M.R. Gigante

Obesity as a risk and severity factor in rheumatic diseases (autoimmune chronic inflammatory diseases)

The growing body of evidence recognizing the adipose tissue (AT) as an active endocrine organ secreting bioactive mediators involved in metabolic and inflammatory disorders, together with the global epidemic of overweight and obesity, rise obesity as a hot topic of current research. The chronic state of low-grade inflammation present in the obese condition and the multiple pleiotropic effects of adipokines on the immune system has been implicated in the pathogenesis of several inflammatory conditions including rheumatic autoimmune and inflammatory diseases. We will discuss the main relevant evidences on the role of the AT on immune and inflammatory networks and the more recent evidences regarding the effects of obesity on the incidence and outcomes of the major autoimmune chronic inflammatory diseases.

MicroRNA-155 influences B-cell function through PU.1 in rheumatoid arthritis

MicroRNA-155 (miR-155) is an important regulator of B cells in mice. B cells have a critical role in the pathogenesis of rheumatoid arthritis (RA). Here we show that miR-155 is highly expressed in peripheral blood B cells from RA patients compared with healthy individuals, particularly in the IgD-CD27- memory B-cell population in ACPA+ RA. MiR-155 is highly expressed in RA B cells from patients with synovial tissue containing ectopic germinal centres compared with diffuse synovial tissue. MiR-155 expression is associated reciprocally with lower expression of PU.1 at B-cell level in the synovial compartment. Stimulation of healthy donor B cells with CD40L, anti-IgM, IL-21, CpG, IFN-α, IL-6 or BAFF induces miR-155 and decreases PU.1 expression. Finally, inhibition of endogenous miR-155 in B cells of RA patients restores PU.1 and reduces production of antibodies. Our data suggest that miR-155 is an important regulator of B-cell activation in RA.

Biomolecular features of inflammation in obese rheumatoid arthritis patients: management considerations

ABSTRACT Adipose tissue is an active organ playing a role not only in metabolism but also in immune and inflammatory processes, releasing several pro-inflammatory mediators. This can explain the possible association between obesity and rheumatoid arthritis (RA) and its role in the progression of the disease. Adipose and synovial tissues share common histological features of local inflammation in terms of activation of target tissues infiltrating cells (i.e. myeloid cells). Among the so-called adipocytokines, PEDF and Chemerin orchestrate the cellular cross-talk between adipose and myeloid cells, being possible biomarkers to monitor the effect of weight loss or the decrease of adipose tissue in patients with RA. Moreover, dietary intervention has been demonstrated to reduce Chemerin as well as IL-6 and MCP-1 expression. Finally, epigenetic regulators such as micro-RNAs (i.e. miR-155) are key regulators of myeloid cells activation in RA and obesity as well as in adipocytes. In this review, we will summarize the biological link between obesity/overweight state and RA focusing on pathophysiological mechanisms, consequences and management considerations.

A3.21 MicroRNA-34a and microRNA-155 in Systemic Sclerosis: possible epigenetic biomarkers of endothelial dysfunction in VEDOSS and long-standing disease

