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In this study, we explore the role of nuclear survivin in maintaining the effector phenotype of IFNγ-producing T cells acting through the transcriptional control of glucose utilization. High expression of survivin in CD4

Significance Identifying signaling pathways contributing to resistance to anti-TNF therapy in rheumatoid arthritis is crucial for the development of new therapeutic strategies for refractory rheumatoid arthritis. Th17 cells, a subset of proinflammatory CD4+ T cells, are implicated in the pathogenesis of the disease. We analyzed the gene expression profiles of Th17-enriched CD4+ T cells in anti-TNF–treated patients with rheumatoid arthritis and found that the elevated expression levels of transcription factor USF2 in anti-TNF refractory patients were associated with increased proinflammatory signaling of Th17 cells. USF2-knockdown experiments in Th17 cells revealed that USF2 promotes the pathogenicity of Th17 cells. These findings have implications for the development of new therapeutic strategies for refractory rheumatoid arthritis., IL-17–producing Th17 cells are implicated in the pathogenesis of rheumatoid arthritis (RA) and TNF-α, a proinflammatory cytokine in the rheumatoid joint, facilitates Th17 differentiation. Anti-TNF therapy ameliorates disease in many patients with rheumatoid arthritis (RA). However, a significant proportion of patients do not respond to this therapy. The impact of anti-TNF therapy on Th17 responses in RA is not well understood. We conducted high-throughput gene expression analysis of Th17-enriched CCR6+CXCR3−CD45RA− CD4+ T (CCR6+ T) cells isolated from anti-TNF–treated RA patients classified as responders or nonresponders to therapy. CCR6+ T cells from responders and nonresponders had distinct gene expression profiles. Proinflammatory signaling was elevated in the CCR6+ T cells of nonresponders, and pathogenic Th17 signature genes were up-regulated in these cells. Gene set enrichment analysis on these signature genes identified transcription factor USF2 as their upstream regulator, which was also increased in nonresponders. Importantly, short hairpin RNA targeting USF2 in pathogenic Th17 cells led to reduced expression of proinflammatory cytokines IL-17A, IFN-γ, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as transcription factor T-bet. Together, our results revealed inadequate suppression of Th17 responses by anti-TNF in nonresponders, and direct targeting of the USF2-signaling pathway may be a potential therapeutic approach in the anti-TNF refractory RA.

MicroRNAs (miRs) are non-translated RNA sequences that elicit negative control over protein expression. The adipose tissue (AT) is considered the major producer of miRs and inflammatory interleukin 6 (IL-6). This study aims to investigate the relationship between production of IL-6 and miRs in AT.IL-6 gene expression was analysed in RNA extracts from subcutaneous AT of 75 patients with rheumatoid arthritis (RA), with qPCR. Genome-wide profile of human miRs (2565 miRs, 96.6%) was analysed in 35 AT samples on 3D microarray. The miR-processing proteins Dicer, Drosha and DGCR8 were analysed with qPCR. In silico prediction of protein targets for the differentially expressed (DE) miRs (p0.05; log2FC±0.5) was conducted by DIANA software. Seven AT samples were stimulated in vitro with IL-6 or IL-6+IL-6R antibody tocilizumab and analysed for the miR processing proteins.We identified 30 DE miRs between AT with high and low IL-6 mRNA, of which 26 miRs were inversely related with IL-6 levels. DE miRs were predicted to interfere in oestrogen (p=0.001), FoxO (p=0.006) and insulin (p=0.03) signalling pathways. High expression of IL-6 in AT was associated with significantly higher expression of Dicer (p=0.04) and Drosha (p=0.04), while inhibition of IL-6 signalling with tocilizumab decreased the levels of total miRs processing enzymes (p=0.003).IL-6 mRNA production in AT has a negative effect on the miRs expression profile and it increases miR-production capacity.

Interferon-gamma (IFNγ) producing T cells develop metabolic adaptation required for their effector functions in tumour biology, autoimmunity and antiviral defence.Using sorted CD4+cells we demonstrated that glycolytic switch and high glucose uptake in IFNγ-producing cells was associated with survivin expression. Inhibition of survivin restored glycolysis by upregulating the transcription of phosphofructokinase PFKFB3 and reducing glucose uptake. Integration of the whole-genome sequencing of the chromatin immunoprecipitated with survivin with transcription changes in CD4+cells after survivin inhibition revealed co-localization of survivin, IRF1 and SMAD3 in the regulatory elements paired to the differentially expressed genes. Western blot demonstrated direct binding of survivin to IRF1 and SMAD3. Functionally, inhibition of survivin repressed IFNγ signalling and activated SMAD3-dependent protein remodelling, which resulted in the effector-to-memory transition of CD4+cells. These findings demonstrate the key role of survivin in IFNγ-dependent metabolic adaptation and identify survivin inhibition as an attractive strategy to counteract these effects.

Adiposity is strongly associated with cardiovascular (CV) morbidity. Uncoupling protein 1 (UCP1) increases energy expenditure in adipocytes and may counteract adiposity. Our objective was to investigate a connection between UCP1 expression and cardiovascular health in patients with rheumatoid arthritis (RA) in a longitudinal observational study. Transcription of UCP1 was measured by qPCR in the subcutaneous adipose tissue of 125 female RA patients and analyzed with respect to clinical parameters and the estimated CV risk. Development of new CV events and diabetes mellitus was followed for five years. Transcription of UCP1 was identified in 89 (71%) patients. UCP1 positive patients had often active RA disease (p = 0.017), high serum levels of IL6 (p = 0.0025) and were frequently overweight (p = 0.015). IL-6hiBMIhi patients and patients treated with IL6 receptor inhibitor tocilizumab had significantly higher levels of UCP1 compared to other RA patients (p &lt, 0.0001, p = 0.032, respectively). Both UCP1hi groups displayed unfavorable metabolic profiles with high plasma glucose levels and high triglyceride-to-HDL ratios, which indicated insulin resistance. Prospective follow-up revealed no significant difference in the incidence of new CV and metabolic events in the UCP1hi groups and remaining RA patients. The study shows that high transcription of UCP1 in adipose tissue is related to IL6-driven processes and reflects primarily metabolic CV risk in female RA patients.

