing Th1 cells.References:-Disclosure of Interests:Malin Erlandsson: None declared, Karin ME Andersson: None declared, Nisha Nair: None declared, Anastasius Damdimopoulos: None declared, Sofia Töyrä Silfverswärd: None declared, Rille Pullerits: None declared, Anne Barton Consultant of: AbbVie, Maria I Bokarewa: None declared

Background:The oncogenic protein survivin is a marker of severe rheumatoid arthritis (RA). High serum levels of Survivin predict progressive joint damage1and poor treatment response2.Objectives:To study the role of survivin in the transcriptional regulation of phenotype in CD4+T cells.Methods:CD4+T cells of RA female patients were isolated from the perpheral blood. Activated CD4+cells were treated with survivin inhibitor YM155. Transcriptional analysis was done by RNAseq (Illumina) and conventional qPCR. Chromatin of CD4 cells was immunoprecipitated using polyclonal antibodies to survivin and subjected to deep sequencing (survivin ChIPseq, Hiseq2000, Illumina) and aligned to GRCh38. Statistical analysis of differentially expressed genes (DEG) was done in R-studio using Benjamini-Hochberg adjustment for multiple testing (Bioconductor, DESeq2 package).Results:Survivin ChIPseq of the activated CD4+T cells was enriched with the genes engaged in regulatory transcription factor specific DNA binding (GO:0000987, adj p=0.0005) and RNA polymerase II regulatory transcription (GO:0000978, adj p = 0.0004). Among survivin targets were the genes of HOX-B cluster and TALE family proteins MEIS, PKNOX and PBX1 controlling early leukopoesis and T cell maturation. Inhibition of survivin in PBMC resulted in significant upregulation of PBX1 (p=0.023), MEIS3 (p=0.0036), similar tendency was observed for HOXB6 and HOXC4 genes. RNAseq analysis CD4 cells of RA patients with different transcription of PBX1, identified 1636 genes (adj p+cells with low PBX1 (p=0.0005). Among the core transcription factors of T helper cell differentiation, we identifed NF-kB1 and NF-kB2, TBX21, IRF4, IRF8 and STAT3, BATF and BATF3. This followed by significantly higher TNF, IFNg and IL17A and IL17F in PBX1lo CD4 T cells. The pathway enrichment analysis of DEG identified strong over-representation of cytokine-specific genes (GO:005125, GO:0005126, GO:0048018, GO:0030545, FDR q-values 10-12-10-9). The genes of IL4, IL5, IL13, IL9, IL3 and CSF2 located within the chromosome 5 were common for all GO-lists, and were higher in PBX1lo, but none of those genes was identified by survivin-ChIPseq or PBX1-ChIPseq. Analysis of ChIPseq data identified the genes of STAT3, IRF4, IRF8 and BATF as common targets for PBX1 and survivin.Conclusion:This genome-wide analysis indicates that survivin regulates transcription of the TALE family protein PBX1 in CD4+ T cells, which has essential effect for differentiation and phenotype of Th subsets. Low PBX1 in RA patients is associated with terminally differentiated effector CD4+ T cells.References:[1]Svensson, B.et.al.Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study.Arthritis Res Ther16, R12 (2014).[2]Levitsky, A.et.al.Serum survivin predicts responses to treatment in active rheumatoid arthritis: a post hoc analysis from the SWEFOT trial.BMC Med13, 247 (2015).Disclosure of Interests:None declared

Background:Depression and cognitive impairment have been frequently reported in rheumatoid arthritis (RA) (1). Studies of the molecular mechanisms behind these phenomena attract increasing attention. We previously reported that signaling through the insulin-like receptor is impaired in RA and has consequences for pain processing (2).Objectives:We investigated the central and peripheral footprint of the major neurotrophin in the central nervous system, brain-derived neurotrophic factor (BDNF), on pain and mood perception of RA patients.Methods:Pain symptomatology was assessed in 216 female RA patients (mean age 52y, mean disease duration 10 years) by a visual analogue scale (VAS), 18 tender points count (TPC), and by pressure-induced pain threshold measurement. The mood was patient-reported based on the Hospital Anxiety and Depression Scale (HADS). Clinical RA activity was assessed by DAS28. Serum levels of BDNF, IL6, IL1b, IL10 and IFN-gamma were measured by ELISA. Transcription of FOXO1 and FOXO3 was measured by RT-PCR in whole-blood RNA. Effect of BDNF signaling in leukocytes was assessed by differentially expressed gene (DEG) analysis in RNAseq of 24 female RA patients (R-studio, Bioconductor). High-resolution brain MRI was performed in a representative selection of 16 patients. Brain volumes were analyzed with MAPER software for accurate measurement of 83 anatomical regions (3) and compared between two groups of patients with high and low serum BDNF, respectively.Results:In RA patients, high serum levels of BDNF were associated with low TPC (4.1 vs 5.3, p=0.04) and higher pain threshold (kPa, 416 vs 382, p=0.09). No connection between BDNF and mood measures was evident. High BDNF was associated with high serum VEGF (pConclusion:We conclude that high serum BDNF was associated with larger volumes of nociception-related brain regions and lower pain perception, acting independently of IGF1 and systemic inflammation.References:[1] Dougados M, Curr OppRheumatol 2016[2] Andersson, Wasen, PNAS2017[3] Heckemann, NeuroImage 2010Disclosure of Interests:None declared