Background and Objectives MicroRNAs (miRs) are a novel class of post-transcriptional regulators that have been implicated in the pathogenesis of Systemic sclerosis (SSc). MiR-34a and miR-155 were found to be related to endothelial senescence and inflammation. The aim of this study was to investigate the expression of miR-34a and miR-155 in peripheral blood mononuclear cells (PBMCs) in SSc. Methods Twenty-seven consecutive patients with Raynaud phenomenon (RP) were enrolled in this exploratory study. Particularly, patients were divided into 3 different study cohorts: patients with primary RP with normal capillaroscopic findings and without any autoantibodies (n = 8), SSc patients fulfilling the Very Early Diagnosis Of Systemic Sclerosis (VEDOSS) criteria (n = 9) and SSc patients fulfilling the 1987 ACR criteria (n = 10) respectively. Gender and age matched healthy individuals (n = 7) were enrolled as controls. Expression of miR-34a and miR-155 was evaluated by qPCR on PBMCs isolated by gradient hystopaque technique from peripheral blood (PB) of all patients. To identify miR-34a HumanTargetScan cross-referenced was employed. Identified targets were experimentally verified by qPCR. VEGF, VEGF-RII, IL-6 and IL-6R plasma levels were determined through ELISA. Results MiR-155 is overexpressed either in VEDOSS (p = 0.0002) and SSc (p = 0.04) patients compared to healthy controls. MiR-34a expression is increased only in SSc compared to healthy controls (p = 0.01). Patients with primary RP did not differ according to miR-155 and miR-34a expression from healthy controls (p>0.05) Dividing SSc and VEDOSS patients according to the autoimmune profile, anti-Scl70 + have higher expression of miR-34a compared to anti-centromere + patients (p = 0.04). Furthermore, stratifying SSc patients according to the clinical vascular manifestations, SSc patients with active and/or previous digital ulcers have significantly higher miR-34a expression compared to patients without ulcers history (p = 0.01). Finally the expression of miR-34a directly correlated with the skin score value (R = 0.52, p = 0.032) in both SSc and VEDOSS patients and with IL-6 plasma levels (R = 0.42, p = 0.01). Using HumanTargetScan cross-referenced methodology, IL-6 receptor (IL6-R) was selected as target of miR-34a. IL6-R gene expression was found to be significantly higher in VEDOSS compared to SSc patients (p = 0.02). VEGF plasma levels were significantly higher in SSc patients compared to healthy controls (p = 0.01) as well as IL-6 plasma levels (p = 0.02), whereas no significant differences were found in VEGF-RII and IL-6R plasma levels comparing the different patients’ cohorts. Conclusions Epigenetic factors, as miR-34a and miR-155 are deregulated in SSc and VEDOSS. Their expression analysis could help to differentiate different disease phases and between primary and SSc associated RP.

OP0178 The Body Mass Index: A Determinant of Remission in Early Rheumatoid Arthritis

Background There are evidences that obesity determines higher disease activity status in rheumatoid arthritis (RA) and that obesity possibly affects the response to therapy in long standing RA. There are only few data about early RA (ERA), showing that obesity associates with a less erosive disease, with no clear explanation. Objectives To evaluate whether the body weight could influence the outcomes in patients with ERA in terms of disease remission and treatment after 6 and 12 month of follow-up. Methods 346 patients with ERA (symptoms duration <12 months), treated according to a treat-to-target strategy aiming at remission (strict follow-up visits, treatment with methotrexate up to 25 mg/week±steroids; then a combination with a TNF blocker if at least a good response according to EULAR criteria was not obtained), were enrolled. At each visit the ACR/EULAR core data set was registered. Baseline BMI was collected. Clinical remission was evaluated according to DAS and CDAI (Clinical Disease Activity Index) values. The BMI was categorized into three classes, as a BMI <25 Kg/m2 (normal weight), 25-30 (overweight) and >30 (obese), according to the NIH classification. Logistic regression models were applied to determine the influence of the independent variables that reached the value of p<0.25 at the univariate analysis, on the dependent variables “DAS and CDAI remission at 12th month and anti-TNF therapy at 12th month”. Results Of the 346 ERA patients (76.3% female, age 54.6±14.0 years, 32.9% very ERA, 70.2% seropositive, baseline DAS 3.6±1.1), 168 (48.6%) were normal weight, 135 (39%) overweight and 43 (12.4%) obese. The BMI values correlated with age (r=0.23, p<0.001), baseline inflammatory markers (ESR: r=0.14, p=0.009, CRP: r=0.19, p<0.001), DAS (r=0.18, p=0.001), CDAI (r=0.14, p=0.01), HAQ (r=0.17, p=0.001). Overweight and obese patients reached a lower rate of remission, both with DAS and CDAI criteria, at 6 and 12 month follow-up visits (sustained DAS remission at 12th month: 49.1% in normal, 28.7% in overweight, 34.1% in obese, p=0.008; CDAI remission at 12th month: 50%, 37.1%, 31% in normal, overweight and obese, respectively, p=0.07). Moreover, an higher percentage of obese and overweight ERA patients were under anti-TNF treatment after 12 months of follow-up (28.1% of obese, 28.8% of overweight, 16.2% of normal weight). At the multivariate analysis, the independent baseline variables associated with the risk of “not obtaining a sustained DAS remission at 12th month follow-up” were female gender (OR (95% CI): 2.44 (1.56-21.45)), baseline HAQ≥1.5 (OR: 1.56 (1.47-5.94)) and a BMI≥25 (OR: 2.22 (1.13-4.1)), whereas the variables identifying the “non-CDAI remission at 12th month” were female sex (OR: 1.97 (1.04-3.73)), baseline HAQ≥1.5 (OR: 1.76 (1.0-3.15)) and a BMI≥25 (OR: 1.81 (1.04-3.13)). The independent variables associated with the probability of being in anti-TNF therapy at 12th month follow-up were an age <55 years (OR: 2.7 (1.4-5.3)), baseline DAS≥3.7 (OR: 2.27 (1.19-4.34) and BMI≥25 (OR: 2.36 (1.21-4.59)). Conclusions Data suggest that in ERA patients, not only obesity but also overweight, associates with a lower percentage of success in obtaining remission. Overweight ERA subjects required 2.4 times more anti-TNF therapy, than normal weight to achieve the outcomes. BMI is one of the few modifiable variables influencing the major outcomes in RA. Disclosure of Interest None Declared

