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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 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

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

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.