Background Rheumatoid arthritis (RA) is a joint destructive disease, which leads to physical disability. There is no cure of RA today. Thus, recognition of RA at preclinical stages is essential for successful treatment. Objectives To validate and improve known prediction rules aiming at early recognition of clinically suspect arthralgia and diagnosis of RA. Methods Medical records of 241 first-time patients with clinically suspect joint pain were evaluated for RA diagnosis by the 2010 EULAR/ACR criteria (1) and by the RA prediction rules (13-points, Amsterdam (2)); and 16-points clinical assessment (Leiden (3)) and prospectively followed during 29–47 months. All the patients were a part of the inception cohort of 5455 patients whose samples were tested for RF and/or ACPA during 12 consecutive months at the Laboratory of Clinical Immunology at the University Hospital of Gothenburg. Patients with known diagnosis of RA, other rheumatic disease and chronic pain conditions were excluded. The utility of arthritis-specific autoantibody status (ACPA and/or RF) and the family history for RA prediction were analyzed. Results The RA criteria applied to the records of the first visit identified 24% of patients as RA (EULAR score mean 7.6), 30% as undifferentiated arthritis (UA, EULAR score mean 3.6), while the remaining 45% of the patients were characterized as arthralgia (EULAR score mean 2.3). The 13-point and 16-point prediction rules allocated RA patients to the high-risk group with comparable sensitivity of 78% and 91%. The significant correlation between the two models was present in UA (r=0.398; p=0.0005) and arthralgia (r=0.411; p Conclusions Both prediction rules are shown to be sensitive identify RA patients at assessment. The clinical 16-point assessment has prospective sensitivity for new cases. This is shown independently of autoantibody status and family history of RA. References Cader MZ et al. Performance of the 2010 ACR/EULAR criteria for rheumatoid arthritis: comparison with 1987 ACR criteria in a very early synovitis cohortAnn Rheum Dis. 2011;70:949–55. van de Stadt LA et al. A prediction rule for the development of arthritis in seropositive arthralgia patients. Ann Rheum Dis. 2013;72:1920–6. Krabben A et al. Risk of RA development in patients with unclassified arthritis according to the 2010 ACR/EULAR criteria for RA. Rheumatology (Oxford). 2013;52:1265–70. Disclosure of Interest None declared

Background Recognition of rheumatoid arthritis (RA) at preclinical stages is essential for successful treatment, long-term functional ability and survival. Available serological markers of the disease are restricted to acute-phase reactants and presence of autoantibodies - rheumatoid factor (RF) and ACPA. Although specific, these markers cover less than a half of the patients with RA. Oncoprotein survivin has been recently identified as a marker of severe RA frequently associated with progressive joint damage and poor response to antirheumatic treatment (1). A pilot study on the samples from the Nordic Biobank showed that survivin could predict development of RA several months ahead of clinical symptoms (2). Objectives To validate the utility of survivin measurements for early recognition of clinically suspect arthralgia and diagnosis of RA in the epidemiological setting. Methods The first-visit patients with joint pain from the inception cohort of 5455 subjects tested for RF and/or ACPA during 12 consecutive months at the Laboratory of Clinical Immunology at the University Hospital of Gothenburg, were evaluated for RA diagnosis by the 2010 EULAR/ACR criteria (3). Patients with known diagnosis of RA, other rheumatic disease and chronic pain conditions were excluded. The subjects not fulfilling RA diagnosis at the first visit were prospectively followed during 29–49 months. The utility of survivin measurement and arthritis-specific autoantibody status (ACPA and/or RF) for RA diagnosis were analyzed. Results The total of 385 patients were included in the study. RA criteria applied to the records of the first visit identified 17% of patients as RA. Additional 21% of patients were recognized as undifferentiated arthritis (UA), while the remaining 62% of the patients were characterized as arthralgia. Majority of the RA patients were survivin positive (52%) with an overlap for antibodies (RF and/or ACPA) in 88%. The UA and arthralgia groups had low prevalence of patients positive for survivin (p=0.018) and for autoantibodies (p=0.002). During the follow-up period, 23 cases of new RA and 5 new UA were registered. The incidence of new cases was significantly higher among the survivin-positive patients and corresponded to the increased risk compared to survivin-negative (p=0.008). The combination of survivin and autoantibodies increased further the risk for development of the disease compared to negative patients (RR 4.05[ 1.73–9.20], p=0.002). The presence of isolated survivin was also associated with higher risk of RA (RR 2.55 [1.14–5.71], p=0.028), while no increase in RA risk could be shown for the isolated presence of autoantibodies. Conclusions This prospective study showed in the epidemiological setting that the presence of survivin was associated with an increased risk of RA. Combination of survivin and autoantibodies further increased this risk. References Svensson B et al. Increased expression of proto-oncogene survivin predicts joint destruction and persistent disease activity in early RA. Ann Med. 2010;42:45–54. Bokarewa M et al. Survivin but not Flt3 ligand is up-regulated before the onset of RA: a pilot study. Arthritis Res Ther. 2014;16:R45. Cader MZ et al. Ann Rheum Dis. 2011;70:949–55. Disclosure of Interest None declared

