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MicroScale Thermophoresis (MST) follows the movement of fluorescent-labelled biomolecules with different sizes along a temperature gradient.

To analyze the safety and effectiveness of abatacept (ABA) given in routine care to patients with systemic sclerosis (SSc).Retrospective multicenter observational study that enrolled patients with SSc treated with ABA. We collected epidemiological data and clinical outcomes. First, we analyzed the frequency of adverse effects. Secondly, we compared the evolution of different organ manifestations during ABA treatment. We collected data from 6 months before start of therapy to the last follow-up the following parameters: modified Rodnan Skin Score (mRSS), joints, lung and gastrointestinal involvement, concomitant medications, and laboratory tests.Data on twenty-seven patients with SSc were collected (93% females; 67% limited SSc). Rheumatoid arthritis was the most frequent concomitant autoimmune disease. ILD was present in 15 patients. Anti-Scl 70 antibodies were present in 13 patients and rheumatoid factor and ACPA antibodies were present in eight and seven patients respectively. The main indication to use abatacept was joint involvement (59%) followed by myositis (26%). A total of 16 adverse effects were reported in 28 months of abatacept treatment including five that required hospitalization. Most of them occurred in the first 3 months after starting abatacept. After 12 months, the number of tender and swollen joints decreased compared to baseline (p0.03 and p0.02 respectively). Moreover, a beneficial effect of abatacept on HAQ-DI at 3 and 6 months (p0.05) and on morning stiffness at 6 and 12 months (p0.03) was observed. We also observed a decrease in the modified Rodnan skin score (p0.05). No changes in lung or gastrointestinal involvement were found.ABA demonstrated a good safety profile and seems to have some effectiveness on joint involvement and related disability in SSc patients treated in routine care.

Vertically oriented graphene (VG) has attracted attention for years, but the growth mechanism is still not fully revealed. The electric field may play a role, but the direct evidence and exactly what role it plays remains unclear. Here, we conduct a systematic study and find that in plasma-enhanced chemical vapor deposition, the VG growth preferably occurs at spots where the local field is stronger, for example, at GaN nanowire tips. On almost round-shaped nanoparticles, instead of being perpendicular to the substrate, the VG grows along the field direction, that is, perpendicular to the particles' local surfaces. Even more convincingly, the sheath field is screened to different degrees, and a direct correlation between the field strength and the VG growth is observed. Numerical calculation suggests that during the growth, the field helps accumulate charges on graphene, which eventually changes the cohesive graphene layers into separate three-dimensional VG flakes. Furthermore, the field helps attract charged precursors to places sticking out from the substrate and makes them even sharper and turn into VG. Finally, we demonstrate that the VG-covered nanoparticles are benign to human blood leukocytes and could be considered for drug delivery. Our research may serve as a starting point for further vertical two-dimensional material growth mechanism studies.

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.

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.

Osteoporosis is a major cause of age-related fractures. Healing complications in osteoporotic patients are often associated with increased mortality and morbidity. Stimulation of the implant-adjacent bone could be beneficial in terms of the surgical outcome. Over the past decade, numerous investigations have implicated insulin in normal bone growth, and recent studies have described the advantages of administering insulin locally to increase bone formation. Therefore, we hypothesized that insulin-coated titanium implants would increase bone formation in osteoporotic animals. The aim of this study was to evaluate the effects of insulin delivered from an implant surface on bone-related gene expression and bone formation in osteoporotic rats. Characterizations of the surfaces of insulin-coated and control implants were performed using ellipsometry and interferometry. Forty ovariectomized and four healthy Sprague Dawley rats were used and implants were inserted in the tibias. The systemic effect of insulin was assessed by measuring the blood glucose levels and total body weight. The animals were sacrificed either 1 day or 3 weeks postimplantation. Implant-adherent cells were analyzed by quantitative real-time PCR, and the bone adjacent to the implants was examined by microcomputed tomography and histomorphometry. The insulin-coated implants had no systemic effects. The insulin-coated samples demonstrated significantly lower expression of the gene for interleukin 1β (p = 0.019) at 1 day, and significantly exhibited more periosteal callus (p = 0.029) at 3 weeks. Locally delivered insulin has potential for promoting bone formation and it exerts potentially anti-inflammatory effects in osteoporotic rats. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2472-2480, 2018.