Background: Cardiovascular disease (CVD) causes premature mortality in rheumatoid arthritis (RA). Levels of soluble (s)RAGE change with aging, hypertension and hypercholesterolemia. We assessed whether sRAGE was associated with increased risk of CVD in RA patients.Methods: Serum sRAGE was measured in 184 female RA patients and analyzed with respect to CVD risk estimated by the Framingham algorithm (eCVR), metabolic profile and inflammation. Levels of sRAGE in 13 patients with known cardio-metabolic morbidity defined the cut-off for low sRAGE. Prospective 5-year follow-up of new CV and metabolic events was completed.Results: Low sRAGE was significantly associated with previous history and with new imminent cardiometabolic events in the prospective follow-up of RA patients. In both cases, low sRAGE reflected higher estimation of CVR in those patients. Low sRAGE was attributed to adverse metabolic parameters including high fasting plasma glucose and body fat content rather than inflammation. The association of sRAGE and poor metabolic profile was prominent in patients younger than 50 years.Conclusions: This study points at low sRAGE as a marker of metabolic failure developed during chronic inflammation. It highlights the importance for monitoring metabolic health in female RA patients for timely prevention of CVD.Trial registration:ClinicalTrials.gov with ID NCT03449589. Registered 28, February 2018.

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ−IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases.

Background Signalling through insulin-like growth factor 1 receptor (IGF-1R) is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated and supports expansion of the inflamed synovia. Aim In the present study, we assess if disruption of IGF-1R signalling resolves arthritis. Material and methods Clinical associations of IGF-1R expression in leukocytes of the peripheral blood were studied in 84 RA patients. Consequences of the IGF-1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of insulin receptor substrates. Results In RA patients, high expression of IGF-1R in leukocytes was associated with systemic inflammation as verified by higher expression of NF-kB, serum levels of IL6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF-1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF-1R and STAT3 in synovia, and alleviated arthritis and joint damage in mice. It also reduced expression of IGF-1R and despaired ERK and Akt signalling in spleen T cells. This limited IL-6 production, changed RoRgt/FoxP3 balance and IL17 levels. Conclusion IGF-1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF-1R on the level of insulin receptor substrates alleviates arthritis by restricting IL6-dependent formation of Th17 cells and may open for new treatment strategies in RA.

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: Adipose tissue is an important player in cardiovascular (CV) morbidity. Thermogenic brown adipocytes, rich with uncoupling protein 1 (UCP1), increase metabolic and CV health. (1) Objectives: Study the impact of anti-rheumatic treatment on production of UCP1 in subcutaneous fat of RA patients. Methods: Samples of subcutaneous fat were collected from 125 female RA patients by aspiration from periumbilical region. Expression of UCP1 and a reverse cholesterol transport protein ABCA1 were measured by qPCR and analysied with respect to anti-rheumatic treatment and clinical disease activity. Bt treatment, the patient comprised 4 major groups including tocilizumab (Toci, n=14), anti-TNF (n=29), methotrexate monotherapy (n=47) and methotrexate-sulfasalazine-hyroxychloroquine (tripple therapy, n=15). CV risk was estimated with the Framingham risk algorithm. Results: Measurable expression of UCP1 was found in 54.6% of the studied fat tissue samples. Patients on Toci had measurable expression of UCP1 in 79%, which was significantly more often than among TNFi-treated (45%, p=0.04) and MTX-treated patients (42%, p=0.02). Patients on triple therapy had also often measurable UCP1 levels compared to other groups (69% vs 43%, p=0.035). Toci patients have more lean body mass than patients treated with TNFi. This was based on lower BMI in Toci and TNFi treated patients compared to triple therapy (24.1 vs 27.1, p=0.041; 23.6 vs 27.1, p=0.017; respectively). Additionally, the estimated mucle mass by creatinin/height ratio was significantly lower in TNFi than in triple therapy (p=0.034) and Toci (p=0.008). Clinically, the treatment groups were similar in age, disease activity DAS28 and disease duration with the except for Toci. Toci patients were older (65 vs 57, p=0.04) and had numerically longer disease duration (17y vs 7y) and lower DAS28 (1.98 vs 3.11). Notably, Toci patients had significantly higher TC compared to TNFi (p=0.027), and triple therapy (p=0.041). Triple therapy had the lowest TC levels (p=0.017). The differences were due to LDL, here patients on Toci had higher LDL than TNFi (p=0.09) and triple therapy (p=0.015). Serum HDL was similar. These differences in serum lipids were not related to expression of ABCA1 or UCP1. Despite the difference in the serum lipid profile, the estimated CV risk was significantly lower in Toci compared to MTX patients (4.1[0.87-5.75] vs 6.6[3.9-9], p=0.041). Conclusion: In this study is Toci treatment is associated with persistent UCP1 production by adipose tissue. This was followed by lower estimated CV risk and favourable body composition in female RA patients. Reference: [1] Carpentier, A. C., Blondin, D. P., Virtanen, K. A., Richard, D., Haman, F., & Turcotte, E. E. (2018). Brown Adipose Tissue Energy Metabolism in Humans. Frontiers in endocrinology, 9, 447. doi:10.3389/fendo.2018.00447 Disclosure of Interests: None declared

