S. Canestri

PTPN22 1858C>T Polymorphism Distribution in Europe and Association with Rheumatoid Arthritis: Case-Control Study and Meta-Analysis

Objective The PTPN22 rs2476601 polymorphism is associated with rheumatoid arthritis (RA); nonetheless, the association is weaker or absent in some southern European populations. The aim of the study was to evaluate the association between the PTPN22 rs2476601 polymorphism and RA in Italian subjects and to compare our results with those of other European countries, carrying out a meta-analysis of European data. Methods A total of 396 RA cases and 477 controls, all of Italic ancestry, were genotyped for PTPN22 rs2476601 polymorphism. Patients were tested for autoantibodies positivity. The meta-analysis was performed on 23 selected studies. Results The PTPN22 T1858 allele was significantly more frequent in RA patients compared to controls (5.7% vs. 3.7%, p = 0.045). No clear relationship arose with the autoantibodies tested. The 1858T allele frequency in Italian RA patients was lower than the one described in northern European populations and similar to the frequency found in Spain, Turkey, Greece, Tunisia. A clear-cut North-South gradient arose from the analysis. Conclusions The PTPN22 T1858 allele is associated with RA in the Italian population. A North-South gradient of the allele frequency seems to exist in Europe, with a lower prevalence of the mutation in the Mediterranean area.

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.

Porphyromonas gingivalis and the pathogenesis of rheumatoid arthritis: analysis of various compartments including the synovial tissue

IntroductionWe evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed.MethodsPeripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA.ResultsNo differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability or positivity for autoantibodies.ConclusionsThe presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.

Polymorphisms of the IgH enhancer HS1.2 and risk of systemic lupus erythematosus

Objective To determine whether the allelic frequency variation of the HS1.2 enhancer of the immunoglobulin heavy chain (IgH) 3′ regulatory region (3′RR-1) locus represents a risk factor for systemic lupus erythematosus (SLE) and to identify a possible functional difference in the two most frequent alleles (*1 and *2) in binding nuclear factor- κB (NF-κB) and Sp1. Methods The frequency of the enhancer HS1.2 alleles was determined in two cohorts of patients with SLE (n=293) and in 1185 controls. Electrophoretic mobility shift assays (EMSA) were carried out with B cell nuclear extracts with different probes of HS1.2 alleles *1 and *2 to map the consensus binding sites of the nuclear factors. A confirmatory cohort of 121 patients with SLE was also included. Results The frequency of allele *2 of the HS1.2 enhancer was significantly increased in patients with SLE compared with controls (OR 1.60, 95% CI 1.33 to 1.92, p<0.001). EMSA experiments showed the presence of the Sp1 binding site in both alleles whereas only allele *2 carried the consensus for the NF-κB factor. The presence versus absence of allele *2 in patients with SLE correlated with a higher concentration of IgM levels and with the expression of B cell activating factor receptor (BAFF-R). Conclusions The increased frequency of allele *2 in patients with SLE identifies a new genetic risk factor for SLE. A possible biological effect of the polymorphism could be the difference observed in the localisation of an NF-κB binding site which is specific for allele *2 and absent in allele *1. These observations suggest a functional effect of the HS1.2 enhancer in this disease.

Memory B cell subsets and plasmablasts are lower in early than in long-standing Rheumatoid Arthritis

BackgroundAlterations of B cell subset distribution have been described in the peripheral blood (PB) of rheumatoid arthritis (RA) patients, but no data are available on differences between the onset and the established phases of the disease. The purpose of the study was to clarify whether a peculiar distribution of B cell subsets characterizes RA onset, thus leading to a more favorable clinical response to treatment, and to evaluate the possible association of a particular B cell subpopulation with response to therapy.Results122 RA patients were enrolled: 25 had symptom duration less than 3 months and were defined as having “very early RA” (VERA), and 43 had symptom duration from more than 3 months up to one year (early-RA: ERA). The other 54 RA patients had long-standing RA (LSRA). At baseline and at 6-month follow-up visit peripheral blood samples were collected and analyzed by flow cytometry for the distribution of circulating B cell subsets by staining with surface markers CD45, CD19, CD38, CD27 and IgD and intracellular marker ZAP70.VERA and ERA patients showed higher percentages and absolute counts of circulating antigen inexperienced naïve B cells (IgD + CD27-) and lower percentages and absolute numbers of double negative (IgD-CD27-) memory B cells and plasmablasts (CD38 + CD27+) compared to LSRA patients. At the multivariate analysis, a higher frequency of naïve B cells (IgD + CD27-) at baseline arose as significant predictor of CDAI remission, together with “having VERA disease” and a low disease activity at baseline.ConclusionsThe onset of RA is characterized by higher percentages and absolute numbers of naïve B cells and lower numbers of plasmablasts and double negative memory B cells compared to established RA. naïve B cells could represent a promising biomarker of outcome.

