Anna Radziszewska

MicroRNA 497 modulates interleukin 1 signalling via the MAPK/ERK pathway

The MAPK/ERK signalling pathway has been described to mediate IL‐1 induction of target genes and is known to be regulated by microRNAs (miRNA). We describe a novel miRNA regulating the expression of the MEK1 gene and how it impacts IL‐1 induced IL‐6 transcription. miR‐497 was predicted to target MEK1 3′UTR using bioinformatic tools. Transfection of miR‐497 into HeLa cells inhibited MEK1 protein expression by 50%. In transient transfection experiments, the luciferase activity of a MEK1 3′UTR luciferase reporter construct was reduced in the presence of miR‐497, and mutation of the predicted miR‐497 binding site restored activity. miR‐497 also decreased protein levels of RAF1 and ERK1 but not ERK2. Addition of miR‐497 was further shown to inhibit IL‐1 induced IL‐6 gene transcription.

Oxidation of β2-glycoprotein I associates with IgG antibodies to domain I in patients with antiphospholipid syndrome

Domain I (DI) of beta-2-glycoprotein I (β2GPI) contains the immunodominant epitope for pathogenic antiphospholipid antibodies (aPL). DI is exposed in the linear form of the molecule but not in the circular form that comprises 90% of serum β2GPI. The majority of circulating β2GPI is biochemically reduced with two free thiols in Domain V. However, increased levels of oxidised β2GPI are found in patients with antiphospholipid syndrome (APS). It is not known whether oxidation of β2GPI favours the linear form of the molecule and thus promotes development of anti-DI antibodies. We investigated whether the proportion of oxidised β2GPI associates with the presence of anti-DI in APS patients. Serum samples from 44 APS patients were screened for IgG, IgM and IgA anti-DI, anti-β2GPI, anti-cardiolipin (anti-CL) and biochemically reduced β2GPI. A negative correlation was found between the proportion of β2GPI in the biochemically reduced form and IgG anti-DI levels (r = -0.54, p = 0.0002), but not with IgM or IgA anti-DI. Moreover, the proportion of β2GPI in the reduced form was lower in IgG anti-DI positive than anti-DI negative APS patients (p = 0.02). The relative amount of reduced β2GPI was no different between patients who were positive or negative for IgG, IgM and IgA anti-β2GPI or anti-CL. This study demonstrates that oxidised β2GPI lacking free cysteine-thiol groups most closely associates with IgG anti-DI positivity compared to IgG anti-CL and anti-β2GPI. Future studies are required to ascertain the directionality of this association to define causation.

OP0084 Oxidation of β2-Glycoprotein I is Strongly Associated with the Presence of Anti-Domain I Antibodies in Patients With Antiphospholipid Syndrome

Background Beta-2 glycoprotein I (β2GPI) represents the major antigenic target for antiphospholipid antibodies (aPL), the hallmark of antiphospholipid syndrome (APS). β2GPI contains five homologous domains, with domain I (DI) being identified as the main antigenic epitope for pathogenic anti-β2GPI (aβ2GPI). It has been reported that APS patients show an elevated amount of total β2GPI as compared with healthy donors and other autoimmune disease control groups. Moreover, in healthy individuals, β2GPI mainly exists in its biochemically reduced form whilst the post-translational oxidized structure (fish-hook linear configuration) is elevated in patients with APS. Antibodies targeting β2GPI-DI (aDI) represent a key pathogenic sub-population of aPL, thus their detection may allow the identification of patients at highest clinical risk. Objectives Our hypothesis is that the different redox state of β2GPI, from reduced (circular) to oxidised (linear), might be responsible for the exposure of DI, thus providing the antigenic drive for the aDI antibodies. However, no study has investigated the association between the circulating oxidized form of β2GPI and the presence of aDI in APS patients. Methods Serum samples from 40 patients who fulfilled the classification criteria for APS were tested (34 primary and 6 secondary APS). A sandwich ELISA for quantifying total β2GPI levels within serum was performed for all samples. An in-house standard (consisting of pooled serum from 10 healthy controls) was used to obtain a standard curve in each ELISA. Biochemically reduced β2GPI was detected via labelling free thiols within serum proteins with a biotinylated free-thiol binding reagent and then, via coating on a streptavidin plate, detecting the presence of labelled β2GPI with an aβ2GPI antibody, based on published methods. Then samples were screened for IgG aDI, aβ2GPI and anti-cardiolipin (aCL). Results The positivity for aDI, aβ2GPI and aCL IgG was 60%, 75% and 80% respectively. The relative proportion of reduced β2GPI was expressed as a percentage of that observed in the in house-standard after correction for the total amount of β2GPI. A significant negative correlation was observed between proportion of reduced β2GPI and aDI levels (Spearman coefficient r = -0.409, p=0.008) and aCL titre (r = -0.317, p=0.045). There was no significant correlation between proportion of reduced β2GPI and aβ2GPI (r = -0.059, p=0.713). Conclusions The strong association between the oxidative state of β2GPI and aDI positivity supports the hypothesis that modulation of the redox state of β2GPI affects the production of antibodies against the DI epitope, which become exposed in the oxidised protein. However, it remains to be proven if the exposure of DI within the oxidised (hence linearized) form of β2GPI acts as the antigenic drive for development of aDI autoantibodies or whether the presence of these antibodies stabilizes the antigen-antibody complex maintaining the linear configuration of the molecule. Disclosure of Interest None declared