Background Insulin-like growth factor (IGF-1) signaling is essential for development and function of the brain. It helps to improve neuronal plasticity in response to stress, and recently it has been reported to mediate depression [1]. In rheumatoid arthritis (RA), depression is a serious comorbidity with a tight connection to functional disability and early mortality [2]. The serum levels of IGF-1 and the local expression of its receptor (IGF-1R) in inflamed joints are affected in RA [3]. However, the brain IGF-1R signaling in arthritis and its consequences for behavior has not been studied previously. Objectives In this study we analyze how arthritis affects IGF-1R expression and activity in the brain and its relation to behavioral changes. Methods Arthritis was induced in DBA/1 mice by immunization with collagen type II. Development of arthritis was followed clinically. One group was treated with shRNA targeting IGF1R, second group obtained control shRNA, and naive healthy siblings comprised the control group. Behavioral pattern of each mouse was registered by filming at different stages. Protein and mRNA levels of IGF-1R, CD68 and IL-1b were measured in different parts of the brain with IHC and real-time PCR. Serum IGF-1 and IL6 were analyzed with a sandwich ELISA. We validated the findings from experimental arthritis with a cohort of 235 patients with established RA. Results In RA patients, the arbitrary measure of pain (VAS) was mainly associated with depression index (FIQ, r=0.46), fatigue (r=0.44), and disability index (r=0.68) rather than to RA activity (DAS28, r=0.36). In mice, arthritis decreased locomotion and rearing, corresponding to the depressive-like behavioral pattern. Severity of arthritis and decreased mobility correlated with the increased mRNA levels of inflammatory cytokine IL-1, and a marker of activated microglia CD68. We observed a decreased expression of IGF-1R and a remarkable reduction in the number and regularity of the Purkinje cells in the cerebellum accompanied by an accumulation of phosphorylated tau along these cells. Arthritis increased serum levels of IGF-1, which correlated with behavioral pattern in arthritic mice. When the IGF1R signaling was inhibited with shIGF1R-treatment, the arthritic mice showed aggravation of the depressive-like behavior, including increased immobility time, which was no longer correlated to severity of arthritis. Conclusions Depression and functional disability aggravate pain experience in RA patients. Arthritis-induced inhibition of IGF-1R expression and signaling provide molecular mechanism of depression in RA. References Kopczak A, et al. IGF-I in major depression and antidepressant treatment response. Eur Neuropsychopharmacol. 2015 Jun; 25(6):864–872. Ang DC, et al. Comorbid depression is an independent risk factor for mortality in patients with rheumatoid arthritis. J Rheumatol. 2005 Jun; 32(6):1013–1019. Bostrom EA, et al Resistin and insulin/insulin-like growth factor signaling in rheumatoid arthritis. Arthritis Rheum. 2011 Oct; 63(10):2894–2904. Disclosure of Interest None declared

Background Smoking is the most extensively studied environmental risk factor of rheumatoid arthritis (RA). We have recently shown that smoking is associated with high serum levels of survivin (Svensson, Hafstrom et al. 2014). Survivin is a novel biomarker for RA that can predict severe joint destruction as well as treatment outcome for RA patients (Levitski et al, 2015). We hypothesized cytotoxic activity to be the reason for the increased serum levels of survivin observed in RA patients and we wished to further investigate possible molecular mechanisms behind cytotoxicity in RA. Objectives We have studied the effect of nicotine on cytotoxic activity of T lymphocytes during RA paying special attention to the expression of PD-1, a receptor that inhibits activation of T cells. Methods We used female Balb/c mice that received 0.03% nicotine in drinking water or regular tap water as controls, after 18 days mice were immunized with collagen and subsequently developed arthritis (CIA). At the end of experiment bone marrow, lymph nodes and serum were collected and analyzed using flow cytometry (FACS), qPCR and ELISA. To confirm nicotine-dependent nature of experimental findings, we analyzed blood samples of female RA patients and healthy women with known smoking status. Results The proportion of CD8+ cells was 1.9 times larger in nicotine treated mice compared to control (p=0.00020). Meanwhile, the nicotine treated mice had a 2.3 times less PD-1 expressing CD8+ cells in the bone marrow (p=0.032), but PD-1 expression in the lymph nodes was not affected by nicotine. This resulted in an accumulation of the CD8+PD-1- population in the bone marrow of the nicotine treated mice (p=0.0074). The nicotine treated mice had higher levels of serum survivin (0.0 vs 0.29 ng/ml, p=0.017) which also correlated to the size of CD8+PD-1- population in the bone marrow (r=0.52, p=0.024). The decrease in PD-1 expression of CD8+ T cells caused by nicotine was confirmed in smoking women. Smokers had a 1.7 times less PD-1 positive CD8+ T cells compared to non-smokers (p=0.041). In patients, the serum levels of survivin correlated to the expression of the cytotoxic marker CD107 on CD8+ T cells (r=0.52, p=0.047). Conclusions Nicotine exposure supports cytotoxic phenotype of CD8+ T cells characterized by high CD107 and low PD-1 expression in experimental arthritis and RA patients. Increased cytotoxic activity of CD8+ T cells is a plausible mechanism for the enrichment of survivin in serum in RA patients. See graphical abstract. References Svensson B, Hafstrom I, Erlandsson MC, Forslind K, Bokarewa MI. Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study. Arthritis Res Ther. 2014;16(1):R12. doi: 10.1186/ar4438. Levitsky A, Erlandsson MC, van Vollenhoven RF, Bokarewa MI, Serum survivin predicts responses to treatment in active rheumatoid arthritis: a post hoc analysis from the SWEFOT trial. BMC Med. 2015;13:247. doi: 10.1186/s12916-015-0485-2. Disclosure of Interest None declared