FRI0021 Weight Loss in Obese Rheumatoid Arthritis Patients Improves Disease Activity Without Modifying RA Treatment

Background Obesity is one of the potentially preventable risk factors for RA being associated with RA onset, disease severity and poor response to therapy. New evidence suggests that weight loss may lead to outcome improvement in obese osteoarthritis and psoriatic arthritis patients. Objectives To evaluate whether, in obese RA patients with a low-moderate disease activity, a weight loss obtained with a controlled nutritional intervention may lead to an improvement of disease activity and to a reduced need of increasing therapeutic regimens, without modifying RA treatment during the study period. Methods 64 consecutive obese RA patients (BMI>30 kg/m2) with DAS>1.6, treated according to a treat-to-target strategy and in a stable therapy with conventional DMARDs (cDMARDs) and/or biological DMARDs (bDMARDs) for at least 12 weeks, were enrolled. All patients underwent a scheduled diet under a Nutritionist guide, aimed at a weight loss >5% at 6 months (T6), maintaining unchanged the RA therapy. Patients were evaluated by rheumatologist and nutritionist every 2 months and at each visit clinical and laboratory data and the ACR/EULAR core data set was registered. Disease activity was evaluated by Disease Activity Score on 44 joints (DAS) and Simplified Disease Activity Index (SDAI). Results Of the 64 RA patients (82.8% female, age 56.5±12.5 years, disease duration 8.1±8.1 years, 66.9% seropositive, baseline (T0) DAS 2.8±0.7, baseline BMI 35.3±4.3), 36 (56.3%) were under cDMARDs-only therapy and 28 (43.8%) under biologic bDMARDs therapy ± cDMARDs. At now, 56 patients reached the 6 months follow-up. At T6, the mean reduction in percentage of body weight was 6.6±5.5% and of DAS was 25.4±28.5% (DAS T6: 2.0±0.7, p<0.01 vs DAS T0). Moreover, at T6 patients showed a significant improvement of SDAI with respect to baseline (p<0.01), of tender (p<0.01) and swollen (p<0.01) joint count, of systemic inflammatory parameters (ESR p=0.001, CRP p=0.003), GH (p<0.01), VAS pain (p<0.01) and HAQ (p<0.01), without any change of RA therapy nor corticosteroids need. Dividing patients according to the percentage of weight loss at T6, the 34 (60.1%) RA patients reaching a weight reduction >5% of the baseline body weight obtained higher rates of DAS remission than patients with a weight reduction <5% (DAS remission at T6: 35.3% vs 13.6%, respectively, p=0.07), as well as of SDAI remission (33.3% vs 4.5%, p=0.02). The differences were even more significant when considering a weight reduction >10% (DAS remission at T6: 57.1% in patients reaching 10% weight reduction vs 16.7% in not reaching patients, p=0.01; SDAI remission at T6: 57.1% vs 9.8%, p=0.001). Results were similar between patients under cDMARDs-only and bDMARDs treatment. Conclusions A weight loss obtained with a controlled diet in obese still active RA patients can allow to obtain a better disease control without changing the treatment of RA, and in particular a weight loss >10% permits to reach disease remission in a significant percentage of patients, and therefore reduces the need of an increase in therapy. The effects of weight loss based on a nutritional intervention, and so applicable at the population level, on the RA disease course appears to be crucial in terms of potential clinical and pharmacoeconomics perspectives. Disclosure of Interest None declared