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 analyse the relation between serum levels of IGF1 and cardiovascular risk (CVR) in women with rheumatoid arthritis (RA). Methods The risk of dying of CV disease within 5 years was calculated using the strategy proposed by Pocock et al. BMJ 2001 in 185 women with RA (mean age 51.7 years) with no previous history of CV events. The CVR and characteristics related to it were analysed with respect to serum IGF1. IGF1 levels below the median of the total cohort were considered low. Results The RA women with low IGF1 (n=91, mean 104 pg/ml) had significantly higher CVR compared to those with normal IGF1 (n=94, mean 194 pg/ml) with the predicted risk of 0.51% and 0.17%, respectively (p>10–5). Among the traditional CVR factors, the low IGF1 group was 10 years older (mean 56.8 vs. 46.9 y, p=10–5), lower in height (165 vs. 168 cm, p=0.013, 21% of the patients being The low IGF1 group displayed the unfavourable metabolic profile with higher BMI (p=0.002) and obesity in 22%, higher predicted body fat content (mean 39.5 vs. 35%, p=10–5), and higher total and LDL cholesterol (p=0.0014 and p=0.0053, respectively). The levels of adiponectin (p=0.032) and HDL-cholesterol (p=0.25) tended to be higher in the low IGF1 group, which resulted in the comparable total cholesterol to HDL ratio between the groups. This could also explain that the prevalence of diabetes mellitus and metabolic syndrome were low. With exception of the disease duration, the groups displayed no significant difference in the RA-related CVR factors such as the disease activity measured by DAS28 (3.29 vs 2.99), systemic inflammation measured by serum IL6 (mean 8.46 vs. 5.99 pg/ml) and IL1β (mean 19.47 vs. 23.1 ng/ml), and the prevalence of seropositivity (91% vs. 92%). The prevalence of treatment with MTX monotherapy was higher in the low IGF1 group (56% vs. 39%, p=0.024), while the use of TNF-inhibitors and other biologic and synthetic anti-rheumatic drugs was similar within the groups. Conclusions Serum IGF1 levels in the normal low range are associated with higher CVR in RA female patients. This increase in CVR seems to be independent of the RA-related characteristics. The combination of low height and hypertension argues for the important role of congenital factors in defining serum IGF1 levels in the studied RA women. 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

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

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19 + PC, CD19 − PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR low Ki-67 − CD95 low CD28 + CD56 +/− , with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin V H rearrangements of CD19 − BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19 − PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19 + and CD19 − PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection.

Introduction Smoking is a risk factor for the development of rheumatoid arthritis (RA) and associates with positivity for disease specific anti cyclic citrullinated peptide (aCCP) antibodies. It is not known exactly how smoking promotes autoimmunity but we have previously demonstrated that smoking limits the expression of the T cell co-inhibitory receptor programmed death-1 (PD-1).1 Objectives To investigate if smoking can interfere with the interaction between PD-1 and its ligand by influencing the serum levels of soluble PD-1 ligand 1 (sPD-L1). Methods Serum samples were collected from 254 RA patients and 168 healthy controls with known smoking status and analysed with ELISA for levels of sPD-L1 and inflammatory cytokines IL-6 and IL-1b. Fc-receptor mRNA expression analysis of peripheral blood monocytes (PBMC) from a group of 10 healthy controls and 15 RA patients was done by qPCR. Results In RA patients current smokers had 5 times lower median serum levels of sPD-L1 compared to never smokers (p=0.027). This difference persisted in former smokers that quit smoking Furthermore, aCCP positivity in RA patients was associated with higher levels of sPD-L1 (p=0.0036). We speculate that the presence of antibodies might influence the levels of sPD-L1 through the stimulation of Fc-receptors expressed by PD-L1 producing cells. In PBMC depleted of T cells, we saw that smokers had lower mRNA expression of the stimulatory FcgRIIIA (p=0.028) and predominance of the inhibitory FcgRIIB in the FcgRIIB/FcgRIIIA ratio (p=0.0004). Conclusions Smoking decreases the serum levels of the inflammation limiting protein sPD-L1, but levels were restored by treatment with TNF-inhibitors. aCCP positive RA patients had higher levels of sPD-L1, possibly due to activation of Fc-receptors expressed by PD-L1 producing cells. Reference . Wasen C, et al. J Autoimmun2017. Disclosure of interest None declared