Antibodies to Cyclic Citrullinated Peptides in Patients With Juvenile Idiopathic Arthritis and Patients With Rheumatoid Arthritis: Shared Expression of the Inherently Autoreactive 9G4 Idiotype

Antibodies to cyclic citrullinated peptides (anti‐CCP) in rheumatoid arthritis (RA) can express the inherently autoreactive gene VH4–34, detected using the rat monoclonal antibody 9G4. Patients with the polyarticular subtype of juvenile idiopathic arthritis (JIA) share some but not all of the features of adult patients with RA. This study was undertaken to compare serologic findings for rheumatoid factor (RF), anti‐CCP, and 9G4‐expressing anti‐CCP in a large JIA cohort with a cohort of adult RA patients.

Antibodies to citrullinated peptides in patients with juvenile idiopathic arthritis and rheumatoid arthritis: Shared expression of the inherently autoreactive 9G4 idiotype

Antibodies to cyclic citrullinated peptides (anti‐CCP) in rheumatoid arthritis (RA) can express the inherently autoreactive gene VH4–34, detected using the rat monoclonal antibody 9G4. Patients with the polyarticular subtype of juvenile idiopathic arthritis (JIA) share some but not all of the features of adult patients with RA. This study was undertaken to compare serologic findings for rheumatoid factor (RF), anti‐CCP, and 9G4‐expressing anti‐CCP in a large JIA cohort with a cohort of adult RA patients.

CD19+CD24hiCD38hi B Cells Are Expanded in Juvenile Dermatomyositis and Exhibit a Pro-Inflammatory Phenotype After Activation Through Toll-Like Receptor 7 and Interferon-α

Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of the CD40–CD40L pathway (known to induce B cell IL-10 and IL-6) revealed similar expression of IL-10 and IL-6 in B cells cultured with CD40L from both JDM patients and controls. In conclusion, JDM patients with active disease have a significantly expanded immature transitional B cell population which correlated with the type I IFN signature. Activation through TLR7 and IFNα may drive the expansion of immature transitional B cells in JDM and skew the cells toward a pro-inflammatory phenotype.

THU0484 Ro60 expression decreases with age in peripheral blood mononuclear cells of children and adolescents and correlates with tlr7 stimulation in pdcs of prepubertal children