Background: Recent meta-analysis reported that rheumatoid arthritis (RA) is associated with high frequency of hypertension and stroke (1). IGF1 is an important angioprotector and its deficiency predisposes to development of ischemic stroke (2). Objectives: Since levels of active IGF1 are affected by systemic inflammation, we analyze if low IGF1 is associated with increased cardiovascular disease (CVD) in women with RA. Methods: The CVD risk was estimated (eCVR) in 184 female RA patients (median age 53 years, range 21-71) using the Framingham algorithm. A 5-year prospective follow-up for new CVD events, type II diabetes and medication for hypertension and hyperlipidemia was completed in all the patients. The event-free survival curves were built and the Mantel-Cox analysis was performed with respect to serum IGF1, where IGF1 levels below or equal to the median 139 ng/ml were considered low. Results: Low IGF1 was clinically significant. These patients were recognized by high prevalence of hypertension (26% vs. 7.9%, p=0.001), overweight (19% vs 6.8%, p=0.016) and hypercholesterolemia (71% vs 48%, p=0.0025), which resulted in a higher eCVR in these RA patients (7.2% and 3.3%, p 10 years indicated 80.5% specificity for development of new CV events. Conclusion: We identified low normal levels of IGF1 to be associated with higher prevalence of CVD events in RA patients. Importantly, low IGF1 appeared to be an independent predictor of hypertension in middle-aged female patients. References: [1] Wiseman SJ, Ralston SH, Wardlaw JM. Cerebrovascular Disease in Rheumatic Diseases: A Systematic Review and Meta-Analysis. Stroke. 2016;47(4):943-50. [2] Laughlin GA, Barrett-Connor E, Criqui MH, Kritz-Silverstein D. The prospective association of serum insulin-like growth factor I (IGF-I) and IGF-binding protein-1 levels with all cause and cardiovascular disease mortality in older adults: the Rancho Bernardo Study. J Clin Endocrinol Metab. 2004;89(1):114-20. Disclosure of Interests: None declared

Background Rheumatoid arthritis (RA) is more prevalent in females. It is reported to be alleviated during pregnancy, and increase in severity during menopause, which implies estrogen as an important contributor in RA pathogenesis. Micro-RNA (miR) are short, non-coding RNAs, that act within a RNA-induced silencing complex. miRs have recently emerged as important epigenetic controls of leukocyte maturation and function. Objectives To study the epigenetic effect of estrogen on the transcription of microRNA and their bio-processor enzymes in RA patients. Methods The leukocytes of 145 female RA patients split for estrogen receptor alpha (ERα) (dCT 9.57) were analyzed for the expression of Dicer, Drosha and DGCR8 mRNA by RT-PCR and serum levels of TGFb-dependent cytokines IL4 and IL9. Micro-RNA transcription array was performed by 3D-Gene™ microarray measuring >2560 miRs (TATAA Biocenter, Gothenburg) in human primary leukocytes, fat tissue and plasma. The samples were split by expression of estrogen receptor alpha (ERα, dCT 9.57), a proxy of an active estrogen signaling, and used to identify miRs of interest. Bioinformatic analysis was performed using DIANA, miRDB, miRTarBase and Ensembl, using microRNA nucleotide sequences obtained from miRBase, to predict signaling pathways and gene targets of miR. To confirm estrogens effect on miR processing, leukocyte cultures of RA patients were exposed to estrogen and subjected to miR, gene and protein analysis. Results Comparing leukocytes with different ERα, we identified 214 miRs with high and 7 miRs with low expression when ERα was high. Cross-analysis in the fat and serum sample miR array identified most of those miRs. Bioinformatic analysis of the upregulated miRs confirmed that 16miRs were involved in the estrogen signaling pathway (p=0.0063) and 15 TGFb signaling pathway (p= To study if transcription of these predicted targets was dependent on estrogen signaling, we took advantage of the clinical RA material. Patients with high ERα were significantly younger (51y vs 61y, p Exposure of leukocytes to estrogen in culture induced a significant reduction in IL9 (p=0.0078), which suggests a suppression of TGFb signaling. Conclusion High ERα expression can be considered a significant regulator of miR transcription in leukocytes. miR and bio-processors regulated by estrogen could be of importance in the pathogenesis on RA by targeting genes responsible for regulation of inflammation and the non-protective elements of RA development; explaining the ameliorating effects of estrogen. 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 Oestrogen has ameliorating effects on rheumatoid arthritis (RA). Oestrogen receptor (ER) signalling affects micro-RNA (miR) expression and processing in breast cancer, while its effect on miR profile in RA have never been studied. Objectives To study the effect of the oestrogen replacement therapy and oestrogen receptor signalling on the miR transcription and bioprocessing in RA patients. Methods The expression of the key miR processing enzymes Dicer, Drosha and DGCR8 was measured in the leukocytes of the peripheral blood of 145 female RA patients (age 53 years, disease duration 9.8 years) by RT-PCR and analysed with respect to the levels of ERα, used as surrogate marker of active ERα signalling. Total RNA was prepared from the serum samples of 46 postmenopausal female RA patients who received treatment with oestradiol (E2), noretisterone acetate, vitamin D3 and calcium (n=22, mean E2 levels 229.4±143.2 pg/ml) or vitamin D3 and calcium supplementation only (n=24, mean E2 levels 30.8±8.7 pg/ml). Serum was obtained after 12 and 24 months of treatment and was stored at −70 °C until the time of analysis. MicroRNA transcription was performed by 3D-Gene microarray measuring >2560 miRs (TATAA Biocenter, Gothenburg). Results A higher ERα expression in RA female patients was associated with increased transcription of the major miR processing proteins Dicer (p=0.0057), Drosha (p=0.019) and DGRC8 (p=0.0095). This activated transcription of miR biomachinery could indicate a higher demand and a facilitated miR maturation. The E2-treatment significantly changed the profile, but not the levels, of miRs in serum, where only 50% of the miR transcripts were present both in E2-treated and control samples. Also the profile of miRs related to RA and of those regulating translation of ERα and Dicer, Drosha, and DGCR8 proteins was affected. Extensive bioinformatic analysis of the miR-targeting genes affected during E2-treatment, revealed a reduction of gene clusters aiding autoimmune pathology and the RA-associated molecular processes including DNA replication (p=2.6x10–4), peptidyl-serine phosphorylation (p=2x10–4), protein localization to nucleus and nuclear import (p=5x10–4). Conclusions Activation of ERα significantly enhances the miR processing, and affects the profile of miR transcription in female RA patients. The change in miR profile during E2-treatment could contribute to a significantly change in the miR landscape and disposition of intracellular processes in RA. Disclosure of Interest None declared

