Chi Wong

Histone deacetylases are dysregulated in rheumatoid arthritis and a novel histone deacetylase 3–selective inhibitor reduces interleukin‐6 production by peripheral blood mononuclear cells from rheumatoid arthritis patients

OBJECTIVE To characterize the role of histone deacetylase (HDAC) activity in rheumatoid arthritis (RA) and to evaluate the effects of MI192, a novel HDAC-3-selective inhibitor, compared with the established nonselective HDAC inhibitor trichostatin A (TSA), on proinflammatory cytokine production. METHODS Activity of HDAC and histone acetyltransferase was measured in peripheral blood mononuclear cells (PBMCs) from RA patients by spectrophotometric assay, prior to and after 12 weeks of etanercept therapy. The effects of HDAC inhibitor treatment on cytokine production in both RA and healthy PBMCs were assessed by enzyme-linked immunosorbent assay. RESULTS RA PBMCs exhibited significantly increased HDAC activity (P = 0.007) compared to PBMCs from healthy individuals, and the increase was unaltered after 12 weeks of etanercept therapy. TSA was a potent inhibitor of tumor necrosis factor (TNF) and interleukin-6 (IL-6) production in both RA and healthy PBMCs and of interferon-γ (IFNγ) production in healthy PBMCs; IFNγ was not produced by RA PBMCs. MI192 inhibited TNF production at high concentrations and dose-dependently inhibited IL-6 in RA PBMCs but not healthy PBMCs, across a dose range of 10 μM-5 nM. CONCLUSION HDAC activity is dysregulated in RA PBMCs and is a potential target for therapeutic intervention, as it is not affected by conventional anti-TNF treatment with etanercept. Both the selective and the nonselective HDAC inhibitors (MI192 and TSA, respectively) were found to regulate cytokine production from PBMCs, but their effects were cell type and compound specific. HDAC inhibitors have potential in the treatment of RA, and HDAC-selective inhibition may improve the therapeutic margin of safety; however, further clinical characterization and evaluation for adverse effects is needed.

Evidence of NLRP3-inflammasome activation in rheumatoid arthritis (RA); genetic variants within the NLRP3-inflammasome complex in relation to susceptibility to RA and response to anti-TNF treatment

Background The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). Methods Relative gene expression of NLRP3-inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6 months, with secondary expression quantitative trait loci (eQTL) analyses. Results At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. Conclusions This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.

TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes.

X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF.

Differential effects of infliximab on absolute circulating blood leucocyte counts of innate immune cells in early and late rheumatoid arthritis patients

Anti‐tumour necrosis factor (TNF) biologics have revolutionized therapy of rheumatoid arthritis (RA). We compared the effects of infliximab on numbers of circulating leucocyte subsets in early RA (disease/symptom duration of ≤1 year) and late RA patients (>1 year). A control group consisted of early RA patients treated with a combination of methotrexate (MTX) and methylprednisolone. Blood samples were obtained at baseline (pre‐therapy) from all RA patients, divided into three groups: (i) late RA receiving infliximab/MTX, (ii) early RA–infliximab/MTX, (iii) early RA–steroid/MTX, and also from follow‐up patients at 2 and 14 weeks. Significant differences in absolute counts of monocytes and granulocytes were observed between healthy controls and RA patients. At baseline CD14bright monocytes and CD16+ granulocytes were increased in both early RA and late RA patients. CD4+ T cells, CD8+ T cells and B cells were all increased at baseline in early RA, but not in late RA. At 2 weeks following infliximab treatment decreased granulocytes were observed in both early and late RA and decreased natural killer (NK) cells in late RA. CD16+ granulocytes and NK cells were also decreased at 14 weeks post‐infliximab in early RA. Biotinylated infliximab was used to detect membrane‐associated TNF (mTNF)‐expressing leucocytes in RA patients. CD16+ granulocytes, NK cells and CD14dim monocytes all expressed higher levels of mTNF in RA patients. In summary infliximab is associated with decreased CD16+ granulocyte and NK cell counts, possibly through binding of mTNF. Differential effects of infliximab between early and late RA suggest that pathogenic mechanisms change as disease progresses.