ZAP-70+ B Cell Subset Influences Response to B Cell Depletion Therapy and Early Repopulation in Rheumatoid Arthritis

Objective. To define the role of ZAP-70+ B cells (CD19+/ZAP-70+) as a biomarker of response to B cell depletion therapy (BCDT), their relationship with clinical outcome, and their behavior during repopulation of peripheral blood in patients with rheumatoid arthritis (RA). Methods. Thirty-one patients with RA underwent BCDT and were followed for 12 months. Disease activity was assessed with the European League Against Rheumatism (EULAR) criteria. Cytofluorimetric analysis of peripheral blood B cell subsets at baseline and at 6- and 12-month intervals after BCDT was performed using surface markers (CD45, CD3, CD56, CD19, IgD, CD38, CD27) and intracellular ZAP-70. Results. A moderate/good EULAR response was achieved in 66.6% of the RA cohort. The baseline percentage of CD19+/ZAP-70+ cells was lower in good responder patients (1.8% ± 1.7%) compared to poor responders (5.6% ± 4.9%; p = 0.02). A decrease of plasmablasts (IgD-CD27+CD38+) and pre-switch memory (IgD+CD27+) B cells occurred after BCDT. Recovery of B cells in peripheral blood after the first course of BCDT was characterized by the reappearance of B cell subtypes that showed a naive, activated phenotype, coupled with a decrease in memory cells. B cells carrying intracytoplasmic ZAP-70 increased significantly from the baseline value of 4.4% ± 4.5% to 12.4% ± 9.2% (p = 0.001) at the 6-month and to 9.4% ± 6.4% (p = 0.002) at the 12-month followup. Conclusion. Baseline percentage of CD19+/ZAP-70+ cells is associated with the clinical outcome after BCDT in patients with RA. Depletion of plasmablasts and pre-switch memory B cells and increase of CD19+/ZAP-70+ cells are features of the recovery of the B cell pool after BCDT.

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.

THU0089 Biomarkers of Erosive Disease in a Cohort of Early-Rheumatoid Arthritis Patients Treated According to the Treat to Target Strategy

Background Seropositive and seronegative rheumatoid arthritides (RA) have been considered as two different clinical entities, and traditionally autoantibodies have been associated to worse prognosis. Objectives To assess differences in disease characteristics, presence of erosions and outcome between anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF)-IgM and -IgA seropositive and seronegative early rheumatoid arthritis (ERA) patients. Methods A total of 386 ERA patients with a disease duration of less than 12 months were enrolled in the study. Seronegative ERA patients were defined as those negative for ACPA, RF-IgM and -IgA [113 (29.3%)]; all the other subjects were defined as seropositive [273 (70.7%)]. ERA patients fulfilled the 2010 ACR criteria for RA and were followed according to the treat-to-target strategy. Subjects with symptom duration less than 3 months were defined as having “very early RA” (VERA). At baseline, and every three months, the ACR/EULAR core data set [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), swollen joint count (SJC), tender joint count (TJC), physician and patient global assessment, pain, health assessment questionnaire (HAQ)] were recorded. At each visit, clinical improvement and remission were evaluated according to EULAR criteria. Results At diagnosis, seronegative patients were older (p=0.002) and had shorter disease duration, comprising a greater number of VERA (p=0.04), compared to seropositive ones, and at baseline had higher average of SJC (p<0.0001), TJC (p=0.004) and consequently DAS28 (p=0.02), CDAI (p=0.001) and SDAI (p=0.001). There were no differences in gender and presence of erosion between the two analyzed cohorts. Similar percentages of good-EULAR response and DAS-remission were seen over time between seropositive and seronegative patients. Dividing patients according to the positive autoantibody, between ACPA seropositive and seronegative subjects we observed the same previously reported differences. RF-IgM and RF-IgA seropositive patients had higher ESR value (p=0.03) and were more smokers (p=0.03), compared to seronegative ones. Moreover, a higher percentage of RF-IgA seropositive patients had erosions at baseline (p=0.03), a longer disease duration (p=0.03) and BMI>30 (p=0.02), compared to seronegative subjects. At 12th month of follow-up, patients that developed new erosions or had a worsening of Sharp erosion score (13.1%, of which 6.9% were seronegative and 20.0% were seropositive, p=0.02), differed from non erosive subjects only for ACPA seropositivity (p=0.04). At the multivariate analysis, RF-IgA seropositivity [OR (95%CI): 2.25 (1.32-3.82)] and age at diagnosis [OR (95%CI): 1.02 (1.01-1.04)] arose as significant predictors of erosive disease at baseline. At 12th month of follow-up, ACPA seropositivity [OR (95%CI): 3.61 (1.38-9.45)] and age at diagnosis [OR (95%CI): 1.03 (1.01-1.06)] were the variables significantly associated to development of new erosions and to worsening of Sharp erosion score. Conclusions Even though seronegative early RA had higher disease activity scores at diagnosis, the presence of RF-IgA and ACPA represent the strongest risk factors for being erosive and developing new erosions. 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