Background Ligand to the inducible T cell costimulator (ICOSL) on B cells is essential for the ICOS-dependent follicular recruitment of activated T cells. In patients with rheumatoid arthritis (RA) the IGF1-IGF1R axis is altered. Inhibition of IGF1R alleviated arthritis by reducing IL-6-dependent formation of Th17 cells.1 Here we study the role of IGF1R on CD21 +cells in experimental arthritis. Methods Female Balb/c mice were immunised with methylated BSA or with CII. Consequences of the IGF-1R inhibition for arthritis were studied in mBSA and CII-immunised mice treated with NT157 compound promoting degradation of insulin receptor substrates or using shRNA producing construct (shIGF1R). At termination three sub-populations of CD21 +cells were analysed: follicular dendritic cells (FDc, CD21 +CD19-CXCR5-); marginal zone B cells (MZBc, CD21 +CD19+CXCR5-); follicular B cells (FBc, CD21 +CD19+CXCR5+). Supernatants of LPS-stimulated splenocytes were analysed for production of cytokines, chemokines using Cytokine Array. Serum levels of antigen specific and autoantibodies were measured in an ELISA. Results In spleen of mBSA-immunised mice, ICOSL expression on CD21 +cells correlated to IGF1R (r=0.70, p=0.007). Inhibition of IGF1R induced a 20% reduction in ICOSL expression in all CD21 +subsets (p=0.007) followed by an increase in the number of MZBc (p=0.003), while FDc and FBc were unchanged. Inhibition of IGF1R had no effect on the expression of ICOS +on CD4 T cells or the subset of CXCR5 +follicular T cells. Reduction of the ICOSL +CD21+B cells was associated with lower production of IL-13. Inhibition of IGF1R signalling by NT157 and by shRNA, reduced production of CXCL13 and CXCL12, the chemokines essential for B cell migration towards follicules. In contrast, the production of chemokines CCL5 and CXCL12 preventing intra-follicular migration was increased, which explains the increase of MZBc. Additionally, the insufficient ICOSL signalling significantly reduced the production of IL-7 and IL-4, regulating class switching of B cells in germinal centres and differentiation of B cells into plasma cells. The described disbalance in the cytokines aiding B cell development led to the reduced production anti-inflammatory IL-10 and of mBSA-specific IgM (p=0.005) and increased production of autoreactive RF-IgM levels (p=0.001). Conclusions The study shows that IGF1R controls B cell development through the expression of ICOSL on CD21 +cells. Insufficient ICOSL signalling disturbs a balance between antigen-specific response and autoantibody production in experimental arthritis. Reference [1] Erlandsson MC, Toyra Silfversward S, Nadali M, Turkkila M, Svensson MND, Jonsson IM, et al. IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis. Biochim Biophys Acta. 2017;1863:2158–70. Disclosure of Interest None declared

Introduction Immunoglobulin D (IgD) remains an enigmatic molecule due to the limited understanding of its function both in healthy and in patients with autoimmune diseases. Objectives In this study we analysed serum IgD (sIgD) levels in patients with rheumatoid arthritis (RA) and healthy controls, paying special attention to differences related to age, gender, smoking and presence of autoantibodies Methods sIgD levels were measured in 416 individuals: 248 (184 female, 65 male) RA patients randomly selected at Sahlgrenska and Uddevalla Hospitals, and 169 (95 female, 73 male) healthy controls selected from the OLIN epidemiological study. Sandwich ELISA was developed by using mouse monoclonal antibodies for capture and goat polyclonal for detection (both SouthernBiotech). Serum samples were tested in dilution 1:5000 and sIgD was quantified after serial dilution of human serum with known IgD levels (Siemens, OTRD). The detection limit was 0.08 µg/ml. Smoking information was collected from all RA patients and controls by self-reported questionnaires. ACPA and RF were measured by automatic multiplex method (anti-CCP2) and rate nephelometry technology. Statistical analysis was performed using the Mann-Whitney test. Results Median sIgD was 38 µg/ml (IQR 14–97) in RA and 43 µg/ml (IQR 19–108) in healthy controls. sIgD was lower in RA females than RA males (p=0.039) whereas healthy controls had no gender differences in sIgD. Among the females, healthy controls 50 y (p=0.009), and to RA patients 50 y (p=0.046). sIgD was not related to the disease activity, however, RA females producing RF alone (p=0.009) and in combination with ACPA (p=0.028) had low sIgD compared to non-producers. In RA, female smokers had high sIgD compared to never (p=0.022) and former smokers (p=0.003). Smoking was not affecting sIgD in healthy controls. No consistent difference in sIgD was found in males. Conclusions The present study indicates that sIgD is influenced by age, gender, smoking and presence of autoantibodies. Low sIgD levels seem to be pathological due to their association with RA and the presence of RF and indicate a potential link between serum IgD and disease severity. 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