Objectives To improve understanding of fat-related molecular mechanisms behind the increased cardiovascular (CV) morbidity in patients with rheumatoid arthritis (RA). Methods Transcription of uncoupling protein 1 (UCP1) was measured in the subcutaneous fat tissue and serum levels of lipoproteins, adipokines, and inflammation markers in 185 middle-aged female patients (mean age 51 years) with RA and compared between the groups stratified by the total cholesterol (TC) levels and the body mass index (BMI). The risk of dying of CV disease within 5 years was calculated electronically using the strategy proposed by Pocock et al.1 Results CVR was highest (risk score 27.76, 5 year CVR 0.67%) in the patients combining high TC (>5.1 mmol/L) and high BMI (>25 kg/m2), while those with low levels of TC and BMI had lowest CVR (risk score 10.82, CVR 0.11%). CVR was significantly decreased if either TC (TCloBMIhi) or BMI (TChiBMIlo) was low (p=0.017 and p=0.014, respectively). With the exception of TCloBMIlo group, these groups had no difference with respect to age, disease duration, inflammation defined by serum IL6 and IL1, and disease activity measured by DAS28. TCloBMIhipatients had an overall increase in fat expression of UCP1 (p=0.047) that has the cholesterol lowering capacity and may explain low TC levels in this group. In contrast, TChiBMIlopatients had high prevalence of cases with unmeasurable UCP1 expression and higher levels of serum adiponectin (p=0.053) and HDL (p Measurable expression of UCP1 was found in 79%. In total cohort, the patients with measurable UCP1 had higher inflammation and RA activity presented by IL-6 (p=0.0001), IL1b (p=0.037) and DAS28 (p=0.0086), compared to those with no UCP1 expression. TCloBMIhipatients had an overall increase in fat expression of UCP1 (p=0.047) and lowest prevalence of cases with no UCP1 expression (6.2%). Conclusions The study shows that UCP-1 expression in subcutaneous fat may be a CV protective mechanism in RA patients. The inflammation seems to be the driving force of UCP1 expression in RA. Reference [1] Pocock SJ, McCormack V, Gueyffier F, Boutitie F, Fagard RH, Boissel JP. A score for predicting risk of death from cardiovascular disease in adults with raised blood pressure, based on individual patient data from randomised controlled trials. BMJ2001;323:75–81. Disclosure of Interest None declared

Background Oncoprotein survivin emerged as an important player in the pathogenesis of rheumatoid arthritis (RA). Results of genome-wide study suggest that survivin may take part in transcription stimulation of the RA-specific genes. Objectives To identify and describe survivin-dependent differences in transcription pattern between CD4+ T-cells of RA patients and healthy subjects focusing in particular on a subset of genes involved in maturation of Th1 and Th17 cells. Methods CD4+ T-cells were isolated from PBMC of 3 RA patients and 5 non-smoking and 2 smoking healthy controls using a positive selection and activated by Pam3cys+Concanavalin A+LPS. Chromatin immunoprecipitation (ChIP) was done using rabbit polyclonal anti-Survivin, purified DNA was prepared into libraries using ThruPLEX (Rubicon) and sequenced using Hiseq 2000 (Illumina). Resulting fastq sequencing files were mapped to the human reference genome (hg38) using the STAR aligner. Peaks were associated with the closest transcription start site. Enriched peak regions (p Results We identified 11 145 survivin-bound chromatin sequencies. Out of them, GO technique indicated 770 genes in RA samples (7.3%) and 766 genes in healthy controls (19.5%) which were annotated and enriched (>log-5) in GO terms. In the screening for genes regulating maturation of Th1/Th17 cells, CBLB, IRF1, STAT3, SGK1 and TNFSF14 were identified among the genes enriched in RA samples, while EGR3 and ETS1 were enriched only in healthy controls. Additionally, transcription factors Ezh2, Rad21, Ctbp2 and Suz12 were identified as common for both RA and healthy groups genes, associated with significant GO enrichment. Conclusions This study confirms the role of survivin as a transcription mediator in CD4 +T cells and is suggested to influence multiple genes involved in RA pathology. References [1] Andersson KME, et al. Pathogenic Trans differentiation of Th17 Cells Contribute to Perpetuation of Rheumatoid Arthritis during Anti-TNF Treatment. Mol. Med. 2015;21:536–43. [2] Hu D, et al. Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis. Nat. Commun. 2017;8:1600. Disclosure of Interest None declared