THU0525 Metaflammation, PEDF and Chemerin: Potential Systemic Factors Which Link Obesity to Response to Therapy in Early Rheumatoid Arthritis

Background Obesity per se is a systemic, low-grade inflammatory state and the adipose tissue is an endocrine organ that releases bioactive substances, including pro-inflammatory cytokines, like TNFα and IL6 and specific adipokines. There are only few data about early RA (ERA), suggesting that obesity associates with disease outcomes. In this work we aimed to evaluate whether the body weight, and fat metabolic (PEDF-Pigment Epithelium-Derived Factor) and meta-inflammatory parameters (Chemerin), could be associated with the outcomes in terms of disease remission and treatment in ERA patients (symptoms duration <12 months). Methods 166 ERA patients, treated according to a treat-to-target strategy, were enrolled. At each visit the ACR/EULAR core data set was registered. Baseline BMI was collected and baseline interleukin-6, PEDF and Chemerin plasma levels were evaluated by ELISAs methods. PEDF gene expression was evaluated in adipose tissue of overweight and obese subjects (30 ERA and 10 osteoarthritis (OA) patients as control cohort). Results Of the 166 ERA patients (75.9% female, age 55.4±14.6 years, 34.3% very ERA, 66.9% seropositive, baseline DAS 3.4±1.0), 76 (45.8%) were normal weight, 67 (40.4%) overweight and 23 (13.9%) obese. Overweight and obese patients showed a higher disease activity at baseline compared to normal-weight patients (DAS: 3.6±1.0 vs 3.3±0.9, p=0.02). At baseline, BMI values correlated with baseline PEDF (r=0.33, p<0.001) and chemerin (r=0.31, p<0.001) plasma levels. Moreover, chemerin plasma levels correlated with age (r=0.31, p<0.001), baseline inflammatory markers (IL-6: r=0.28, p<0.001; ESR: r=0.39, p<0.001, CRP: r=0.35, p<0.001), swollen joint count (r=0.26, p<0.001), tender joint count (r=0.23, p<0.001), HAQ (r=0.24, p=0.002), DAS (r=0.34, p<0.001), CDAI (r=0.28, p<0.001) and SDAI (r=0.32, p<0.001). On the other hand, PEDF plasma levels correlated only with age (r=0.35, p<0.001) and baseline inflammatory markers (ESR: r=0.17, p=0.03, CRP: r=0.22, p=0.01). At 6 and 12 months none of the patients had a significant reduction of body-weight. A significant reduction of the disease activity at 6 and 12 months follow-up was observed in the three subgroup of ERA patients (normal-weight, overweight and obese patients). In the same manner, circulating chemerin levels significantly decreased over time; conversely, the circulating levels of PEDF remained unchanged. These findings were observed both in patients reaching and not reaching remission at 12 months of follow up. In adipose tissue, PEDF relative gene expression was 1.2±0.6 times higher in ERA patients compared to OA. Conclusions In ERA patients, PEDF and chemerin seem to be biomarkers of obesity and metaflammation, respectively. Chemerin seems to be linked to RA disease activity and to treatment response, irrespective of body weight cathegory, supporting its dual role in inflammation and metabolism and providing a link between chronic inflammation and obesity. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4936

Synovial predictors of differentiation to definite arthritis in patients with seronegative undifferentiated peripheral inflammatory arthritis: microRNA signature, histological and ultrasound features.