Adipose tissue (AT) is a complex endocrine organ with profound effects on body physiology. Research of recent years generated remarkable volume of information on metabolic and immune functions of AT. The lecture will focus on topics of AT biology that shed light on areas of interest linking metabolic, immune and rheumatic conditions. AT is a recognized master regulator of energy balance and nutritional homeostasis. Cells of AT are active part in establishment of autoimmunity, and also osteoarthritis and osteoporosis. Macrophages enrich AT, change its phenotype and mediate inflammation, enhance antigen presentation. Th17 and regulatory T cells reside and operate in AT, having distinct transcriptional and functional characteristics from counterparts of the lymphoid organs. IL6 and IL33 signaling contribute to this distinction. Adipocytes communicate with other cell types by means of signal molecules adipokines, interleukins, and fatty acids and may affect blood pressure, muscle functions, wound healing, bone formation, hematopoiesis and tumor growth. Localization of AT plays important role for its properties including cellular composition, their gene expression profile and secretome, response to external forces and their therapeutic potential. Subcutaneous AT and bone marrow AT are most available and studied in humans. Adiposity has been revealed as a risk factor for development of multiple autoimmune conditions including type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis and thyroiditis. It also associates with low response rate to both biologic and non-biologic anti-rheumatic treatment. Notably, cumulative evidence show a surprising protective action of AT for radiographic joint damage in arthritis. Antirheumatic drugs have remarkable effect on the body fat composition and serum lipid profiles, and could therefore be considered as AT-targeting agents. Impact of these drugs on the composition and function of AT localized subcutaneously and in the bone marrow remains in focus of research and will provide new insights in mechanisms of insulin resistance, cardiovascular risk, bone metabolism and autoantibody production. Production of adipokines has been proposed a mechanism through which AT fuels inflammation and severity of autoimmune diseases however direct evidence still awaits. Several adipokine targeting and adipokine stimulating therapies have been designed and experimentally tested in the models of collagen-induced arthritis, autoimmune diabetes and insulin resistance, encephalitis, and osteoporosis induced by estrogen-deficiency. In human settings, the experience of leptin substitution in anorexia neurosa and in lipodistrophy revealed positive effects on bone metabolism and improved cognition. In conclusion, AT attracts increasing attention as an active contributor of inflammation and autoimmunity. Further studies are needed to confirm and specify the effects and pathogenic links within AT. Novel insights in mechanisms regulating the diversity of AT composition and functions may assist conversion of AT into a teammate of anti-rheumatic processes. 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.

Background In patients with rheumatoid arthritis (RA), osteolysis, osteopenia and osteoporosis are predictive of a severe disease phenotype. CCL3 is a chemokine associated with inflammatory osteolytic lesions and bone resorption by osteoclasts in patients with multiple myeloma. CCL3’s role in modulating bone destruction during inflammatory arthritis has not been established. The purpose of this study was to assess pharmacological blockade of CCL3 as a strategy to prevent or inhibit osteoclast-associated bone damage during inflammatory arthritis. Method Fully differentiated osteoclasts were formed from human CD14-positive mononuclear cell precursors cultured with macrophage-colony stimulating factor (m-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Collagen-induced arthritis (CIA) was selected for in vivo studies. Antibodies (anti-CCL3 and isotype control) were tested for their potency to modulate; (i) osteoclast differentiation and resorption of bone substrate in vitro and (ii) arthritis severity, synovial inflammation by histology and bone erosion by x-ray. Results The concentration of CCL3 in supernatants increased in a time-dependent manner over the 14-day osteoclast culture period (p Conclusion Targeted inhibition of CCL3 protect the synovial joint against arthritis-associated pathology by potent inhibition osteoclast formation and bone erosion. Our data may help guide future innovations for the diagnosis and treatment of inflammatory arthritis.

Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including increased regulatory T cells (Tregs), prevention of auto-immune disease and ability to reduce growth of human cancer cells. We have previously shown in large cohorts that elevated serum mannose levels are associated with future development of type 2 diabetes (T2D) and cardiovascular disease. However, potential direct effects of mannose on insulin sensitivity in vivo or in vitro are unknown. We here show that administration of mannose (0.1 g/kg BW twice daily) for one week in man did not elicit negative effects on meal-modified glucose tolerance, markers of inflammation or insulin levels. Tregs number and insulin signaling in human liver cells were unchanged. These data suggest that mannose is a marker, and not a mediator, of insulin resistance. To verify this, we examined serum mannose levels during long-term euglycemic hyperinsulinemic clamps in non-diabetic and T2D individuals. Mannose was reduced by insulin infusion in proportion to whole-body insulin sensitivity. Thus, mannose is a biomarker of insulin resistance which may be useful for the early identification of diabetic individuals with insulin resistance and increased risk of its complications.

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.

Fibromyalgia (FM) is characterized by generalized chronic pain and reduced pain thresholds. Disturbed neuroendocrine function and impairment of growth hormone/insulin-like growth factor-1 is common. However, the pathophysiology of FM is not clear. MicroRNAs are important regulatory factors reflecting interface of genes and environment. Our aim was to identify characteristic microRNAs in FM and relations of specific microRNAs with characteristic symptoms. A total of 374 circulating microRNAs were measured in women with FM (n = 20; median 52.5 years) and healthy women (n = 20; 52.5 years) by quantitative PCR. Pain thresholds were examined by algometry. Pain [fibromyalgia impact questionnaire (FIQ) pain] levels were rated (0-100 mm) using FIQ. Fatigue (FIQ fatigue) was rated (0-100 mm) using FIQ and multidimensional fatigue inventory general fatigue. Sleep quantity and quality (1-4) rated from satisfactory to nonsatisfactory. Higher scores indicate more severe symptoms. Eight microRNAs differed significantly between FM and healthy women. Seven microRNAs, miR-103a-3p, miR-107, let-7a-5p, miR-30b-5p, miR-151a-5p, miR-142-3p and miR-374b-5p, were lower in FM. However, levels of miR-320a were higher in FM. MiR-103a-3p correlated with pain (r = 0.530, p = 0.016) and sleep quantity (r = 0.593, p = 0.006) in FM. MiR-320a correlated inversely with pain (r = -0.468, p = 0.037). MiR-374b-5p correlated inversely with pain threshold (r = -0.612, p = 0.004). MiR-30b-5p correlated with sleep quantity (r = 0.509, p = 0.022), and let-7a-5p was associated with sleep symptoms. When adjusted for body mass index, the correlation of sleep quantity with miR-103a and miR-30b was no longer significant. To our knowledge, this is the first study of circulating microRNAs in FM. Levels of several microRNAs differed significantly in FM compared to healthy women. Three microRNAs were associated with pain or pain threshold in FM.

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.