Introduction In addition to inflammation of the joints, rheumatoid arthritis (RA) has a neurological part consisting of pain, fatigue, depression and cognitive deterioration. These symptoms are critical for the patients’ ability to cope with daily life, but are not alleviated completely with modern antirheumatic drugs. Sufficient IGF1R signalling is important for neurogenesis in the hippocampus. Its misbalance correlates with depression and anxiety. Objectives The aim of this study was to evaluate the pathological changes in the brain during experimental RA and investigate their connexion to IGF1R. Methods Characteristics of pain, depression and anxiety were collected among 214 RA patients and analysed in relation to IGF1R expression in WBC. Experimental RA was induced by immunisation with collagen II. Inhibition of IGF1R was achieved by injection of shRNA-producing lentiviral construct. The behavioural pattern of each mouse was recorded by filming. The hippocampus was analysed morphometrically, and gene and protein expression were analysed by qPCR and immunohistochemistry, respectively. Results The RA patients’ perception of depression and anxiety was associated with high IGF1R expression in WBC. This group of patients was also less physically active. In experimental RA, an enrichment of IBA1 +microglia and high expression of CD68 and IL-1b was found in the hippocampus. This was followed by an increased density of IGF1R+cells in cornu ammoni, and a decreased neurogenesis by limited expression DCX in the subgranular layer of the dentate gyrus. This results in a significant reduction of the hippocampus area. These changes in the brain were associated with immobility in RA mice. Treatment with shRNA targeting IGF1R improved arthritis, but led to increased immobility. Conclusions RA induces remote inflammation in the hippocampus reducing neurogenesis and physical activity. The neurological symptoms in patients and in experimental RA are connected to IGF1R expression and signalling, and further expands our knowledge of neurological processes in RA. Disclosure of interest None declared

Background Rheumatoid arthritis (RA) has recently been linked to an exhausted state of CD4+ T cells in peripheral blood of patients [1]. Exhaustion of CD4+ T cells limits their proliferation and increases cell death. In CD8+ T cells smoking counteract exhaustion, which may lead to increased cytotoxic activity exemplified by targeting of cells with high expression of the anti-apoptotic protein survivin [2]. Exhaustion of CD4+ T cells coincides with expression of interferon (IFN) response genes [3], referred to as the IFN signature. The development of the IFN signature has been suggested to predate RA [4]. Objectives We investigated how smoking affect the CD4+ T cell population in peripheral blood of RA patients with focus on the exhaustion marker programmed cell death-1 (PD-1). Methods Blood samples were collected from RA patients and healthy women with different smoking status and analysed for PD-1 and survivin expression using flow cytometry and qPCR. Sorted Th17 cells from peripheral blood were analysed for expression of 18 genes up regulated during exhaustion [3], herein referred to as the exhaustion set, and serum levels of survivin were assessed by ELISA. Peripheral blood CD4+ cells were analysed for their expression of seven IFN response genes [4]. The role of survivin in the formation of exhausted CD4+ T cells was studied in collagen-induced arthritis (CIA), where mice were treated with nicotine or vaccinated with survivin peptides. Results High frequency of exhausted PD-1+CD4+ cells was found in smoking RA patients. The numbers of PD1+CD4+ cells correlated inversely with the PD-1 expression by cytotoxic CD8+CD107+ cells (r=-0.62, p=0.01). Additionally, the frequency of PD-1+CD4+ cells increased with reduction of the CD4+ population (r=-0.71, p=0.002). The IFN signature was found exclusively among smoking RA patients. The patients with the IFN signature all had CD4+ cells with low survivin production. Th17 cells from RA patients with high serum survivin were enriched in genes of the exhaustion set. CD4+ cells with high survivin expression were negative for PD-1, while PD-1hi cells had low expression of survivin. In CIA mice the survivinhiPD-1- CD4+ cells were reduced by nicotine treatment (p=0.03) or survivin vaccination (p=0.009). Conclusions Smoking associates with exhaustion of CD4+ T cells in RA by increasing the frequency of PD-1+CD4+ cells and supporting the IFN signature. Balancing T cell exhaustion and preventing the IFN signature are potential future treatment strategies for RA. References Frenz T, et al. J Allergy Clin Immunol 2016. 138(2): 586–589. Wasen C, et al. J Autoimmun 2017. DOI: 10.1016/j.jaut.2016.12.00. Crawford A, et al. Immunity 2014. 40(2): 289–302. Lubbers J, et al. Ann Rheum Dis 2013. 72(5): 776–780. Disclosure of Interest None declared

Background Insulin-like growth factor (IGF) 1 receptor is essential for cell energy metabolism. It plays a key role linking glucose and lipid metabolism of active cells and may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated supporting expansion of the inflamed synovia. Objectives The aim of the present study was to understand the place of IGF-1R expression and signalling for development of adaptive immune responses in the setting of prospective RA cohort and in experimental arthritis. Methods Clinical associations of IGF1R expression in leukocytes of the peripheral blood were studied in 84 female RA patients with mean disease duration 8 years, all treated with methotrexate. Consequences of the IGF1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of the insulin receptor substrates IRS-1/2. Results In RA patients, high expression of IGF1R in leukocytes was associated with systemic inflammation as verified by higher transcription factor NF-kB (p=0.033), serum levels of IL-6 (p=0.006) and erythrocyte sedimentation rate (p=0.003), and higher pain perception (VAS, p=0.019). Additionally, women with high IGF1R were often in need of combined DMARD treatment (p=0.045). Phosphorylated IGF1R and STAT3 enrich T cells infiltrate in RA synovia. Treatment with NT157 rendered no metabolic consequences to mouse body weight and serum glucose levels. NT157 inhibited the phosphorylation of IGF1R and STAT3 in synovia, and alleviated arthritis and erosions in mice. It also reduced IGF1R+ T cell population in spleen and despaired ERK and Akt signalling. This limited the ability of mBSA-specific T cells to produce IL-6 (p=0.013), IFNg (p=0.024) and IL-17 (p=0.0097). The reduction of IL-6 and IL-17 levels was associated with changed RoRgt/FoxP3 balance. Conclusions IGF1R signalling contributes to severe RA by triggering T cell dependent inflammation in arthritis. Inhibition of IGF1R on the level of insulin receptor substrates alleviates arthritis by restricting IL-6 dependent formation of Th17 cells and may open for new treatment strategies in RA. Disclosure of Interest None declared