SAT0151 Imaging and Histopathological Changes To Tocilizumab in Patients with Moderate To Severe Ra: A Single Centre Randomized Double-Blind Placebo-Controlled Study

Background Tocilizumab (TCZ) is a well-established biologic therapy in the treatment of rheumatoid arthritis (RA). There is limited data on imaging and synovial tissue histology changes. Objectives To evaluate level of response as defined by power Doppler (PD) ultrasound (US) and synovial tissue histology changes. Methods Patients with RA, inefficacy to minimum one DMARD +/− TNFi, with DAS28≥3.2 and knee synovitis amenable to synovial biopsy were recruited to this open-label 48-week study. Patients randomised to TCZ/methotrexate (MTX) for 48 weeks or placebo (PBO)/MTX for the first 16 weeks followed by TCZ/MTX until week 48. Clinical and US hand, wrist and knee assessments with US-guided knee synovial biopsy at baseline (BL), weeks 12 and 48 (biopsy optional). US was scored accrording to OMERACT 0–3 grey scale and PD synovitis scoring system. Synovial tissue was assessed for synovial inflammatory infiltrate, stromal cell density, synovial lining on 0–3 scale and overall synovitis (0–9 scale) determined. Results 15 patients recruited: 12 (80%) females; 9 received TCZ/MTX, 6 PBO/MTX. 2 patients withdrew at each arm, one due to TCZ infusion reaction. Week 16: 33% (3/9) TCZ/MTX achieved DAS28ESR-rem vs 0 PBO+MTX, latter remaining in moderate/high disease activity. Week 48: 92% (12/13) whole group in DAS28ESR-rem. US response: 38% (3/8) TCZ/MTX group who had BL PD synovitis in hand/wrist had absence of PD synovitis at week 16 vs none PBO/MTX group. All patients with abnormal BL PD in knees (median (IQR) PD score of 2 (0–9)) had improved week 48 PD score (median (IQR) PD score of 0 (0–1)). 13 patients (8 TCZ/MTX, 5 PBO/MTX) had synovial biopsies obtained weeks 0 & 12; 21/26 samples (80%) samples useable. No difference between pre- and week 12 synovitis score in both groups. Median (IQR) total synovitis score at BL and after week 12 respectively of 3 (2.75, 4.25) and 3 (2.5–4) in the TCZ/MTX group vs 6 (4,7) and 6 (4,6) in PBO/MTX group. BL total synovitis score did not predict early or late response. Conclusions TCZ/MTX was associated with significant clinical and imaging improvement compared to MTX alone. An absence of change in synovial infiltrate with TCZ/MTX at 12 weeks suggests a different mechanism for response compared to other anti-cytokine therapies such as the TNFi. Further histochemistry analysis and investigation may potentially determine mechanism and indicators of response on a tissue level. Disclosure of Interest None declared

Inositol-Requiring Enzyme 1-Mediated Downregulation of MicroRNA (miR)-146a and miR-155 in Primary Dermal Fibroblasts across Three TNFRSF1A Mutations Results in Hyperresponsiveness to Lipopolysaccharide

Tumor necrosis factor (TNF)-receptor-associated periodic fever syndrome (TRAPS) is a rare monogenic autoinflammatory disorder characterized by mutations in the TNFRSF1A gene, causing TNF-receptor 1 (TNFR1) misfolding, increased cellular stress, activation of the unfolded protein response (UPR), and hyperresponsiveness to lipopolysaccharide (LPS). Both microRNA (miR)-146a and miR-155 provide negative feedback for LPS-toll-like receptor 2/4 signaling and cytokine production, through regulation of nuclear factor kappa B (NF-κB). In this study, we hypothesized that proinflammatory cytokine signaling in TRAPS downregulates these two miRs, resulting in LPS-induced hyperresponsiveness in TRAPS dermal fibroblasts (DFs), irrespective of the underlying genetic mutation. Primary DF were isolated from skin biopsies of TRAPS patients and healthy controls (HC). TNFR1 cell surface expression was measured using immunofluorescence. DF were stimulated with LPS, interleukin (IL)-1β, thapsigargin, or TNF, with and without inositol-requiring enzyme 1 (IRE1) inhibitor (4u8C), following which miR-146a and miR-155 expression was measured by RT-qPCR. IL-1β, IL-6, and TNF secretion was measured by enzyme-linked immunosorbent assays, and baseline expression of 384 different miRs was assessed using microfluidics assays. TNFR1 was found to be expressed on the surface of HC DF but expression was deficient in all samples with TRAPS-associated mutations. HC DF showed significant dose-dependent increases in both miR-146a and miR-155 expression levels in response to LPS; however, TRAPS DF failed to upregulate either miR-146a or miR-155 under the same conditions. This lack of miR-146a and miR-155 upregulation was associated with increased proinflammatory cytokine production in TRAPS DF in response to LPS challenge, which was abrogated by 4u8C. Incubation of HC DF with IL-1β led to downregulation of miR-146a and miR-155 expression, which was dependent on IRE1 enzyme. We observed global dysregulation of hundreds of other miRs at baseline in the TRAPS DF. In summary, these data suggest a mechanism whereby IL-1β, produced in response to activation of the UPR in TRAPS DF, downregulates miR-146a and miR-155, by inducing IRE1-dependent cleavage of both these miRs, thereby impairing negative regulation of NF-κB and increasing proinflammatory cytokine production.

