Sofia Töyrä Silfverswärd

Pathogenic Transdifferentiation of Th17 Cells Contribute to Perpetuation of Rheumatoid Arthritis during Anti-TNF Treatment.

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

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.

Smoking Functions as a Negative Regulator of IGF1 and Impairs Adipokine Network in Patients with Rheumatoid Arthritis

Objectives. Smoking is pathogenic for rheumatoid arthritis (RA) being tightly connected to the genetic and serological risk factors for this disease. This study aims to understand connections between cigarette smoking and serum levels of IGF1 and adipokines in RA. Methods. Serum levels of IGF1 and adipokines leptin, adiponectin, resistin, and visfatin were measured in two independent cohorts of RA patients from Gothenburg (n = 350) and Leiden (n = 193). An association of these parameters with smoking was tested in a direct comparison and proved by bivariate correlation analysis. The obtained associations were further tested in multivariate regression models where the confounders (age, gender, disease duration, and BMI) were controlled. Results. The smokers had significantly lower serum levels of IGF1, adiponectin, and leptin compared to never smokers. In regression analysis, smoking and low leptin, but not adiponectin, were associated and predicted low IGF1. Additionally, high disease activity and high BMI increased the probability of low leptin. Conclusions. The study indicates cigarette smoking as an important cause of a relative IGF1 and leptin deficiency in RA patients. This novel association between smoking and hypoleptinemia may be of importance for long-term prognosis of RA and for prediction of comorbidities.

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.

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.

High Expression of STAT3 in Subcutaneous Adipose Tissue Associates with Cardiovascular Risk in Women with Rheumatoid Arthritis

Despite the predominance of female patients and uncommon obesity, rheumatoid arthritis (RA) is tightly connected to increased cardiovascular morbidity. The aim of this study was to investigate transcriptional activity in the subcutaneous white adipose tissue (WAT) with respect to this disproportionate cardiovascular risk (CVR) in RA. CVR was estimated in 182 female patients, using the modified Systematic Coronary Risk Evaluation scale, and identified 93 patients with increased CVR. The overall transcriptional activity in WAT was significantly higher in patients with CVR and was presented by higher serum levels of WAT products leptin, resistin and IL-6 (all, p < 0.001). CVR was associated with high WAT-specific transcription of the signal transducer and activator of transcription 3 (STAT3) and the nuclear factor NF-kappa-B p65 subunit (RELA), and with high transcription of serine-threonine kinase B (AKT1) in leukocytes. These findings suggest Interleukin 6 (IL-6) and leptin take part in WAT-specific activation of STAT3. The binary logistic regression analysis confirmed an independent association of CVR with IL-6 in serum, and with STAT3 in WAT. The study shows an association of CVR with transcriptional activity in WAT in female RA patients. It also emphasizes the importance of STAT3 regulatory circuits for WAT-related CVR in RA.

01.13 Igf1R signalling contributes to T cell dependent inflammation in rheumatoid arthritis

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.