Survivin is a protein functionally important for cell division, apoptosis, and possibly, for micro-RNA biogenesis. It is an established marker of malignant cell transformation. In non-malignant conditions, the unique properties of survivin make it indispensable for homeostasis of the immune system. Indeed, it is required for the innate and adaptive immune responses, controlling differentiation and maintenance of CD4+ and CD8+ memory T-cells, and in B cell maturation. Recently, survivin has emerged as an important player in the pathogenesis of autoimmune diseases. Under the conditions of unreserved inflammation, survivin enhances antigen presentation, maintains persistence of autoreactive cells, and supports production of autoantibodies. In this context, survivin takes its place as a diagnostic and prognostic marker in rheumatoid arthritis, psoriasis, systemic sclerosis and pulmonary arterial hypertension, neuropathology and multiple sclerosis, inflammatory bowel diseases and oral lichen planus. In this review, we summarise the knowledge about non-malignant properties of survivin and focus on its engagement in cellular and molecular pathology of autoimmune diseases. The review highlights utility of survivin measures for clinical applications. It provides rational for the survivin inhibiting strategies and presents results of recent reports on survivin inhibition in modern therapies of cancers and autoimmune diseases.

Background Signalling through insulin-like growth factor (IGF) 1 receptor is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated supporting expansion of the inflamed synovia. The aim of the present study was to understand the place of IGF-1R expression and signalling for development of adaptive immune responses in the setting of prospective RA cohort and in experimental arthritis. Material and methods Clinical associations of IGF1R expression in leukocytes of the peripheral blood were studied in 84 female RA patients with mean disease duration 8 years, all treated with methotrexate at the time of enrolment. Consequences of the IGF1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of IRS-1/2. Results In RA patients, high expression of IGF1R in leukocytes was associated with and systemic inflammation as verified by higher transcription factor NF-kB, serum levels of IL-6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF1R and STAT3 in synovia, and alleviated arthritis and erosions in mice. It also reduced expression of IGF1R in spleen T cells and despaired ERK and Akt signalling. This limited the ability of mBSA-specific T cells to produce IL-6, IFNg and IL-17. The reduction of IL-6 and IL-17 levels was associated with changed RoRgt/FoxP3 balance. Conclusion IGF1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF1R signalling alleviates arthritis by restricting IL-6 dependent formation of Th17 cells and may open for new treatment strategies in RA.

Background Exhausted CD4+ T cells are recognised by low proliferation, increased cell death and high expression the inhibitory co-receptor programmed cell death-1 (PD-1). CD4+ T cells in patients with rheumatoid arthritis (RA) have recently been reported to show signs of exhaustion.1 Exhaustion of CD4+ in experimental infection was associated with expression of interferon (IFN) response genes,2 referred to as the IFN signature. In human, the IFN signature has been suggested to predict rheumatoid arthritis (RA).3 We investigated if CD4+ T cells with an exhausted phenotype and IFN signature are accumulating in peripheral blood of RA patients. We study a connexion between CD4+ T cell exhaustion and survivin expression in RA. Materials and methods IFN response genes, PD-1 and survivin expression were analysed in peripheral blood of RA patients and healthy women with different smoking status using flow cytometry and qPCR. The role of survivin in the formation of exhausted CD4+ T cells was studied in the collagen-induced arthritis model, where mice where treated with nicotine or vaccinated with survivin peptides. Results High frequency of exhausted PD-1+CD4+ cells was found in smoking RA patients. The numbers of PD1+CD4+ cells correlated inversely with the PD-1 expression by cytotoxic CD8+CD107+ cells (r=−0.62, p=0.01). Additionally, the frequency of PD-1+CD4+ cells increased with reduction of the CD4+ population (r=−0.71, p=0.002). The IFN signature was found exclusively among smoking RA patients. The patients with the IFN signature all had CD4+ cells with low survivin production. Sorted Th17 cells from RA patients with high serum levels of survivin had higher transcription of exhaustion related genes compared to healthy controls. CD4+ cells with high survivin expression were negative for PD-1. In mice, the survivinhiPD-1- population of CD4+ cells was reduced in nicotine-treated mice (p=0.03) and in response to activation of survivin targeting cells through vaccination (p=0.009). Conclusions We show that smoking associates with exhaustion of CD4+ T cells in RA. Smoking increases the frequency of PD-1+CD4+ cells with the IFN signature by activating survivin targeting mechanisms. References Frenz, T, et al, CD4+ T cells in patients with chronic inflammatory rheumatic disorders show distinct levels of exhaustion. Journal of Allergy and Clinical Immunology, 2016;138(2):p.586–589.e10. Crawford, A, et al, Molecular and Transcriptional Basis of CD4+ T Cell Dysfunction during Chronic Infection. Immunity2014;40(2):p.289–302. Lubbers, J, et al, The type i IFN signature as a biomarker of preclinical rheumatoid arthritis. Annals of the Rheumatic Diseases2013;72(5):p.776–780.