Background MicroRNA (miR) represent a part of epigenetic contol of autoimmunity and gain increasing attention in the pathogenesis of RA. Genome-wide and candidate miR surveys indicated deregulation of selected miRs in RA blood and synovial tissue. Smoking belongs to enviromental factors with the ability to reprogram epigenetic control. It is also the most studied risk factor in RA being tightly connected to the production of autoantibodies, high levels of oncoprotein survivin, and resistance to antirheumatic therapy (1,2). Objectives The aim of this study was to determine effect of smoking on the expression of miRs in leukocytes of RA patients. Methods Leukocytes of the peripheral blood of 46 female RA patients (age 21–69 years, disease duration 1–22 y) were used as a source of miR. Information about smoking status was collected via a questionnaire and identified current smokers (CS, n=12), former smokers (FS, n=21) and never smokers (NS, n=13). We used the Toray 3D-Gene microRNAs assay-based approach to compare the levels of 2598 miRs between the RA patients with different smoking status. For the comparison between the groups we used only miRs with abundant (raw values >100) expression in leukocytes. Smoking and RA-associated miRs were retrived from the available comprehensive reviews (3,4). Results The number of detectable miR was highest in the PBMC samples of NS. Former and current smoking was associated with a step-wise decreased global miR expression being most severe in CS. We identified 267 miRs, which had an abundant expression in NS. The expression of 93 of these miRs was significantly affected by smoking being 1.75-times lower in FS and CS (p=0.0002). Analyses of miRs, which had previously been reported sensitive to smoking showed that FS+CS had lower expression of the miRs belonging to the let-7 family compared to NS (2.43 times, p=0.034). Additionally, FS had low expression of the miR-17/92 cluster, where miR-17 family was affected most, reduced 2.05 times (p=0.066), followed by miR-19 and 18 families (1.74 times) and miR-92 family (1.56 times). FS had also 2.1 times lower expression of the miR-16 family. The analysis of miRs described in relation to RA showed that 20 of 30 RA-related miRs were abundantly expressed in the studied samples. The expression of miR-16–5p, miR-126–3p, miR-142–3p, miR-150–5p, miR-22–3p and miR-221–3p showed more then 1.75 times difference between FS and NS. Conclusions Smoking is associated with a global reduction in miR expression in RA patients, where the members of let-7; miR-17/92 and miR-16 families with key roles in regulation of cell cycle progress, tissue development and immune responses clusers are most affected. References Klareskog L et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA-DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum. 2006;54(1):38–46 Svensson B et al. Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study. Arthritis Res Ther. 2014;16(1):R12 Churov AV et al. MicroRNAs in rheumatoid arthritis: altered expression and diagnostic potential. Autoimmun Rev. 2015;14(11):1029–37 Vrijens K et al. MicroRNAs as potential signatures of environmental exposure or effect: a systematic review. Environ Health Perspect. 2015;123(5):399–411 Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is an established risk factor for premature development of cardiovascular disease. Adipokines are signal molecules originating from adipose tissue and exhibit strong proinflammatory effects. We have previously shown that resistin utilizes IGF-1R (1) and TLR4 (2) pathway to induce inflammation in RA leukocytes and synovia. Objectives In the present study we evaluated the role of adipokines and adipokine-related genes on the cardiovascular risk in female RA patients. Methods The estimated cardiovascular risk was calculated in 187 female RA patients, using the modified Systematic Coronary Risk Evaluation (mSCORE). Serum levels of adipokines and IGF-1 and transcription of genes affected by Resistin and IGF-1 signalling in peripheral blood leukocytes and in subcutaneous fat tissue were analyzed in relation to mSCORE. To assess the predictive value of adipokines and gene transcription for the increased mSCORE, two independent binary logistic regression models were designed where high mSCORE or high resistin were chosen as the dependent variables. Results mSCORE was directly related to the age, disease duration, smoking habits and blood fat profile (cholesterol and triglycerides). These parameters were higher in the group with mSCORE≥1 (n=96) indicative for the increased cardiovascular risk. The patients with mSCORE≥1 had significantly higher levels of leptin (p=0.007), and resistin (p=0.03), while the levels of IGF-1 were significantly lower (p The logistic regression analysis with mSCORE as a dependent variable showed that high serum levels of resistin and leptin were independently associated with the increased cardiovascular risk. Similar associations were observed for adipocyte expression of resistin-triggering genes TLR4 (OR 1.15 [95% CI 1.01-2.15]; p=0.03) and STAT3 (OR 2.39 [95% CI 1.00-5.74]; p=0.05). The logistic regression model with high serum resistin as a dependent variable revealed an association to high transcription of STAT3 (OR 1.12 [95% CI 1.00-3.30]; p=0.02), and low transcription of AKT1 in leukocytes and in adipocytes. Conclusions The results of our study indicate that transcriptional activity of subcutaneous adipose tissue in women with RA is associated with the increased cardiovascular risk. High transcriptional activity of resistin-triggering genes may be a sufficient force driving premature cardiovascular disease in RA patients. References Bostrom EA, Svensson M, Andersson S, Jonsson IM, Ekwall AK, Eisler T, Dahlberg LE, Smith U, Bokarewa MI. Resistin and insulin/insulin-like growth factor signaling in rheumatoid arthritis. Arthritis Rheum. 2011,63:2894-904 Tarkowski A, Bjersing J, Shestakov A, Bokarewa MI. Resistin competes with lipopolysaccharide for binding to toll-like receptor 4. J Cell Mol Med. 2010,14:1419-31. Disclosure of Interest None declared