OBJECTIVES: Primary Sjogren's syndrome (pSS) is an autoimmune disease affecting the exocrine glands and internal organs including the central nervous system (CNS). The fms-related tyrosine kinase 3 ligand (Flt3L) is a maturation factor essential for brain homeostasis. Blood levels of Flt3L are increased in inflammatory diseases including the inflamed salivary glands in pSS. The present study evaluated the role of Flt3L in the CNS of patients with pSS and in two non-autoimmune conditions, fibromyalgia (FM) and Alzheimer's disease (AD). METHOD: Levels of Flt3L were measured in cerebrospinal fluid (CSF) and serum of patients with pSS (n = 15), FM (n = 29), and AD (n = 39) and related to CNS symptoms and to markers of inflammation and degeneration. RESULTS: Levels of CSF Flt3L in pSS and AD were significantly lower than in FM (p = 0.005 and p = 0.0003, respectively). Flt3L in pSS correlated to tau proteins [total tau (T-tau), r = 0.679; phosphorylated tau (P-tau), r = 0.646] and to a marker for microglia activation, monocyte chemoattractant protein 1 (MCP-1). Similar correlations were present in FM and AD patients. One-third of pSS patients had low levels of CSF Flt3L. This group had decreased levels of amyloid precursor protein metabolites (Aβ40 and Aβ42) in CSF, which was not seen in FM patients. CONCLUSIONS: This study shows a strong correlation between CSF Flt3L and tau proteins in pSS patients suggesting ongoing degradation/remodelling in the CNS. In pSS patients, low levels of Flt3L were linked to changes in amyloid turnover and may represent processes similar to those in AD.

The aim of the study was to evaluate long-term clinical and immunological effects of anti-B cell treatment in patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis refractory to conventional immunosuppressive treatment. Rituximab (RTX) was added to the ongoing immunosuppressive treatment in 29 patients with refractory ANCA-associated vasculitis. The disease activity was measured using Birmingham Vasculitis Activity Score/Wegener’s granulomatosis (BVAS/WG score), and clinical laboratory variables were recorded. The median BVAS/WG score before treatment was 6 (IQR 3–8), and 28 patients (97%) had disease flare classified either severe (62%) or limited (34%). Six of 29 patients (21%) achieved a complete remission, and 12 (41%) had a treatment response with ≥50% decrease in BVAS/WG score at 6 months. Fourteen patients (64%) with kidney involvement achieved remission, and in seven patients (50%), no flare was seen during the follow-up period. Three patients had renal flare and were successfully re-treated with RTX. Seventeen patients had disease symptoms from airways and eyes at RTX initiation, whereas only 29% displayed ≥50% treatment response. Limited clinical improvement was seen in patients with endobronchial lesions and trachea-subglottic granulomatous disease. RTX is a potent therapeutic option for ANCA-associated vasculitis refractory to conventional treatment. Best response may be expected in patients with vasculitic manifestations.

The presence of autoantibodies against C-reactive protein (anti-CRP) has been reported in association with autoimmunity and histopathology in chronic hepatitis C virus (HCV) infection. Resistin could play a role in the pathogenesis of hepatitis, although results on HCV infection are ambiguous. Here we retrospectively analyzed anti-CRP and resistin levels in the sera of 38 untreated and well-characterized HCV patients at the time of their first liver biopsy. HCV activity and general health were assessed by a physician at least yearly until follow-up ended. Anti-CRP and resistin were also measured in patients with autoimmune hepatitis (AIH) and nonalcoholic fatty liver disease (NAFLD). Anti-CRP antibodies were registered in all HCV patients, whereas only a few AIH (11%) and NAFLD (12%) sera were positive. Anti-CRP levels were related to histopathological severity and were highest in patients with cirrhosis at baseline. Resistin levels were similar in HCV, AIH, and NAFLD patients, but high levels of resistin were associated with early mortality in HCV patients. Neither anti-CRP nor resistin predicted a response to interferon-based therapy or cirrhosis development or was associated with liver-related mortality. We conclude that anti-CRP antibodies are frequently observed in chronic HCV infection and could be a useful marker of advanced fibrosis and portal inflammation. Funding Agencies|Swedish Society for Medical Research||Professor Nanna Svartz Foundation||King Gustaf V 80-Year Foundation||Sweden-America Foundation||County Council of Ostergotland||Clas Groschinsky||byggmastare Olle Engkvist||apotekare Hedberg