A2.40 Investigating IL-6 pathway signalling kinetics in peripheral blood single cell subsets with tocilizumab therapy in patients with early rheumatoid arthritis

Background and objectives Rheumatoid arthritis (RA) arises in individuals with a genetic predisposition, triggered by environmental influences, leading to dysfunctional immune responses. The importance of pro-inflammatory cytokines, such as TNF and IL-6, in RA is well recognised, and the successful use of biologic agents inhibiting their action is widely established. The study of the intracellular effect of cytokine ligation to their receptors is of interest in elucidating mechanisms of action and potentially response prediction. This project focuses on the IL-6 signalling pathway and its blockade, using tocilizumab (TCZ; IL-6 receptor monoclonal antibody) to (i) determine the relative roles of the 3 arms of the pathway (JAK-STAT but also, PI3K/Akt and MAPK/ERK) in T-cells, B-cells and monocytes; (ii) examine whether there is heterogeneity in the predominant IL-6 intracellular signalling pathway and whether this associates with response to TCZ (iii) the effect of TCZ therapy on intracellular pathways. Materials and methods Multiparameter phosflow cytometry method to identify phosphorylation intensities of transcription factor STAT3 and tyrosine kinases Akt and Erk that cover the entire IL-6 pathway is being undertaken. Twenty patients with treatment-naïve, early RA; 10 of whom are receiving TCZ monotherapy and 10 receiving combined methotrexate and TCZ have been recruited. Peripheral blood mononuclear cells (PBMCs) have been isolated at baseline, weeks 24 and 48 after treatment and cryopreserved. Healthy individual samples have been used as control. PBMC are either unstimulated, or stimulated with IL-6 or PMA, in order to activate the pathway. Median fluorescence intensity (MFI) is being measured using LSRII (BD Biosciences), and data are analysed using BD FACSDiva software. Results After adequate optimisation and using a gating strategy to identify immune cell subsets, including lymphocytes (T, B and NK cells) and monocytes (CD14 and CD16 subsets), the phosphorylation kinetics of p-STAT3, p-Akt, p-Erk1/2 are being monitored. Preliminary data suggest that there are differences in immune cell signalling between healthy individuals and RA patients. In addition, following stimulation, differences in the MFI have been observed in the cell subsets. The phosflow cytometry and data analyses will be completed shortly and presented as heat maps to illustrate baseline differences and changes following TCZ. Conclusions Comprehensive evaluation of IL-6 intracellular signalling within immune cells from RA patients will provide insights into disease pathophysiology and heterogeneity, TCZ drug mechanism of action and possibly prediction of outcome/response.

A7.2 Identification of predictive micro-RNAs of progression from PRE-RA systemic autoimmunity to development of RA using matched serum samples

Background and objectives Recent EULAR recommendations for terminology of rheumatoid arthritis (RA) at-risk groups recognise those with systemic autoimmunity associated with pre-clinical RA, characterised by the presence of ACPA. Identification of predictors of progression from this pre-RA state to the RA syndrome would have clinical utility. MicroRNA (miRNA) are highly conserved small, non-coding RNA that serve as transcriptional negative regulators; with growing evidence that they contribute to the pathogenesis of human diseases, including RA. Our study aimed to identify serum miRNA that predict progression from pre-RA to RA using global profiling. Materials and methods Matched serum samples were used from 12 subjects presenting with CCP positivity only (CCP+) that were followed to the point of development of RA (Very Early RA, VERA). Samples were also obtained from 9 subjects with Early RA (ERA), symptom history of ≤ 12 months; 12 healthy controls formed a control group (HC). TaqMan low density MicroRNA Array Cards was applied to examine the expression of 754 human miRNAs and controls in the 4 cohorts: miRNA with a 4-fold change in expression (down or up-regulation) were selected as candidate biomarkers for RA progression. Heatmaps were plotted using unsupervised hierarchical clustering analysis, permitting separation of cohorts into groups according to miRNA expression levels. A validation analysis of the miRNAs of interest was conducted on a further 12 HC, 12 CCP+ individuals who did not progress and 12 CCP+ individuals that did progress, using custom miRNA array cards. Results After adequate normalisation and data analysis, serum miRNA profiling identified 22 candidate miRNA that were dysregulated when comparing their relative expression from one of the studied cohorts to another. We have identified a four-miRNA signature dysregulated between the matched CCP+ to VERA cohorts. In addition, hierarchical clustering revealed a clear differential miRNA expression between the cohorts. The validation study consists of evaluating expression levels of the 22 miRNA and also 9 miRNA that have been previously implicated in RA (such as miR-155 and miR-146a) using custom array cards in order to confirm their involvement in RA pathogenesis; the results of which are awaited. Conclusions Using a comprehensive MicroRNA array approach this study is a first to use matched pre-RA and VERA serum samples to identify dysregulated miRNA expression as biomarkers predictive of progression along this transition phase. We have identified 4 miRNAs in particular that could be potential diagnostic markers of RA, which we are about to validate further.