Background Survivin is a sensitive biomarker predicting development of rheumatoid arthritis (RA) several years ahead of clinical symptoms (1). In early RA, more than 50% of the patients have high serum levels of survivin (2), which recognises the patients with poor treatment responses and progressive joint damage (3). Objectives In this study we evaluated predictive potential and specificity of serum survivin for early recognition of RA. Methods All serum samples of individuals >18 years of age referred during the period of 12 months to the Laboratories of Clinical Immunology at the University Hospitals of Gothenburg and Umea in Sweden were enrolled in the analysis of survivin, rheumatoid factor (RF) and/or anti-citrullinated peptide (ACPA) antibodies. Serum levels of survivin were measured using a sandwich ELISA, and the levels >0.45 ng/ml were considered positive. Medical records of the individuals positive for survivin and RF/ACPA antibodies were reviewed by independent experts to identify cases with newly-diagnosed RA, established RA, and unexplained arthralgia. Results The total of 8868 (5422 Gothenburg, 3446 Umea) adult individuals were analysed and the prevalence of survivin-positive samples was 11.2% and 14.7%, respectively. Among 921 survivin-positive samples, 46% were also positive for RF and/or ACPA vs 10% of survivin-negative, and indicated that survivin-positive individuals had increased estimated risk for being also RF/ACPA positive (RR 4.44[4.01-4.92], p -7 ). Survivin levels were significantly higher in patients with combined positivity of survivin and RF/ACPA (p -7 ). This difference between the cohorts was due to the increasing prevalence of newly-diagnosed RA compared to arthralgia cases in survivin-negative-RF/ACPA-positive cohort (15.3% vs 7.2%, p=0.0004), while this prevalence did not change in surviving-positive cohort (27% vs 21%, respectively). Conclusions In the unselected serum samples, high levels of survivin were associated with RF and/or ACPA antibodies. Cases with arthralgia and newly-diagnosed RA were accumulated within the Survivin-positive samples and suggest a predictive potential of survivin early in the disease course. References Bokarewa M et al., Arthritis Res Ther 2014;16(1):R45 Svensson B et al., Ann Med 2010;42:45-54 Svensson B et al., Arthritis Res Ther 2014;16(1):R12 Disclosure of Interest None declared

Background Survivin is an oncological biomarker. In rheumatoid arthritis (RA), elevated serum survivin is common and has been used to predict disease onset and progressive joint damage. Objectives We investigated the predictive capacity of survivin in clinical disease activity and/or response to various antirheumatic treatments in patients with early RA. Methods Survivin levels from serum were measured using ELISA at baseline in 302 patients enrolled in the Swedish pharmacotherapy (SWEFOT) trial with follow-up at 3, 12 and 24 months. After methotrexate (MTX) monotherapy for 3 months, responders (DAS28≤3.2) remained on MTX, while non-responders were randomized to triple therapy (MTX+sulfasalazine+hydroxychloroquine) or anti-TNF (MTX+infliximab). Survivin levels >0.45 ng/mL were considered positive. Based on survivin status over 24 months, core-set outcomes (i.e. DAS28, HAQ, pain & global VAS) were evaluated at 3, 12, and 24 months. Results Over one-third of all patients (n=114) were survivin -positive at baseline. S urvivin -positive ever-smokers (51/71 vs. 64/112, OR 1.91 [95% CI 1.01-3.62], p=0.045) and survivin -negative patients who converted to positive over 24 months (13/161 vs. 2/100, OR 4.39 [0.97, 19.88], p=0.037) responded seldom to MTX. At 3 months, survivin- positive patients who converted negative (n=11) had greater reductions in DAS28 (p=0.002), HAQ (p=0.011) and global VAS (p=0.025) vs. those who remained positive (n=28), which were maintained over 24 months. At 12 months, survivin -positive MTX-responders who continued monotherapy had a higher risk of disease re-activation on MTX compared to the survivin -negative patients (12/36 vs. 7/52, OR 3.21 [1.12-9.24], p=0.032) and showed no improvement in HAQ over 24 months. Survivin -positive MTX non-responders who converted to negative (n=32) or remained negative (n=83) had greater improvements from 3 to 12 months than those who converted to positive (n=13) (ΔDAS28>1.2, 63% & 60% vs. 31%, p=0.053, p=0.046, respectively). Among survivin -positive patients on triple therapy, converting to negative (n=19) yielded a lower DAS28 at 12 months (2.34 vs. 4.12, p=0.046) and a high frequency of DAS≤3.2 (86% vs. 37%, p=0.056) at 24 months vs. converting to positive (n=7). They also had lower pain (p=0.048) and global VAS (p=0.015) at 24 months compared to the same subgroup, which was not observed among anti-TNF – where no differences in core-set outcomes were observed between the survivin groups. Survivin -positive patients on anti-TNF had a higher risk to have active disease at 24 months compared to those on triple therapy (16/29 vs. 9/32, OR 3.15 [1.09-9.10], p=0.037). Conclusions Survivin -positive patients have worse 24-month outcomes than survivin -negative patients if treated with MTX monotherapy, but conversion to survivin- negative is associated with a good and stable response to MTX monotherapy. For survivin -positive patients with early RA who fail MTX, triple therapy is associated with a better likelihood for response than anti-TNF therapy. Disclosure of Interest A. Levitsky: None declared, M. Erlandsson: None declared, R. van Vollenhoven Grant/research support from: AbbVie, BMS, GSK, Pfizer, Roche, UCB, Consultant for: AbbVie, Biotest, BMS, Crescendo, GSK, Janssen, Lilly, Merck, Pfizer, Roche, UCB, Vertex, M. Bokarewa: None declared

