Caroline Wasén

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.

Downregulation of toll-like receptor 4 and IL-6 following irradiation of the rat urinary bladder.

The pathophysiology behind radiation cystitis is poorly understood. Here we investigated whether bladder irradiation affects the immune system of the rat urinary bladder. Female rats were sedated and exposed to one single radiation dose of 20 Gy or only sedated (controls) and killed 16 h to 14 days later. Rats were placed in a metabolic cage at 16 h, 3 days, 7 days and 14 days following bladder irradiation. The urinary bladders were harvested and analysed with qPCR, immunohistochemistry and/or Western blot for the expression of interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, nitric oxide synthases (eNOS, iNOS and nNOS), tumour necrosis factor (TNF)‐α and toll‐like receptor 4 (TLR4). Urine was collected and analysed for IL‐6 and nitrite (reflecting nitric oxide activity) with ELISA and the Griess reaction, respectively. Irradiation increased bladder frequency and decreased voiding volumes 14 days following bladder irradiation. Bladder irradiation increased the expression of IL‐10 and collagen in the bladder, while TLR4 and IL‐6 expressions were decreased in the urothelium concomitantly with a decrease in mast cells in the submucosa and urine levels of IL‐6 and nitrite. The present findings show that bladder irradiation leads to urodynamic changes in the bladder and may suppress important immunoregulatory pathways in the urinary bladder.

Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination.

Significance The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development. Recent reports have shown that Flt3 is reexpressed on peripheral B cells during activation, however the function for Flt3 during B-cell activation is poorly understood. The present study describes a role for Flt3 during B-cell activation and antibody production. We show that Flt3 is reexpressed on activated germinal center B cells and that lack of functional Flt3 signaling in peripheral B cells results in a specific deficiency in production of the antibody subclass IgG1. Mechanistic studies indicate that Flt3 synergizes with the IL-4 receptor to promote proper activation of Stat6 and class switch to IgG1. This study provides evidence for a previously unknown function for Flt3 during peripheral B-cell activation. Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6+ germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

Smoking Is Associated With Low Levels of Soluble PD-L1 in Rheumatoid Arthritis

Background Smoking is a risk factor for developing rheumatoid arthritis (RA), but the mechanism remains uncertain. We previously demonstrated that smoking lowers the T cell activation threshold by limiting programmed death protein 1 (PD-1) expression. Aim To investigate how smoking influence the levels of soluble PD-1 ligand (sPD-L1). Method Serum levels of sPD-L1 were measured in 246 RA patients and in 168 healthy subjects. The analysis was done with respect to inflammation, smoking, treatments, and autoantibody status. The effect of therapeutic TNF-inhibiting antibodies (TNFi) on sPD-L1 was studied in 16 RA patients at their first infliximab infusion. The expression of Fcγ-receptor (FcγR) subclass IIB and IIIA was analyzed with quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from 12 RA patients and 15 healthy controls, and in healthy PBMC exposed to IgG containing antibodies to cyclic citrullinated peptides (aCCP). Results The negative association between smoking and sPD-L1 in RA patients was established by multiple logistic regression (OR = 0.52, p = 0.038). Other covariates in the regression model were serum levels of IL-1β representing inflammation (OR = 1.6, p = 0.0076) and aCCP positivity (OR = 1.9, p = 0.047). First infliximab infusion repressed sPD-L1 (p = 0.023) in patients, and low levels of sPD-L1 were found in patients with early RA treated with TNFi (p = 0.018). Treatment with TNFi was associated with higher sPD-L1 in patients with long disease duration (p = 0.041) and restored levels in smokers. In vitro exposure to aCCP+ IgG suppressed sPD-L1 (p = 0.036), but aCCP+ patients with long disease duration had higher sPD-L1 (p = 0.016). High ratio of the inhibitory FcγR subclass IIB over the stimulatory IIIA resulted in low sPD-L1 release (p = 0.029). Smoking was associated with a higher FcγR IIB/IIIA ratio (p = 0.00062) and lower levels of sPD-L1 (p = 0.013). Conclusion In RA, serum sPD-L1 was related to systemic inflammation and aCCP positivity. Smoking altered the expression of FcγRs and limited sPD-L1 in RA patients, permitting inappropriate T cell responses. Differential regulation of sPD-L1 during the early and late RA may indicate transposition from acute to chronic inflammation.

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

AB0084 Nicotine Causes Enrichment of Survivin in Serum by Activating Cytotoxic CD8+ T Cells in Experimental Arthritis and Patients with Rheumatoid Arthritis

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