OP0309 Identification of A Predictive Microrna Signature of Progression from Pre-RA Systemic Autoimmunity to Development of RA Using Matched Serum Samples

Background Recent EULAR [1] recommendations for terminology of rheumatoid arthritis (RA) at-risk groups recognise those with systemic autoimmunity associated with pre-clinical RA, characterised by the presence of ACPA. Identification of predictors of progression from this pre-RA state to the RA syndrome would have clinical utility. We have reported on a number of potential clinical and imaging markers [2]. MicroRNA (miRNA) are highly conserved small, non-coding RNA that serve as transcriptional negative regulators; with growing evidence that they contribute to the pathogenesis of human diseases, including RA. Objectives Our study aimed to identify serum miRNA that predict progression in CCP+ individuals with no synovitis to RA using global profiling. Methods Matched serum samples were used from the following groups: (i) 12 subjects presenting with CCP positivity only (CCP+) (with no synovitis, confirmed on ultrasound) that were followed to the point of development of RA (Very Early RA, VERA) (ii) 9 subjects with Early RA (ERA), symptom history of ≤12 months (iii) 12 healthy controls (control group, HC). TaqMan low density MicroRNA Array Cards was applied to examine the expression of 754 human miRNAs and controls in the 4 cohorts: miRNA with a 4-fold change in expression (down or up-regulation) were selected as candidate biomarkers for RA progression. Heat maps were plotted using unsupervised hierarchical clustering analysis, permitting separation of cohorts into groups according to miRNA expression levels. A validation analysis of the miRNAs of interest was conducted on a further 12 HC, 12 CCP+ individuals who did not progress and 12 CCP+ individuals that did progress, using custom miRNA array cards. Results After adequate normalisation and data analysis, serum miRNA profiling identified 22 candidate miRNA that were dysregulated when comparing their relative expression from one of the studied cohorts to another. We identified a dysregulated four-miRNA signature between the matched CCP+ to VERA cohorts. In addition, hierarchical clustering revealed a clear differential miRNA expression between the cohorts (Figure 1). The validation study consists of evaluating expression levels of the 22 miRNA and also 9 miRNA that have been previously implicated in RA (such as miR-155 and miR-146a) using custom array cards in order to confirm their involvement in RA pathogenesis; the results of which are awaited. Conclusions Using a comprehensive MicroRNA array approach this study is a first to use matched pre-RA and VERA serum samples to identify dysregulated miRNA expression as biomarkers predictive of progression along this transition phase. We have identified 4 miRNAs in particular that could be potential diagnostic markers of RA, which we are about to validate further. References DM Gerlag et al, Ann Rheum Dis, 2012 C Rakieh et al, Ann Rheum Dis, 2014 Disclosure of Interest None declared

A1.52 TLR-Dependent XBP1 activation induces an autocrine loop in rheumatoid arthritis synovial fibroblasts

Background and Objectives Rheumatoid arthritis (RA) is a polygenic disorder usually arising from combined genetic predisposition and environmental influences with associated dysfunctional immune responses. The X-box binding protein 1 (XBP1) transcription factor is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes including RA. XBP1 can also be activated in direct response to Toll-like Receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Our aim was to investigate the relevance of interactions between UPR and TLR signalling pathways in the serum and synovial fibroblasts (SFs) of patients with RA, using samples from healthy individuals and patients with osteoarthritis (OA) as controls Materials and Methods Peripheral blood mononuclear cells (PBMC) were obtained from control groups: healthy individuals (n = 24) and RA patients, comprising active disease (n = 47) and remission (n = 12). SFs from RA and OA patients were isolated by digestion of synovial biopsies. Gene expression profiling was performed using qPCR for the detection of sXBP1 and enzyme-linked immunosorbent assays (ELISA) to quantify levels of pro-inflammatory cytokines, IL-6 and TNF. siRNA targeting of XBP1 was used for knockdown experiments in SFs. Results We investigated the expression of ER stress response genes in patients with active RA and patients in remission. We show that TLR-dependent XBP1 activation is operative in the SFs of patients with active RA. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission (p = 0.01). Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SFs by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent IL-6 and TNF production. We also show that TNF itself induces sXBP1 in SFs, thus generating a potential feedback loop for sustained activation of these cells. Conclusions sXBP1 plays a central role in two different cellular processes that may first appear unconnected. However, linking inflammatory pathways with ER stress provides SFs with a timely, coordinated and protective response. XBP1 activation may therefore be a suitable target in the treatment of RA, since it forms a cornerstone of two different molecular processes implicated in the pathogenesis of RA.