Background: Leptin has been shown to participate in bone remodelling and leptin substitution reported to have a protective effect in experimental septic arthritis. Objective: To assess leptin levels in inflamed joints and plasma of patients with RA. Material and methods: Leptin concentrations were assessed in matched blood and synovial fluid samples from 76 patients with RA. Blood samples from 34 healthy subjects acted as additional controls. Results were analysed and correlated with duration and activity of RA, x ray changes, and treatment at time of sampling. Results: In patients with RA, leptin levels were significantly higher in plasma than in synovial fluid samples obtained simultaneously and higher than in control samples. Plasma and synovial fluid leptin levels correlated strongly. Locally in the joint, leptin levels were related to WBC count. Such a relation was not seen in the bloodstream. Leptin levels were not related to sex, age, or disease duration. Difference between leptin levels in plasma and synovial fluid was greater in non-erosive arthritis (5.1 (SEM 1.2) v 3.7 (0.9) ng/ml, p=0.006), than in patients with erosive joint disease (6.2 (1.0) v 5.4 (0.8) ng/ml, NS). Methotrexate treatment was associated with relatively high plasma leptin levels, while treatment with other DMARDs was associated with lower leptin levels than in patients receiving no DMARD treatment (p=0.0005). Conclusions: Leptin production was significantly increased in patients with RA compared with healthy controls. Synovial fluid leptin levels were significantly lower than in matched plasma samples, suggesting an in situ consumption of this molecule.

Background Rheumatoid arthritis (RA) is an established risk factor for premature development of cardiovascular disease. Adipokines are signal molecules originating from adipose tissue and exhibit strong proinflammatory effects. We have previously shown that resistin utilizes IGF-1R and TLR4 pathway to induce inflammation in RA synovia. Objectives In the present study we evaluated the role of adipokines and adipokine-related genes on the cardiovascular risk in RA. Methods Cariovascular risk was estimated in 146 RA patients, all women (median age 57.5 years, range 21-71, median disease duration 8.0 years, range 1-49 years), using the modified Systematic Coronary Risk Evaluation (mSCORE). Serum levels of adipokines and IGF-1 were analyzed by ELISA. mRNA levels of IGF-1R, TLR4, Akt1, STAT3, RETN, and RELA (NF-kB) were analysed in peripheral blood leukocytes and in subcutaneous adipose tissue, using qPCR. The gene expression analysis was performed in the subgroup of younger RA patients (n=54, mean age 46 years, median mSCORE=1). The adipokine levels and gene expression were analyzed in relation to mSCORE. Results mSCORE was directly related on the age, disease duration, smoking habits and blood fat profile (cholestrol and triglycerides), and not to the DAS28 and BMI of RA patients. mSCORE correlated to the serum levels of leptin, resistin and visfatin. The patients with the increased cardiovascular risk (mSCORE≥1, n=86) had higher levels of adiponectin and visfatin, and low levels of IGF-1 and Akt1 mRNA in the peripheral blood leukocytes compared to the patients where cardiovascular risk was below detection level (mSCORE In the subgroup of younger patients, serum resistin correlated to mSCORE and to the systemic inflammation but not to DAS28. Additionally, mSCORE showed a strong correlation to the adipocyte expression of resistin-triggering receptors (IGF1-R and TLR4) and to the mediators of their transcriptional signalling Akt, STAT3, and RELA. We observed no correlation between mSCORE and the transcriptional activity of the peripheral blood leukocytes. The correlation of mSCORE and transcriptional activity in adipocytes was not dependent on BMI and was stronger in the normal weight patients. Conclusions This pilot study shows a correlation between the cardiovascular risk and the transcriptional activity of the subcutaneous adipose tissue in women with RA. The expression of resistin-related genes in adipose tissue in younger women suggests that resistin may be a sufficient force supporting persistent transcriptional activity of adipocytes and driving premature cardiovascular disease in RA patients. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3893

Background Survivin is recognized as a marker of persistently active and erosive RA. The findings in experimental arthritis suggested that survivin co-ordinates the antigen-dependent leukocyte maturation and function by regulating transcriptional activity of Bcl-6, essential factor of germinal center formation and a factor involved neoplastic transformation to lymphoma. Objectives We studied intracellular expression of survivin and Bcl-6 in the peripheral lymphocytes and its relation to clinical features and treatment of RA patients. Methods The intracellular expression of survivin and Bcl-6 was studied on the effector and memory subsets of CD4+ and CD8+ T-cells and in CD19+ B-cells in 18 RA patients using flow cytometry. Serum and mRNA levels of survivin were analysed in 144 RA patients (age 21-71 years, disease duration 1-49 years) using ELISA and qPCR, respectively. Patients with serum levels of survivin >0.45 ng/mL comprised the serum survivin-positive (sSurv+) group. Results The sSurv+ group (n=76) was characterized by higher frequency of RF+ and aCCP+ patients, as well as by higher levels of aCCP and of differentiation factor Flt3-ligand compared to sSurv – (n=68). The sSurv+ and sSurv- patients were similar with respect to age, disease duration, DAS28 and anti-rheumatic treatment. Intracellular mRNA levels of Bcl-6 were lower in sSurv + compared to sSurv – patients, while RNA of survivin were similar. Flow cytometry showed that survivin was present in >85% of non-stimulated T-cells and B-cells lymphocytes of RA patients. The survivin hi subset comprised 2-14% of T-cells and was enriched in the effector (CD62L neg ) population of CD4+ T-cells compared to central memory and naive CD4+ T-cells. The expression of Bcl-6 was found within 7-38% of survivin + effector CD4+ T-cells and in 21-69% of CD19+ B-cells. The subsets of survivin+ and of survivin+Bcl-6+ T-cells was smaller in the sSurv+ patients compared to sSurv – patients, and was inversely correlated to the levels of serum survivin in these patients. The subset of survivin+Bcl-6+ B-cells was similar in sSurv+ and sSurv- patients. Conclusions In this pilot study we demonstrate that RA patients have co-expression of the two oncoproteins involved in neoplastic transformation to lymphoma, survivin and Bcl-6. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5490