The neuroendocrine impact on rheumatoid arthritis is not yet fully described although numerous neurotransmitters are shown to act as inflammatory modulators. One of these is the excitatory transmitter glutamate (Glu). In this study, the influence of the Glu receptor (GluR)-mediated effects on collagen-induced arthritis (CIA) was investigated. CIA was induced in DBA/1 mice by immunization with chicken collagen type II (CII). Mice were exposed to the following GluR antagonists: group 1, the N-methyl-D-aspartic acid (NMDA) receptor channel blocker memantine; group 2, the metabotropic GluR antagonist AIDA, and group 3, the excitatory amino acid receptor antagonist kynurenic acid (KA). Arthritis was evaluated clinically and histologically and compared to PBS-treated controls. The effects of treatment on T cell populations and the levels of anti-CII and anti-citrullinated peptide antibodies were evaluated. Memantine treatment significantly improved the course of CIA, reducing synovitis (p = 0.007) and the frequency of erosions (p = 0.007). Memantine treatment up-regulated the expression of Foxp3 in spleen CD4+ T cells followed by an increase in CD4+CD25+ regulatory T cells. The other GluR antagonists, AIDA and KA, had no effect on CIA. These results demonstrate that blockade of the NMDA receptor channel with memantine delays and attenuates the development of arthritis, probably by promoting the development of regulatory T lymphocytes.

Resistin is a cysteine-rich protein, which is abundantly expressed at the site of inflammation, and acts as a regulator of the NF-kB-dependent cytokine cascade. The aim of this study was to evaluat ...

Objective. Human resistin has proinflammatory properties that activate NF-kappa B-dependent pathways, whereas its murine counterpart is associated with insulin resistance. The aim of this study was to examine potential cross-talk between resistin and insulin/insulin-like growth factor (IGF) signaling in rheumatoid arthritis (RA). Methods. Levels of IGF-1, IGF binding protein 3, and resistin were measured in the blood and synovial fluid of 60 patients with RA and 39 healthy control subjects. Human RA synovium was implanted subcutaneously into SCID mice, and the mice were treated with resistin-targeting small interfering RNA. Primary synovial fibroblasts from patients with RA, as well as those from patients with osteoarthritis, and the human fibroblast cell line MRC-5 were stimulated with resistin. Changes in the IGF-1 receptor (IGF-1R) signaling pathway were evaluated using histologic analysis, immunohistochemistry, and reverse transcription-polymerase chain reaction. Results. Resistin and IGF-1R showed different expression profiles in RA synovia. Low levels of IGF-1 in RA synovial fluid were associated with systemic inflammation and inversely related to the levels of resistin. Stimulation of synovial fibroblasts with resistin induced phosphorylation of IGF-1R to a degree similar to that with insulin, and also induced phosphorylation of transcription factor Akt. This was followed by gene expression of GLUT1, IRS1, GSK3B, and the Akt inhibitors PTPN and PTEN. Abrogation of resistin expression in vivo reduced the expression of IGF-1R, the phosphorylation of Akt, and the expression of PTPN and PTEN messenger RNA in RA synovium implanted into SCID mice. Conclusion. Resistin utilizes the IGF-1R pathway in RA synovia. Abrogation of resistin synthesis in the RA synovium in vivo leads to reductions in the expression of IGF-1R and level of phosphorylation of Akt. (Less)

BACKGROUND Efficient host defense mechanisms are crucial for survival in sepsis and septic arthritis. S100 proteins are reported to have proinflammatory and bactericidal properties. The aim of this study was to investigate the role of S100A4 in staphylococcal arthritis. METHODS S100A4 knockout mice (S100A4KO) and wild-type counterparts (WT) were intravenously and intra-articularly challenged with Staphylococcus aureus strain LS-1. Clinical and morphological signs of arthritis and sepsis, phagocytosis, bone mineral density (BMD), and bone metabolism were then monitored in S100A4 and WT mice. RESULTS S100A4KO mice had a lower bacterial load in the kidneys than WT mice (P < .05) but developed more severe clinical signs of arthritis (P < .001) and had higher levels of interleukin 6 and L-selectin (P = .002). S100A4KO mice had fewer morphological signs of synovitis and cartilage/bone destruction following intra-articular instillation of bacteria. S100A4KO mice were protected from loss of BMD and had lower levels of RANKL, MMP3, and MMP9 (P < .05). S100A4 was not bactericidal in vitro. CONCLUSIONS In staphylococcal infection, S100A4 regulates bacterial clearance as well as systemic and local inflammatory responses.

RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.