Minna Turkkila

Survivin in autoimmune diseases

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.

Suppressed diversity of survivin splicing in active rheumatoid arthritis

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

Survivin controls biogenesis of microRNA in smokers: A link to pathogenesis of rheumatoid arthritis

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

IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis

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.

Survivin improves the early recognition of rheumatoid arthritis among patients with arthralgia: A population-based study within two university cities of Sweden

OBJECTIVES The aim of this study was to validate the use of survivin for preclinical recognition of rheumatoid arthritis (RA) among patients with unexplained arthralgia. METHODS Serum levels of survivin and the arthritis-specific autoantibodies RF and ACPA were measured in total of 5046 patients with musculoskeletal complains during 12 consecutive months in Gothenburg and in Umeå. Among them, 303 arthralgia patients were identified and prospectively followed. RESULTS After 48 months, 12.2% of the arthralgia patients developed RA. Most of RA cases had high serum survivin, which increased the relative risk for RA (RR = 5.90, p = 3 × 10-7). Combination of survivin with autoantibodies was present in only 4.6% of the arthralgia patients and increased further the risk of RA and shortened time to RA development. Presence of any single autoantibody in the survivin-negative patients was associated with a minor risk for RA and had RA-free survival similar to the reference group. CONCLUSION This study shows that measurement of survivin in serum improves estimation of RA risk and prospectively predicts RA development in patients with arthralgia. Survivin may indicate a phase preceding autoantibody production.

Survivin Measurement improves Clinical Prediction of Transition From Arthralgia to RA—Biomarkers to Improve Clinical Sensitivity of Transition From Arthralgia to RA

Background: Arthralgia often predates development of rheumatoid arthritis (RA). A set of joint symptoms commonly found in patients during their transition from arthralgia to RA, has been recently proposed. Aim: To combine clinical and serological markers and to improve recognition of imminent rheumatoid arthritis (RA) among patients with arthralgia. Methods: The total of 1,743 first-visit patients attending the rheumatology ward in Gothenburg for joint symptoms were identified during 12 consecutive months. Among those, 63 patients were classified as RA, 73 had undifferentiated arthritis and 180 had unexplained arthralgia. New RA cases, which prospectively developed during 48 months, comprised the preclinical (pre) RA group. The joint symptoms of the first-visit were analyzed aiming to distinguish patients with arthralgia and arthritis, and patients with pre-RA, who later developed the disease. The receiver operating characteristics curves were constructed. In the model, symptoms with the odds ratio >2.0 between the arthralgia and pre-RA were combined with information about RA-specific antibodies, C-reactive protein (CRP), and survivin in serum. Results: The proposed set of clinical symptoms distinguished the arthralgia patients from RA and pre-RA. Presence of survivin in serum showed strong association with clinical joint symptoms in arthralgia. A combination of symptoms in several small joint areas, increasing number of joints with symptoms, and patients experience of swelling in small hand joints at the first visit identified pre-RA cases with 93% specificity. Grouping those symptoms with information about survivin, RA-specific antibodies, and CRP (or gender) in the final algorithm achieved 91% specificity and 55.2% of positive prediction for transition from arthralgia to RA. Conclusion: Clinical and serological parameters in combination aid recognition of imminent RA among arthralgia patients with appropriate sensitivity.

SAT0033 Smoking contributes to exhausted state of CD4+ T cells in rheumatoid arthritis

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. Wasén C, et al. J Autoimmun 2017. DOI: 10.1016/j.jaut.2016.12.00. Crawford A, et al. Immunity 2014. 40(2): 289–302. Lübbers J, et al. Ann Rheum Dis 2013. 72(5): 776–780. Disclosure of Interest None declared

SAT0075 Validation of The Rheumatoid Arthritis Prediction Rules for The Disease Development in Patients with Joint Pain from West Sweden

Background Rheumatoid arthritis (RA) is a joint destructive disease, which leads to physical disability. There is no cure of RA today. Thus, recognition of RA at preclinical stages is essential for successful treatment. Objectives To validate and improve known prediction rules aiming at early recognition of clinically suspect arthralgia and diagnosis of RA. Methods Medical records of 241 first-time patients with clinically suspect joint pain were evaluated for RA diagnosis by the 2010 EULAR/ACR criteria (1) and by the RA prediction rules (13-points, Amsterdam (2)); and 16-points clinical assessment (Leiden (3)) and prospectively followed during 29–47 months. All the patients were a part of the inception cohort of 5455 patients whose samples were tested for RF and/or ACPA during 12 consecutive months at the Laboratory of Clinical Immunology at the University Hospital of Gothenburg. Patients with known diagnosis of RA, other rheumatic disease and chronic pain conditions were excluded. The utility of arthritis-specific autoantibody status (ACPA and/or RF) and the family history for RA prediction were analyzed. Results The RA criteria applied to the records of the first visit identified 24% of patients as RA (EULAR score mean 7.6), 30% as undifferentiated arthritis (UA, EULAR score mean 3.6), while the remaining 45% of the patients were characterized as arthralgia (EULAR score mean 2.3). The 13-point and 16-point prediction rules allocated RA patients to the high-risk group with comparable sensitivity of 78% and 91%. The significant correlation between the two models was present in UA (r=0.398; p=0.0005) and arthralgia (r=0.411; p<0.0001) groups, but not in RA (r=0.0007, ns). Specificity for RA diagnosis at the first visit was higher in the 13-point rule (90.5%), however it was not discriminative between the arthralgia and UA groups. The clinical 16-point assessment discriminated successfully between RA/UA and between UA/arthralgia groups. The 16-point assessment allocated 50% of patients with UA to the high-risk group, which affected specificity. During the follow-up period, 23 cases of new RA and 5 UA were registered. The sensitivity of the 16-point assessment at the first visit was 60%, similar for the antibody-positive and for antibody–negative cases. The sensitivity of the 13-point rule was 21% (p=0.003), and was selective for the antibody-positive cases. At the first visit, subjects with family history of RA were over-represented in the arthralgia group (p=0.022). At follow-up only 1 of them developed UA. Conclusions Both prediction rules are shown to be sensitive identify RA patients at assessment. The clinical 16-point assessment has prospective sensitivity for new cases. This is shown independently of autoantibody status and family history of RA. References Cader MZ et al. Performance of the 2010 ACR/EULAR criteria for rheumatoid arthritis: comparison with 1987 ACR criteria in a very early synovitis cohortAnn Rheum Dis. 2011;70:949–55. van de Stadt LA et al. A prediction rule for the development of arthritis in seropositive arthralgia patients. Ann Rheum Dis. 2013;72:1920–6. Krabben A et al. Risk of RA development in patients with unclassified arthritis according to the 2010 ACR/EULAR criteria for RA. Rheumatology (Oxford). 2013;52:1265–70. Disclosure of Interest None declared

SAT0076 Utility of Survivin Measurements for Early Recognition of Clinically Suspect Arthralgia and Diagnosis of Rheumatoid Arthritis in West Sweden

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

FRI0044 Smoking Induces Irreversible Deregulation of Microrna Expression in Rheumatoid Arthritis

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