Background A calcium-binding protein S100A4 regulates non-muscle myosin assembly and activity and plays important part in cell motility and differentiation. The binding of S100A4 to calcium drives conformation changes and permits S100A4 to regulate the activity of its multiple intracellular protein partners placing S100A4 at the crossroad of several intracellular transduction mechanisms. In RA, S100A4 is abundantly expressed in synovial fibroblasts, macrophages and vascular endothelial cells of the inflamed joints and may be measured in synovial fluid and in blood. The clinical consequences of the high levels of S100A4 in RA patients are associated with resistant joint inflammation and high skeletal damage. Objectives In the present study we evaluated the role of intracellular functions of S100A4 for T-cell responses in rheumatoid arthritis. Methods A preclinical model of the methylated BSA induced arthritis was induced in S100A4-deficent mice lacking the entire S100A4 protein (S100A4KO) and in wild-type (WT) counterparts where the S100A4 gene was inhibited with a specific shRNA-lentiviral constructs (S100A4-shRNA). The subsets of Th-cell in synovial infiltrates were analysed by immunohistology and in spleens by flow cytometry and qPCR. Phosphorylation of STAT3 (pTy5705), CD5 (pTyr453), Fyn (pTyr530) and ZA70 (pTyr319/Syk, pTyr352) were analysed by flow cytometry in spleen cell cultures. Kinase activity of Fyn and Lck was analysed by phosphorylation of CD5 peptide. Results Histological analysis of the inflamed joints demonstrated that S100A4-deficient mice had significantly alleviated joint inflammation and damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice presented accumulation of Foxp3 + Treg, while the effector T-cell population and the number of RORγt + and pSTAT3 + cells were reduced. T-cells of S100A4-deficient mice were characterized by a limited formation of Th17-cells, which was a result of low mRNA levels of RORγt and STAT3 (pTyr705) activity in spleen and low production of IL17 and IFNg. In vitro experiments provide evidence that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards TCR inhibitor CD5. Nuclear magnetic resonance analysis demonstrated an interaction between CD5 cytoplasmic domain and EF2-binding sites of S100A4. This suggests that S100A4 is able to disrupt intracellular interactions of CD5 and could modify its inhibition of TCR. Consequently, S100A4-deficiency was translated in low expression of CD5, and increased lymphocyte proliferation and phosphorylation of ZAP-70. Conclusions Here we provide experimental evidence that S100A4 directly binds the Src-kinases Lck and Fyn and reciprocally regulates their kinase activity. S100A4-deficiency results in reduced activity of STAT3 suppressing transcription of RORgt and lineage differentiation of Th17-cells. The immunological events controlled by S100A4 are functionally important for the pathogenesis of arthritis, since the deficiency in S100A4 alleviates experimental arthritis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5414

Background Survivin is a known inhibitor of apoptosis and a regulator of cell division. In rheumatoid arthritis (RA), survivin has been shown to be involved in joint destruction. Here, we investigated survivin in early RA patients from the SWEFOT trial who were administered methotrexate (MTX) monotherapy for 3 months. Non-responders (DAS28 at 3 months >3.2) were randomized to triple therapy (MTX + sulfasalazine + hydroxychloroquine, group A) or anti-TNF (infliximab + MTX, group B). Methods Serum survivin levels were measured using ELISA with samples from 294 patients. Values >0.45 ng/mL were considered positive. Independent Samples t-tests and nonparametric Mann-Whitney U tests were performed where applicable. Results Out of 294 patients, 112 (38%) were positive for survivin at BL. The survivin (+) patients were more often male (30% vs. 22%, p=0.036) and RF (+) (80% vs. 58%, p Out of the 112 survivin (+) patients at BL, 37 (33%) converted to (−) by 3 months. These patients had greater improvements in disease activity compared to (+) and significantly greater reductions in tender joint count (TJC) and ESR (8.4 vs. 3.9, 30.9 vs. 19.0, respectively; p Among MTX responders, the (+/−) converters (n=11) had significantly greater reductions in DAS28 and patient9s global VAS than those who remained (+) (n=28) (3.5 vs. 2.5, 46.6 vs. 23.2, respectively; p Likewise, in the MTX non-responders, disease activity was improved more for (+/−) converters in the randomized group, A, compared to those who remained (+) or (−). Among these patients, those whose survivin status converted from (+) to (−) from 3 to 12 months (n=12) had numerically greater improvements, and a significantly greater reduction in patient9s global VAS than those who remained (+) (n=15) (17.3 vs. −2.9 [an increase], p=0.024). Converters (+/−) among the randomized group, B (n=7), on the other hand, had numerically worse changes for most parameters compared to those who remained (+) (n=16). Conclusions Serum survivin is positive in about one third of patients with early RA and correlates with male gender, RF-positivity, and higher ESR. Survivin seroconversion (+/−) upon MTX is not uncommon and is associated with clinical improvements. Survivin conversion from (+/−) led to greater improvements for patients on triple therapy compared to non-converters. This finding was not observed among patients treated with anti-TNF. Disclosure of Interest : A. Levitsky: None declared, M. Erlandsson: None declared, M. Bokarewa: None declared, R. van Vollenhoven Grant/research support: AbbVie, BMS, GSK, Pfizer, Roche, UCB, Consultant for: AbbVie, Biotest, BMS, Crescendo, GSK, Janssen, Lilly, Merck, Pfizer, Roche, UCB, Vertex DOI 10.1136/annrheumdis-2014-eular.4205