Objectives: To examine synovial tissue (ST) predictors of clinical differentiation in patients with seronegative undifferentiated peripheral inflammatory arthritis (UPIA). Methods: Fourty-two patients with IgA/IgM-Rheumatoid Factor and anti-citrullinated peptide antibodies negative UPIA, naive to Disease-Modifying Anti-Rheumatic Drugs, underwent Gray Scale (GSUS) and power Doppler (PDUS) evaluation and Ultrasound (US) guided ST biopsy. CD68, CD3, CD21, CD20, and CD31 synovial expression was evaluated by immunohistochemistry. Whole ST microRNA expression was assessed using miScript miRNA PCR Array. Peripheral blood (PB) and synovial fluid (SF) IL-6, VEGF-A, and VEGF-D levels were measured by ELISA and ST TNF expression was assessed by RT-PCR. Each patient was prospectively monitored and classified at baseline and within 1 year as UPIA, Rheumatoid Arthritis (RA), Spondyloarthritis (SpA) or Psoriatic Arthritis (PsA), respectively. Results: At baseline, CD68+ cells were the most common cells within the lining layer (p < 0.001) in seronegative UPIA, directly correlating with GSUS (R = 0.36; p = 0.02) and PDUS (R = 0.55; p < 0.001). Synovial CD31+ vessels count directly correlated with GSUS (R = 0.41; p = 0.01) and PDUS (R = 0.52; p < 0.001). During the follow-up, 6 (14.3%) UPIA reached a definite diagnosis (2 RA, 2 SpA and 2 PsA, respectively). At baseline, UPIA who differentiated had higher GSUS (p = 0.01), PDUS scores (p = 0.02) and higher histological scores for CD68+ (p = 0.005 and p = 0.04 for lining and sublining respectively), sublining CD3+ cells (p = 0.002), CD31+ vessels count (p < 0.001) and higher IL-6 PB levels (p = 0.01) than patients who remained as UPIA. MiRNA PCR Array showed that among the 86 tested miRNA species, at baseline, miR-346 and miR-214 were significantly down-regulated (p = 0.02 for both) in ST of UPIA who differentiated than in patients who remained as UPIA, inversely correlating with the lining CD68+ cells IHC score (R = −0.641; p = 0.048) and CD31+ vessels count (R = −0.665; p = 0.036) and with higher baseline ST expression of TNF (p = 0.014). Finally, logistic regression analysis demonstrated that baseline GSUS and PDUS scores ≥1.5 [OR:22.93 (95%CI:0.98–534.30)] and CD31+ vessels count ≥24.3 [OR:23.66 (95%CI:1.50–373.02)] were independent factors associated with the development of definite arthritis. Conclusions: MiRNA signature, histological and US features of ST may help in the identification of seronegative UPIA with high likelihood of clinical differentiation toward definite seronegative arthritis.

CHEMERIN AND PEDF ARE METAFLAMMATION-RELATED BIOMARKERS OF DISEASE ACTIVITY AND OBESITY IN RHEUMATOID ARTHRITIS

Objective: Obesity is a risk factor for Rheumatoid Arthritis (RA) being associated to low grade inflammation. This study aimed to determine whether PEDF and Chemerin are biomarkers of inflammation related to fat accumulation in RA and to investigate whether weight loss associates with clinical disease improvement through the modification of fat-related biomarkers in overweight/obese RA with low-moderate disease. Participants and Methods: Two-hundred and thirty RA patients were enrolled, of whom 176 at disease onset treated according to a treat-to-target strategy (T2T) and 54 overweight/obese RA in stable therapy and low-moderate disease activity. Gene expression of adipokines, interleukin-6 and their receptors were examined in adipose tissue from obese RA. Obese RA with low-moderate disease activity underwent low-calories diet aiming to Body Mass Index (BMI) reduction >5%, maintaining RA therapy unchanged. Chemerin, PEDF and Interleukin-6 plasma values were assessed by ELISA and disease activity was evaluated. Results: At RA onset, PEDF and Chemerin plasma values correlated with BMI (p < 0.001) but only Chemerin plasma values correlated with disease activity (p < 0.001). After adopting a T2T strategy, Chemerin arose as an independent factor associated with remission in early RA [OR(95%CIs):0.49(0.25–0.97)]. Moreover, after low-calories diet, RA with low-moderate disease activity reaching BMI reduction ≥5% (62.6%) at 6 months had significant decrease of PEDF (p < 0.05) and Chemerin (p < 0.05) plasma values, in parallel with the improvement in disease activity. Conclusions: PEDF and Chemerin arose as biomarkers of obesity and metaflammation respectively, providing a link between chronic inflammation and excess of body weight in RA. Therefore, BMI reduction of at least 5% in obese RA allowed better disease control without modifying RA treatment.