Background Auto-antibodies to the RNA binding protein Ro60 are present in patients with autoimmune disorders such as systemic lupus erythematosus (SLE). In addition to its established role as an auto-antigen, Ro60 has been found to bind Alu RNA retroelements whereby it may target Alu retroelement RNA for degradation suggesting a novel putative function of this auto-antigen. If Ro60 modulates the amount of cellular RNA then one may hypothesise an association with toll-like receptor (TLR) 7 stimulation threshold. Objectives To measure the physiological levels of intracellular Ro60 protein in healthy children and adolescents and investigate a possible link between Ro60 protein expression and interferon-α (IFN-α) production after TLR7 stimulation. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from blood from 48 healthy children and adolescents (age range 6.7–17.9 years old). Cell lysates from thawed PBMC samples were tested for Ro60 expression by Western blot. PBMCs were also stimulated for 20 hours with TLR7/8 agonist R848, at 1ug/ml in the presence of brefeldin A, and plasmacytoid dendritic cell (pDC) IFN-α expression was measured using flow cytometry. Statistical tests to measure correlation between IFN-α expression in (pDCs) and PBMC Ro60 expression were performed using SPSS. Results Ro60 expression in PBMCs correlated negatively with age (Spearmans rho = -0.317, p=0.032). When participants were divided into two groups based on their self-reported puberty status, Ro60 expression was higher in the pre-pubertal group (p=0.02). There was, however, no difference in Ro60 expression between males and females (p=0.44) or when sexes were stratified according to pubertal status. pDC IFN-α production after TLR 7 stimulation, did not correlate with ex vivo PBMC Ro60 expression overall (Spearmans rho =0.159, p=0.302) however a moderate positive correlation was observed in pre-pubertal samples only (Spearmans rho =0.554, p=0.021). Conclusions Ro60 expression decreases with age in healthy young people. These findings need to be confirmed in a larger cohort and further studies are necessary to investigate the link between Ro60 expression and TLR7 signalling across different age groups as well as in patients with SLE. Disclosure of Interest None declared

THU0217 DKK-1 Levels Are Elevated in Patients with Enthesitis Related Arthritis without Sacroiliac Joint Fusion

Background Dkk-1 is an inhibitor of the Wnt signalling pathway and therefore plays an important role in bone remodelling. Studies in adults with ankylosing spondylitis have described lower levels of Dkk-1 compared to healthy controls although this is not consistent. Higher levels have been found in some cohorts and particularly in patients with no syndesmophyte formation, suggesting that Dkk-1 may be an important factor in bony ankylosis. There has been no description of Dkk-1 in a younger population with enthesitis related arthritis (ERA). Objectives To test serum Dkk-1 levels in adolescent and young adult patients with ERA and compare between different disease phenotypes and healthy controls. Methods Patients were recruited from the adolescent and young adult clinics at University College London Hospital. Serum samples were stored at -80°C until use. Dkk-1 levels were measured by ELISA (Quantikine, R&D Systems, Minneapolis, MN) as per manufacturers instructions, in duplicate and correlated with clinical features and MRI results. Results Serum from 78 patients with ERA (median age 17 years) and 20 age and gender matched healthy controls was tested. Dkk-1 levels were significantly higher in patients with ERA (median=2971pg/ml, IQR 2258–3511pg/ml) compared to healthy controls (median=1806pg/ml, IQR 1307–2950pg/ml, p=0.0014) (Figure 1A). Patients with ERA who were HLA-B27 positive had higher Dkk-1 levels (median=3081pg/ml, IQR 2337–3680) than those who were HLA-B27 negative (median=2445pg/ml, IQR 1832–3365, p=0.0488) (Figure 1B). Interestingly, there was no significant difference in Dkk-1 levels between patients with axial and those with persistently peripheral ERA or between patients on TNF inhibitors versus those naïve to biologics. There was no correlation between Dkk-1 and CRP, ESR, patient age at date of sample or disease duration. Eleven patients with ERA of the 78 tested had already developed bony fusion of their sacroiliac joints (SIJs); in this group Dkk-1 levels were unexpectedly lower (median=2214pg/ml, IQR 2103–2618pg/ml) compared to those with ERA who had no evidence of bony fusion on MRI scan (median=3042pg/ml, IQR 2321–3607pg/ml, p=0.0355) (Figure 1C). Conclusions In our study, patients with ERA had significantly higher serum levels of Dkk-1 compared to controls. Levels were even higher in those patients who were HLA-B27 positive but there was no correlation with axial disease, patient age, disease duration, CRP, ESR or TNF inhibition treatment. Patients with existing bony fusion of their SIJs had lower levels of Dkk-1, despite the numbers being relatively small. Dkk-1 levels appear elevated in ERA but may reduce with the occurrence of bony fusion. Further larger and longitudinal studies are needed to confirm these findings and to correlate changes in Dkk-1 levels with treatment and MRI findings over time. Disclosure of Interest None declared