Background Cigarette smoking has been recently connected to the pathogenesis of rheumatoid arthritis (RA). The cellular mechanisms and molecular events initiated by smoking and leading to severe inflammation, low efficacy of anti-rheumatic drugs, and autoantibody production, are still poorly understood. Adipokines are signalling molecules originating from adipose tissue and regulating carbohydrate and lipid metabolism. In RA, adipokines are connected to inflammation, disease activity and radiological signs of joint destruction. Effects of adipokines are mediated through insulin receptor/insulin-like growth factor-1 receptor (IGF-1R) complex. Objectives Present study evaluates a link between cigarette smoking, IGF-1 signalling and adipokine cascade in patients with rheumatoid arthritis. Methods One hundred-eleven patients (76 women, 35 men) with established RA (mean disease duration 9.9 years) participated in the structured telephone interview on their smoking habits. Patients were stratified by smoking habits, age, and levels of IGF-1. Levels of adipokines (visfatin, adiponektin, resistin and leptin) were analyzed in blood samples of these patients using commercial ELISAs. Results Thirty-three of 111 patients (29.7%) reported to be current smokers, while 78 patients were non-smokers. Smokers had slightly shorter disease duration and had a prevalence of men, while disease activity, erosivity and presence of RF were similar between the smokers and non-smokers. Smokers had lower levels of IGF-1 compared to non-smokers (median: 2.64 vs 8.08, p=0.035). Levels of IGF-1 are inversely related to age, this enabled distinct analysis of the younger (mean age 50 y, n=62) and older (mean age 69 y, n=49) patient groups. Smoking was associated with low levels of IGF-1 in the younger (5.82 vs 11.12, p=0.030) and in the older patients (1.05 vs 4.28 ng/ml, p=0.05). Levels of adipokines were similar in the total group of smokers and non-smokers, however IGF-1 was inversely related to visfatin levels (r=-0.35, p=0.003). In the absence of IGF-1 stimulation in patients with low levels of IGF-1, visfatin correlated to resistin (r=0.599) and leptin (r=0.450) levels. No such correlation within adipokine network was found in patients with high levels of IGF-1. Direct inhibitory effect of nicotine on IGF-1 levels was proved in mice. Mice supplemented with nicotine (0.02-0.05%) in drinking water had significantly lower levels of IGF-1 in blood compared to their siblings drinking pure water (p=0.0007). Treatment of mice with recombinant IGF-1 resulted in significantly low circulating levels of leptin, visfatin and resistin. Conclusions Smoking functions as a negative regulator of IGF-1 levels in RA patients and has repressive effects on the cascade of adipokine signaling molecules, contributing to inflammation and joint destruction in RA. Disclosure of Interest None Declared

Survivin but NOT fms-like tyrosine kinase ligand (FLT3L) is up-regulated before onset of rheumatoid arthritis

Background Fibromyalgia (FM) is a chronic condition characterized by widespread musle pain and tenderness. Chronic pain is the major symptom that affects patients physically, mentally and socially. Mechanisms of pain in FM are not completely understood. Relation between smoking and pain is contradictive. In epidemiologic studies smoking is associated to chronic pain, while in acute experiment stimulation of nicotine receptors alleviates pain. Objectives The present study evaluates the possible relation between pain, cigarette smoking, and levels of IGF-1 in patients with FM. Methods Pain was characterised in 63 patients with FM (all women, age 52 years) by Fibromialgia Impact Questionnaire (FIQ), tender points count, and pain threshold was assessed by algometer. All patients participated in the structured telephone interview regarding their smoking habits. Levels of IGF-1, leptin, resistin, and adiponectin were measured in blood. The statistical analysis was performed regarding smoking habits, IGF-1 levels and intensity of pain. Patients were categorized by pain (FIQ and threshold) quartiles, and relative risk (95%CI) was calculated. Results Eighteen patients (28.6%) reported to be current smokers, and 45 were non-smokers. Among the non-smokers, 25 patients smoked previously and ceased before the study (median 13 years, range 1-30). Smokers had higher pain experience compared to non-smokers, characterised by higher FIQ (p=0.036), and lower pain threshold (p=0.014). The difference in pain was largest between the current smokers and patients who had ceased smoking (dFIQ 12%, p=0.005). The RR of pain in the upper quartile in smokers was 6.3 (95%CI: 1.41-36.18) compared to previous smokers. Furthermore, previous smokers have lower pain, fewer tender points and a higher pain threshold compared to smokers. Smokers had significantly lower levels of IGF-1 (ng/ml, median: 2.72 vs 4.82, p=0.027) compared to never-smokers and lower levels of leptin (ng/ml, median: 16.9 vs 34.8, p=0.013) compared to non-smokers. Current and previous smokers had frequently low levels of IGF-1 compared to non-smokers (88% vs 12%, p=0.018). Conclusions Smoking in FM patients was associated with high pain experience. Pain experience improved in the patients who had ceased smoking. Current and previous smoking was associated with low levels of IGF-1 suggesting long-term effects of smoking on regulation of IGF-1 levels in FM patients. Disclosure of Interest None Declared

Tumour necrosis factor α (TNFα) has emerged as a potent proinflammatory mediator in the inflammatory arthritides. Studies on synovial tissue from patients with rheumatoid arthritis have shown not only the presence of large amounts of TNFα but also demonstrated its regulatory effect on the whole network of proinflammatory cytokines present in inflamed joints.1,2 Consequently, TNFα antagonists given systemically have proved to be efficient in the treatment of chronic arthritides.3,4 Here we report our experience of an attempt to ameliorate arthritis locally by intra-articular administration of TNFα antagonists. Six patients attending the department of rheumatology at Sahlgrenska University Hospital in Goteborg, who had persistent effusions in the knee joints …