THU0028 Interleukin-6 receptor inhibition, as first-line b-dmard, affects b cell subpopulations distribution through epigenetic modifications in rheumatoid arthritispatients

Background Despite IL-6R inhibition was found to influence B cell subpopulations distribution in Rheumatoid Arthritis (RA), no data are available on the effect on epigenetic signature of RA B cells by this treatment. It is well known that B cell maturation is under control of the microRNA-155 (miR-155)/PU.1 axis significantly influenced by IL-6 stimulation1. Objectives To investigate the effect of IL-6R inhibition on the epigenetic signature of B cells (miR-155/PU.1 axis) in RA patients. Methods Twenty-nine RA patients [18 (62.1%) female; 57.2±14.9 years old; disease duration 1.3±0.7 years] starting IL-6R inhibitor treatment as first b-DMARD, have been enrolled. At study entry and after 3–6–12–18 months follow-up, CD19+ cells were isolated from peripheral blood (PB) by magnetic microbeads (Miltenyi) and B cells subpopulations were assessed through FACS according to the IgD/CD27 classification. MiR-155 and PU.1 endogenous expression was determined in PB-derived CD19+ cells by RT-PCR at baseline and after 3–6–12–18 months follow-up. IL-6 plasma level was assessed by ELISA at study entry for each patient. ACR/EULAR criteria were used to assess the response rate to IL-6R inhibitor treatment for each RA patient. PB-derived CD19+ cells of healthy individuals (HC) were used as comparison group. Results At study entry, RA patients showed higher percentage of IgD-/CD27- CD19+ cells (p<0.05) and IgD+/CD27+ CD19+ cells (p<0.05) than HC. Moreover, IgD-/CD27- CD19+ cells percentage directly correlated with Disease Activity Score (p=0.04) and IL-6 plasma levels (p=0.06) in RA patients. IL-6R inhibition lead to DAS and SDAI remission achievement in 73.9% and 52.2% of RA patients after 18 months follow-up, respectively, and significantly reduced IgD-/CD27- CD19+ cells percentage after 18 months follow-up (p<0.02). Stratifying RA patients based on the remission achievement during the follow-up, RA patients who achieved DAS remission under IL-6R inhibition showed a significant decreased of IgD-/CD27- CD19+ cells percentage compared to patients not achieving this outcome (p<0.05), reaching IgD-/CD27- CD19+ cells percentage comparable to HC (p>0.05). Analysing the epigenetic profile in B cells of RA patients, at baseline, PB-derived CD19+ cells of RA patients showed significantly higher endogenous expression of miR-155 (p=0.04) than HC. Moreover, RT-PCR showed that IL-6R inhibition significantly represses endogenous miR-155 expression in PB-derived RA B cells already after 3 months of treatment (p<0.05) and restores PU.1 expression in PB-derived B cells after 6 months (p<0.05) only in RA patients achieving disease remission. Conclusions IL-6R inhibitor, used as first b-DMARD treatment, acts restoring B cells homeostasis through epigenetic modulation in RA. In particular, IL6-R inhibition significantly represses endogenous expression of miR-155 in PB-derived CD19+ cells conversely restoring PU.1 expression mirrored by the decrease of IgD-/CD27- B cell rate in RA patients achieving disease remission. Reference [1] Alivernini S, Kurowska-Stolarska M, Tolusso B, et al. Nat Commun2016. Disclosure of Interest None declared