Survivin is a proto-oncogene that regulates cell division and apoptosis. It is a molecular marker of cancer. Recently, survivin has emerged as a feature of RA, associated with severe joint damage and poor treatment response. The present study examined if inhibition of survivin affects experimental arthritis, which was induced in mBSA-immunized mice by an injection of mBSA in the knee joint or developed spontaneously in collagen type II-immunized mice. The inhibition of survivin transcription by a lentivirus shRNA construct alleviated joint inflammation and reduced bone damage. The inhibition of survivin reduced the levels of metalloproteinases, β-catenin, and vimentin, limiting the invasive capacity of synovia, while no inhibition of osteoclastogenesis could be found. The inhibition of survivin led to a p53-independent reduction of T cell proliferation and favored the transcription and activity of Blimp-1, which limited IL-2 production and facilitated formation of regulatory Foxp3+CD4+ and effector CD8+ T cells. The study shows that the inhibition of survivin is sufficient to reduce joint inflammation and bone damage in preclinical models of arthritis. Antiarthritic effects of survivin inhibition are related to p53-independent control of lymphocyte proliferation.

S100A4 is a Ca-binding protein participating in regulation of cell growth, survival, and motility. Here we studied the role of S100A4 protein in sex hormone-regulated bone formation. Bone mineral density in the trabecular and cortical compartments was evaluated in female S100A4 knockout (KO), in matched wild-type (WT) counterparts, and in WT mice treated with lentiviral small hairpin RNA construct inhibiting the S100A4 gene transcription or with a nontargeting construct, by peripheral quantitative computed tomography. The effect of sex hormones on bone was measured 5 weeks after ovariectomy (OVX) and/or dehydroepiadrosterone treatment. S100A4KO had an excessive trabecular and cortical bone formation compared with the age- and sex-matched WT mice. S100A4KO had an increased periosteal circumference (P = .001), cortical thickness (P = .056), and cortical area (P = .003), which predicted 20% higher bone strength in S100A4KO (P = .013). WT mice treated with small hairpin RNA-S100A4 showed an increase of the cortical bone parameters in a fashion identical with S100A4KO mice, indicating the key role of S100A4 in the changed bone formation. S100A4KO mice had higher serum levels of osteocalcin and a higher number of osteocalcin-positive osteoblasts under the periosteum. OVX-S100A4 resulted in the loss of the cortical bone supported by high CTX-I levels, whereas no such changes were observed in OVX-WT mice. S100A4KO mice resisted the dehydroepiadrosterone -induced bone formation observed in the WT counterparts. Our study indicates that S100A4 is a regulator of bone formation, which inhibits bone excess in the estrogen-sufficient mice and prevents the cortical bone loss in the estrogen-deprived mice.

Objective S100A4 is a Ca-binding protein that regulates cell growth, survival, and motility. The abundant expression of S100A4 in rheumatiod arthritis contributes to the invasive growth of joint tissue and to bone damage. In the present study, we analysed the role of S100A4 in bone homeostasis. Methods Peripheral quantitative computed tomography and histomorphometric analysis were performed in mice lacking the entire S100A4 protein (S100A4KO) and in wild-type (WT) counterparts treated with shRNA-lentiviral constructs targeting S100A4 (S100A4-shRNA). Control groups consisted of sex-matched WT counterparts and WT mice treated with a non-targeting RNA construct. Results S100A4 deficiency was associated with higher trabecular and cortical bone mass, increased number and thickness of trabeculi combined with larger periosteal circumference and higher predicted bone strength. S100A4 inhibition by shRNA led to an increase in cortical bone in WT mice. S100A4-deficieny was associated with a reduced number of functional osteoclasts. S100A4KO and S100A4-shRNA-treated bone marrow progenitors gave rise to a large number of small TRAP + cells with few nuclei and few pseudopodial processes. Poor osteoclastogenesis and the low resorptive capacity in S100A4Ko mice may be linked to low levels of surface integrins, impaired adhesion capacity, and poor multinucleation in S100A4-deficient osteoclasts, as well as a low content of proteolytic enzymes cathepsin K and MMP3 and MMP9 to break down the organic matrix. Conclusion S100A4 emerges as a negative regulator of bone metabolism potentially responsible for the excessive bone turnover in conditions marked by high levels of S100A4 protein, such as inflammation and rheumatoid arthritis.

MicroRNAs (miRs) represent a part of epigenetic control of autoimmunity gaining increasing attention in rheumatoid arthritis (RA). Since cigarette smoking plays important role in RA pathogenesis and reprograms transcriptional profile of miRNAs, we ask if the onco-protein survivin, a novel biomarker of RA, may provide a link between smoking and miRNA. Studying survivin expression in leukocytes of 144 female RA patients we observed that smoking patients had higher survivin transcription and a remarkable spreading of survivin isoforms. This was associated with restricted pattern and low production of miRs. Additionally, miRNA processing enzymes Dicer and DGRC8 were decreased in the patients with survivin isoform spreading. The direct contribution of survivin in miRs biogenesis was confirmed by a massive increase of miRs production following inhibition of survivin in leukocyte cultures. Dicer is shown to mediate these effects of survivin. Chromatin immunoprecipitation analysis demonstrated binding of survivin to the Dicer promoter region. Dicer expression increased 5-folds following survivin inhibition. Taken together, this study presents experimental evidence of a novel cellular function of survivin, control of miRs biogenesis. Up-regulation of survivin in smokers suggests its role as effector of the adverse epigenetic control in RA.

Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

Inactivating GGTase-I reduces disease phenotypes in a mouse model of K-RAS-induced myeloproliferative disease

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA, RAC1, and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized, they have very different properties, and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study, we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity, disrupted the actin cytoskeleton, reduced cell migration, and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover, the absence of GGTase-I activity reduced lung tumor formation, eliminated myeloproliferative phenotypes, and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly, several cell types remained viable in the absence of GGTase-I, and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies.

In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

Introduction Alternative splicing distinguishes normal and pathologic cells. High levels of oncoprotein survivin recognise patients with severe rheumatoid arthritis (RA). Here, we assess clinical relevance of alternative splicing of survivin in leukocytes of peripheral blood (PBMC) and bone marrow (BM) in RA patients. Method Transcription of survivin wild-type (survivin-WT), survivin-2B and survivin-ΔEx3 was measured in 67 randomly selected RA patients and in 23 patients before and after B cell depletion with rituximab. Analysis was done in relation to disease activity, anti-rheumatic treatment and serum levels of rheumatoid factor (RF) and survivin. Results Survivin-WT was the dominant splice variant equally expressed in T and B cells, while survivin-2B and survivin-ΔEx3 were higher in B cells. High disease activity (DAS28>5.1) was associated with an excess of survivin-WT and low ratios between survivin-2B/WT (p=0.035) and survivin-ΔEx3/WT in PBMC. Depletion of B cells by rituximab caused a decrease in survivin-WT (p=0.005) in PBMC, increasing the ratio between survivin-2B/WT (p=0.009) and survivin-ΔEx3/WT (p=0.001) in BM. This increase in survivin-2B/WT was associated with reduction in CD19+ BM cells (r=0.929, p=0.007), RF (IgM, r=0.857, p=0.024; IgA, r=0.739, p=0.021), and DAS28 (0.636, p=0.054). The increase in survivin-ΔEx3 in BM was associated with a reduction of CD19+ BM cells (r=0.714, p=0.058) and DAS28 (r=0.648, p=0.049), while survivin-ΔEx3/WT was associated with RF (IgG, r=0.882, p=0.016). Conclusion This study demonstrates that the suppressed diversity of survivin splicing in leukocytes may attribute to adverse self-recognition in RA. Depletion of autoantibody producing B cells improves the balance of survivin splicing.

T-helper cells producing interleukin (IL)-17A and IL-17F cytokines (Th17 cells) are considered the source of autoimmunity in rheumatoid arthritis (RA). In this study, we characterized specific pathogenic features of Th17 cells in RA. By using nano-string technology, we analyzed transcription of 419 genes in the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of 14 RA patients and 6 healthy controls and identified 109 genes discriminating Th17 cells of RA patients from the controls. Th17 cells of RA patients had an aggressive pathogenic profile and in addition to signature cytokines IL-17, IL-23 and IL-21, and transcriptional regulators RAR-related orphan receptor gamma of T cells (RORγt) and Janus kinase 2 (JAK2), they produced high levels of IL-23R, C-C chemokine ligand type 20 (CCL20), granulocyte-monocyte colony-stimulating factor (GM-CSF ) and transcription factor Tbet required for synovial homing. We showed that Th17 cells are enriched with Helios-producing Foxp3- and IL2RA-deficient cells, indicating altered regulatory profile. The follicular T-helper (Tfh) cells presented a functional profile of adaptor molecules, transcriptional regulator Bcl-6 and B-cell activating cytokines IL-21, IL-31 and leukemia inhibitory factor (LIF ). We observed that anti-tumor necrosis factor (TNF) treatment had a limited effect on the transcription signature of Th17 cells. Patients in remission retained the machinery of receptors (IL-23R and IL-1R1), proinflammatory cytokines (IL-17F, IL-23, IL-21 and TNF ) and adaptor molecules (C-X-C chemokine receptor 5 [CXCR5] and cytotoxic T-lymphocyte-associated protein 4 [CTLA-4]), essential for efficient transdifferentiation and accumulation of Th17 cells. This study convincingly shows that the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of RA patients harbor pathogenic subsets of Th17 and Tfh cells, which may transdifferentiate from Tregs and contribute to perpetuation of the disease.

Osteoclasts are bone-resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms-like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms-like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms-like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms-like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms-like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin–induced arthritis using mice that lack the gene for Flt3l (Flt3L−/−) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L−/− mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L−/− mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L−/− mice with Fms-like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L−/− mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms-like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

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

Survivin is known as an inhibitor of apoptosis and a positive regulator of cell division. We have recently identified survivin as a predictor of joint destruction in patients with rheumatoid arthritis (RA). Flt3 ligand (Flt3L) is expressed in the inflamed joints and has adjuvant properties in arthritis. Studies on 90 RA patients (median age 60.5 years [range, 24–87], disease duration 10.5 years [range, 0–35]) show a strong positive association between the levels of survivin and Flt3L in blood. Here, we present experimental evidence connecting survivin and Flt3L signaling. Treatment of BALB/c mice with Flt3L led to an increase of survivin in the bone marrow and in splenic dendritic cells. Flt3L changed the profile of survivin splice variants, increasing transcription of the short survivin40 in the bone marrow. Treatment with an Flt3 inhibitor reduced total survivin expression in bone marrow and in the dendritic cell population in spleen. Inhibition of survivin transcription in mice, by shRNA lentiviral constructs, reduced the gene expression of Flt3L. We conclude that expression of survivin is a downstream event of Flt3 signaling, which serves as an essential mechanism supporting survival of leukocytes during their differentiation, and maturation of dendritic cells, in RA.